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PMC5290489

many large asian cities rely on groundwater for their water supply , and the abstraction of groundwater has commonly caused the development of large depression cones . in addition , some of these cities , exemplified by kolkata and hanoi , are situated on top of aquifers that are seriously contaminated by geogenic arsenic . generally , the uppermost holocene aquifers are the most - contaminated by arsenic , while underlying pleistocene aquifers , in most cases , are low in arsenic . because groundwater abstraction for municipal water supply occurs from the deeper pleistocene aquifers , with groundwater low in arsenic , the change in groundwater flow patterns may cause the migration of arsenic - rich groundwater from upper aquifers into the underlying low - arsenic aquifers . the effect of municipal groundwater abstraction pumping also influences the water quality on a regional scale because depression cones extend far beyond the city limits . both hanoi and dhaka are located along major rivers , and hydrogeological studies have shown groundwater abstraction to cause a very significant infiltration of river water into the underlying aquifers . the recharge of aquifers by losing rivers , often called bank infiltration , enhances the available water resource , and managed and monitored bank infiltration is a strategy that has been used extensively , particularly in europe . apart from increasing the quantity of the groundwater resource , bank infiltration also may improve the quality of the infiltrating surface water . during bank infiltration , processes taking place at the surface water groundwater interface can remove nutrients , organic carbon ( including organic contaminants ) , and pathogens such as microbes and viruses . however , the effect of red river bank infiltration on the water quality of the aquifers below hanoi is completely unknown , even though it is considered the main source of groundwater recharge below the city . the city of hanoi has a leaky sewerage system as well as open channels carrying wastewaters and untreated sewage water through the city , both of which are leaking into the underlying aquifers . in addition , the aquifers beneath hanoi are contaminated by arsenic with concentrations exceeding 300 g / l . in contrast , the water of the red river running along the city of hanoi is very low in dissolved arsenic . therefore , one could hope that the large scale infiltration of river water can improve the water quality in the aquifers and also flush out the arsenic . the time scale needed for flushing arsenic from the aquifer is currently unknown but must depend on infiltration rates , groundwater flow patterns , and residence times as well as the geochemical processes controlling the groundwater arsenic content . the latter includes the mobilization of arsenic by reductive dissolution of fe - oxides and the adsorption and desorption of the mobilized arsenite . the objective of the present study is to investigate the rate of infiltration of red river water into the underlying aquifer , clarify the geochemical processes occurring between the infiltrating water and the aquifer sediment , and predict the effect of river water infiltration on the groundwater arsenic content . our field locality at nam du ( 205825.43 n , 1055409.73 e ) is located on the eastern boundary of the city of hanoi , vietnam , directly on the shore of the red river . the geology of the area has been investigated extensively in relation with the establishment of the nam du well field in 2004 . generally , the geological sequence in the area consist of a top clay layer underlain by a up to 40 m thick holocene aquifer consisting of fine- to medium - grained sand , comprising more fine- or coarse - grained layers . the sandy aquifer is underlaid by an aquitard of variable thickness ( 110 m ) followed by a coarse - grained sand to gravel aquifer of pleistocene age . remote sensing shows the nam du area to belong to the meander belts of the red river . a detailed geological profile of our study area , based on natural logging of boreholes and sediment description , is shown in figure 1 . uppermost there is an up to 11 m thick layer of clay and silty clay , which is absent close to the river . below follows an up to 40 m layer of fine- to medium - sized sand . underneath , there is an up to 20 m thick layer of coarse sand and gravel . between the two sand formations , finally , at the bottom , there is a layer of claystone siltstone . in accordance with the geological description of the area , the upper aquifer with fine - grained sand is considered to be of holocene age , while the underlying coarse sand gravel layer is interpreted as being of pleistocene age . the intermediate clay lenses are probably remnants of the aquitard separating the holocene and the pleistocene aquifers , which elsewhere is found as a more - continuous layer . the upper layers of clay and fine medium - grained sand belong to the holocene , the coarse sand gravel to the pleistocene , and the silt the low measured electrical conductivity ( ec ) values ( 200270 s / cm ) indicate the presence of river water in the whole system . arsenic is low to absent in the river water and upper 17 m of the groundwater but is mobilized further down . the depth of the river channel at nam du varies between 13.5 and 17.0 m below the ground level at the nest of boreholes on the shore ( figure 1 ) . the mid channel depth is 15.3 m and is situated within the holocene aquifer . the groundwater below hanoi has been exploited since 1894 , and groundwater abstraction presently amounts to > 900 000 m / day . groundwater is being pumped mainly from six major well fields distributed throughout the city . as a result , large coalescing depression cones have developed below the city of hanoi in both the holocene and pleistocene aquifers , which are partially connected . the well field at nam du the pumping station included in our field site ( figure 1 ) is part of this gallery of pumping stations , but the flow toward the pumping station is the result of the drawdown caused by the entire pumping activity below hanoi . the hydrostatic pressure in our wells screened in the pleistocene aquifer is about 6 m lower than those in the nest screened in the holocene aquifer ( figure 1 ) , indicating a strong downward gradient induced by the pumping . the pumping combined with the less - permeable holocene deposit on top of the highly permeable pleistocene causes predominantly vertically downward groundwater flow , providing the shortest passage through the holocene . the pressure difference between the holocene and pleistocene aquifers is also found elsewhere in the city , but its magnitude varies from locality to locality . to investigate the hydrogeological and geochemical processes , we have collected three sets of data ( figure 1 ) ; ( a ) detailed water sampling of the sediment porewater performed on a sandbank in the river , ( b ) water sampling in a nest of boreholes located on the river shore with screens installed at different depths in the holocene aquifer , and ( c ) water sampling in boreholes and the nearest pumping station screened in the pleistocene aquifer . to sample the interface between river and groundwater , we used a 1 cm diameter drive - point piezometer and a 50 ml syringe for extracting the water . sampling was carried out in shallow water , approximately 25 cm deep , on a sandbank in the river located about 50 m from the shore , with a depth resolution of 0.20.5 m until 3 m below the surface ( figure 1 ) . at each depth the internal volume of the piezometer was emptied at least three times before taking the sample . on the shore , a nest of 11 wells ( figure 1 ) was installed in the depth range 4.940 m. additionally , four wells ranging in depth from 40 to 60 m were installed in the pleistocene aquifer between the pumping station and the nest at the shore ( figure 1 ) . the wells were equipped with 60 mm pvc casings , a 1 m long screen , and 1 m sand trap . a quartz sand filter pack was installed , and the well was sealed using bentonite . the top end of the pvc casing was sealed to prevent the entrance of surface water during flooding . to further avoid leakage along the casing , the top of the wells were embedded in a concrete platform , each well within a protective steel casing and steel screw cap . directly after completion , the wells were pumped to remove the water affected by the drilling operation . sampling of the wells was done more than three months after their installation in 20102011 . o2 , ph , and electrical conductivity ( ec ) were measured with probes in a flow cell mounted directly on the sampling tube . samples for other parameters were collected in 50 ml syringes and filtered through 0.2 m sartorius minisart cellulose acetate filters . aqueous as(v ) and as(iii ) were separated by passing the water through a disposable anion exchange cartridge . the cartridge was , together with the filter , carefully flushed with n2 before use . arsenite was determined as the as concentration in the water filtered through a cartridge , and as(v ) was calculated as the difference between the total as and as(iii ) concentrations . in the field , samples for cations were preserved with 2% of a 7 m hno3 solution and refrigerated until analysis in the laboratory . samples for nh4 , cl and so4 were collected in 20 ml polypropylene vials and frozen immediately after sampling . in the laboratory cations arsenic was determined on the same instrument using a hvg hydride generator and a graphite furnace . anions were analyzed by ion chromatography using a shimadzu lc20ad / hic-20a super . detection limits were as , 0.013 m ; mn , 0.91 m ; ca , 0.50 m ; nh4 , 5.6 m ; po4 , 1.1 m ; no3 , 3.2 m ; and so4 2.1 , m . samples for tritium helium dating were taken in duplicate in clamped copper tubes in 2015 and analyzed by the institut fr umweltphysik , universitt bremen . the accuracy of the dates is given by the standard deviation as 0.8 years ( jrgen sltenfu , personal communication ) tritium helium dating of water sampled from the boreholes in the nest screened in the holocene aquifer ( figure 1 ) is shown in figure 2 . the groundwater is extremely young in screens at all depths , indicating the fast infiltration of river water into the underlying aquifer . this piezometer is located in one corner of the well cluster , and here , a clay layer ( not shown in figure 1 ) was encountered during drilling from 26 to 28 m depth , which was not present in the other drillings just a few meters away . below this clay layer , the groundwater apparently has a larger residence time , yielding a higher groundwater age . the 30 m depth screen also shows a maximum in the dissolved as(iii ) concentration ( figure 3 ) and in the nh4 concentration ( not shown ) , reflecting that the longer groundwater residence time allows more as(iii ) and nh4 to build up . the remaining screens yield groundwater ages ranging from less than one year to 1.3 years in the screen at 40 m depth . duplicate measurements are , except for the 30 m screen ( figure 2 ) , within 0.05 years , indicating a high precision . however , the estimated accuracy of 0.8 years is high compared to the measured ages . in addition there is uncertainty as to whether infiltration occurs from the deeper or the more shallow parts of the channel . using the vertical distance between the mean channel depth of 15.3 m and the groundwater age of 1.3 0.8 years at 40 m yields a vertical transport rate of 19 m / year with an uncertainty range of 1249 m / year . helium groundwater ages , plotted in duplicate at all depths , show the presence of young water down to 40 m depth . the electrical conductivity ( ec ) in the groundwater is close to the value of 205 s / cm in the river water . the partial pressure of co2 is 10 in river water and 10 to 10 in the groundwater . effect of redox processes on the groundwater chemistry in the holocene aquifer at nam du . symbols designate field data , and lines indicate reactive transport model predictions with adsorption of as(iii ) disabled . in the model , the upper cell consumes 0.27 mm doc , which reduces o2 and no3 in the infiltrating water . further down , the oxidation of organic carbon and reduction of as - containing fe - oxides are controlled by rate equations in the model of postma et al . model lines are given for 60 years but are nearly identical for other times < 100 year . a further indicator for river water intruding into the aquifer is the electrical conductivity of the groundwater ( figure 2 ) . shallow groundwater on the shores of the red river 30 km nw of hanoi , unaffected by pumping , has an ec in the range 7501000 s / cm . river water , however , has an ec of around 200 s / cm , and the difference is due to mineral weathering mainly occurring in the soil zone . the strong contrast in ec between river water and normal groundwater makes the ec a good indicator for intrusion of river water into aquifers . as shown in figures 1 and 2 , river water and groundwater in the holocene and pleistocene aquifers , as well as water of the pumping station , all have an ec in the range of 200320 s / cm , indicating rapid recharge from the river to be the dominant source of water in the aquifer . an additional useful tracer parameter is the partial pressure of co2 ( pco2 ) . river water has a low pco2 value of 10 , close to that in the atmosphere ( pco2 = 10 ) . however , water infiltrating through soils , or through organic rich sediment , is exposed to root respiration and organic decay that generate co2 . at our field site 30 km upstream along the red river , the groundwater , therefore , has a pco2 in the range of 10 to 10.figure 2 shows how all the water in the aquifer below the river has a pco2 in the range of 10 to 10 . these low partial pressures of co2 also indicate the recharge of river water into the aquifer without being exposed to extensive organic matter decay or respiration . this is consistent with fast recharge of water through the bottom of the river . the river water has an o2 concentration of 0.24 mmol / l , only slightly below the concentration of 0.27 mol / l of nitrate . at a depth of a few meters below the surface , both oxygen and nitrate have disappeared from the groundwater ( figure 3 ) . at the same time , the river water and one sample at 1 m depth contain about 0.6 mmol / l dissolved organic carbon ( doc ) , while the remaining groundwater has a doc concentration near 0.1 mmol / l . as the river water infiltrates , the doc appears to be filtered by the uppermost sediment . stoichiometric calculation suggest that the oxidation of 0.27 mmol / l doc would be sufficient for the complete removal of dissolved oxygen and nitrate from the infiltrating river water . there is no evidence for fe - oxides functioning as electron acceptor for doc oxidation in the top sediment layers because fe(ii ) appears in the groundwater at greater depth . this pattern of doc filtration by the uppermost sediment layer and concomitant removal of dissolved o2 and nitrate was also observed at other bank infiltration sites . below 10 m depth , the fe(ii ) concentration starts to increase in the infiltrating water ( figure 3 ) , probably because the degradation of sedimentary organic carbon causes reductive dissolution of fe - oxides . similar fe(ii ) increases attributed to reductive dissolution of fe - oxides have been observed at other sites with ongoing bank infiltration . the distribution of arsenic in the river and groundwater is displayed in figure 1 and in detail for the holocene aquifer in figure 3 . the river water and the shallow groundwater are free of arsenic , while it is present in the deeper groundwater of the holocene , the pleistocene aquifers , and the water of the pumping station . this distribution indicates that the infiltrating river water is mobilizing arsenic from the aquifer sediment . found the rate of arsenic mobilization from the sediment into the groundwater to depend on the burial age of the aquifer sediment with the youngest sediments being most reactive . the geochemical processes controlling the groundwater arsenic content were quantified in a 1d reactive transport model , up to a burial age of 6000 years , using the code phreeqc-3 . in the model , the oxidation of organic matter causes the reduction of arsenic - containing fe - oxides , resulting in the release of fe(ii ) and as(iii ) to the groundwater . both organic carbon and fe - oxides become consumed over time and less - reactive as well . the oxidation of organic matter and the reduction of fe - oxides are controlled by separate rate equations that quantify their decreasing rates over time ( eqs 1 and 2 in the supporting information ) . the rate equations were calibrated using the groundwater chemistry of four sites on the red river floodplain 30 km northwest of hanoi , where both the burial age of the holocene aquifer sediment and the vertical flow rate of the groundwater are known . the application of the model developed for the field area 30 km north of hanoi to the nam du field site assumes that floodplain sediment is similar at the two sites . this appears a reasonable assumption given that remote sensing shows that both sites to belong to the red river meander belts , and they also have comparable holocene sediments . accordingly , we have applied the model to the nam du data ( see the supporting information for the phreeqc input file ) . the main difference between the two field sites is the much - faster vertical flow rate at nam du of 19 m / year compared to 0.5 m / year in the undisturbed aquifers north of hanoi . the burial age of aquifer sediments adjacent to the river at nam du is not known , but as an end - member case , we assume the aquifer to be very young ( < 100 years ) . with that assumption , the organic carbon and iron oxides have the highest possible reactivity , and the calculated release rates of fe(ii ) and as(iii ) will accordingly be maximum rates . any older sediment would consequently show lower fe(ii ) and as(iii ) release rates . the composition of the solution entering into the 1d model is that of river water . the results calculated by the model for 60 years of infiltration are shown as the solid lines in figure 3 but would be nearly identical for other times < 100 years . to simulate , in addition , the consumption of doc in the uppermost sediment , we have added a constant rate of organic carbon degradation of 5.1 mm c / year in the upper layer of the model , matching the removal of o2 and no3 in the incoming water by doc oxidation ( figure 3 ) . below the top layer , the oxidation rate of sedimentary organic carbon is calculated from the rate equation , and the main electron accepting process is the reductive dissolution of fe - oxides . figure 3 shows the modeled release of fe(ii ) to the groundwater , which corresponds to a rate of 35 m fe / year . in the bottom 4 m of the profile , a small amount of fe(ii ) becomes precipitated as siderite , but higher up in the profile , all fe(ii ) stays in solution . a comparison with field data shows the modeled values to be slightly higher than the field data , but the assumption that the holocene aquifer at nam du is young appears reasonable . otherwise , the modeled fe(ii ) concentration would be much higher than the measured fe(ii ) values because the fe(ii ) release of an old low - reaction sediment would be significantly lower . fe - oxides being reduced in the model contain 1.2 mmol as(v ) per mol of fe , a value based on analysis of fe - oxides by reductive dissolution in sediment samples from four sites with a different burial age of the aquifer sediment on the red river floodplain . during the reduction of fe - oxides in the aquifer , as(v ) becomes reduced to as(iii ) and is released to the groundwater . using the model , we can evaluate how much of the as measured in the groundwater could possibly be derived from reductive dissolution of as - containing fe - oxides from sediment that is considered to be < 100 years old . to keep all as(iii ) released from reduced fe - oxides in solution , we have disabled the adsorption of as(iii ) in the model . figure 3 compares the measured as(iii ) in the groundwater with the model prediction assuming reductive dissolution of as - containing fe - oxide being the only source of as(iii ) in the water . the modeled as profile in figure 3 corresponds to an as(iii ) release rate of 0.052 m as(iii)/year . for the 19 m / year vertical flow rate , the modeled as(iii ) concentration at 40 m depth is 0.11 m , while the uncertainty range of 1249 m / year in the vertical flow rate corresponds to a as(iii ) concentration range of 0.040.17 m . figure 3 clearly shows the modeled as concentration , released by reductive dissolution of fe - oxides in 70 year old sediment , to be much lower than the as concentration measured in the aquifer . any older sediment would have a lower reactivity and , therefore , a lower as(iii ) release rate therefore , most of the as(iii ) present in the groundwater can not be released by reductive dissolution of as - containing fe - oxides . note that the same release rate of 0.052 m as(iii)/year yields a modeled groundwater arsenic concentration of up to 4 mol as(iii)/l at the dan phuong locality upstream along the red river . because the reduction of fe - oxides , and thereby the release of as , is kinetically controlled , the resulting groundwater concentration will depend on the groundwater flow rate . at the dan phuong site , the downward groundwater velocity is 0.5 m / year , while at nam du , it is 19 m / year . the same as(iii ) release rate therefore produces groundwater with a as(iii ) concentration that is almost 40 times higher at dan phuong than at nam du . the importance of reaction kinetics on arsenic mobilization is also evident from the much - higher arsenic concentration found in porewater in sediment along the shores of the red river , 6 km further downstream at van phuc . vertical flow rates here are 0.13.1 m / year , which is an order of magnitude lower than the vertical flow rate we derive for the bottom of the river channel , and therefore result in a higher arsenic concentration . in fact , the main role of redox processes for arsenic mobility in the nam du aquifer is to maintain anoxic conditions in the aquifer , thereby keeping arsenic in its as(iii ) form . if the oxidation of arsenic to as(v ) should occur , it would cause a much - stronger arsenic retardation because as(v ) adsorbs much more strongly to the aquifer sediment . groundwater must have been high in as(iii ) before water supply pumping by the city of hanoi induced river water infiltration into the aquifer because high as(iii ) concentrations are seen everywhere else on the red river floodplain in aquifers contained in young sediments . dissolved as(iii ) in the groundwater is in equilibrium with as(iii ) adsorbed onto the sediment , and the results of a forced gradient experiment and push pull tests indicate that arsenic sorption rapidly responds to changes in the water chemistry . the infiltration of low - arsenic river water is therefore expected to cause desorption of arsenic from the aquifer sediment . the distribution of as(iii ) in the groundwater over depth , with low as(iii ) in the upper 15 m and higher as(iii ) further down ( figure 3 ) , is consequently interpreted as a leaching front , and its position will be determined by the number of pore volumes of groundwater flushed through the aquifer and the adsorption isotherm . in a previous study , we experimentally investigated the adsorption of as(iii ) onto the holocene aquifer sediment at the nam du field site using a sediment sample from 5.5 to 6 m in depth . the results showed a linear adsorption isotherm up to an aqueous concentration of at least 10 m as(iii ) corresponding to a distribution coefficient , kd of 12.6 l / kg . the adsorption of as(iii ) was also found to be completely reversible . for the measured aqueous concentration of 1 m as(iii ) ( figure 3 ) and a porosity of 0.36 , the isotherm predicts an adsorbed concentration of 59 mol as(iii ) per l of contacting groundwater , corresponding to a retardation of 60 . accordingly , it will take the flushing of 60 pore volumes of groundwater through the holocene aquifer to desorb all adsorbed as(iii ) . starting with an initial concentration of 1 m as(iii ) in the groundwater , close to what is measured in the groundwater , and the corresponding adsorbed as(iii ) concentration of 59 mol / l , we have calculated how the as(iii ) versus depth profile would look like at different times and compared it to the measured as(iii ) concentration ( figure 4 ) . as the water moves downward , the as(iii ) concentration increases along the isotherm until it reaches the concentration that was present in the aquifer before river water infiltration began . because the adsorption isotherm is linear , the shape of the leaching front will be independent of the as(iii ) concentration . in the model , the vertical flow rate is 19 m / year , and the leaching front moves downward with 0.32 m / year , and after 60 years of modeled river water infiltration , there is a reasonable correspondence with the field data ( figure 4 ) . using the uncertainty in the infiltration rate from 12 to 49 m / year , the range in front progression is 0.20 to 0.82 m / year , and the time for the leaching front to reach the same depth is from 96 to 24 years . although groundwater abstraction in hanoi started 100 years ago , we do not know when river water started to infiltrate into the aquifer at nam du . van geen et al . constructed a flow model for van phuc , on the next bend of the river 6 km further south . here , the pumping of the hanoi water supply has caused a flow reversal from a gaining to a losing river . the timing of the flow reversal was estimated to be between 1951 and 1971 . our estimate of 60 36 years of river water infiltration is within the time space of groundwater abstraction by the hanoi water supply but clearly associated with considerable uncertainty . lines are leaching profiles calculated using a kd of 12.6 l / kg and a flow velocity of 19 m / year , derived from tritium helium dating . the calculations were carried out for an initial groundwater concentration of 1 m as(iii ) . groundwater flow modeling suggest that infiltrating red river water is the main source of groundwater recharge into the aquifers beneath hanoi , which are heavily pumped by the municipal water supply . infiltrating river water may be beneficial for the groundwater quality in at least two ways . first , the river water contains no arsenic , and one may hope that its infiltration will lower the groundwater arsenic content over time . it is of major importance to understand the hydrogeological and geochemical processes occurring at the river our data from nam du suggest that a very fast recharge is taking place from the river channel . however , another recent study at the van phuc site located 6 km downstream from our site at nam du suggests much - slower infiltration to occur along the shores of the river . in future work , it would be important to delineate the relative importance of the two modes of recharge , particularly because they seem to result in a different groundwater arsenic content . while we find at nam du that , due to the high infiltration , the kinetically controlled reductive dissolution of as - containing fe - oxides only contributes insignificantly to the groundwater arsenic content , the study at van phuc suggest that the slow infiltration rates allow much - higher arsenic concentrations to build up in the groundwater . an additional reason for the much - higher arsenic concentrations found in porewater of the shoreface sediment could also be that van phuc is located just downstream of the channel at yen my , which drains most of the untreated urban wastewater from the city of hanoi into the red river , providing an ample source of reactive carbon . while the infiltrating river water may readily displace the original groundwater as the result of pumping , our results indicate that a decrease in the arsenic concentration in the pumped water will take much longer . our data indicates that it probably will take another 65 39 years to flush the adsorbed as(iii ) from the holocene aquifer . to reach the screens of the groundwater abstraction wells van geen et al . determined the retardation of as(iii ) in pleistocene sands to be 1620 , which is a factor of 34 lower than what we find in the holocene . in addition , many pleistocene sediment layers consist of highly permeable gravels that probably have an even - lower retardation . flushing of arsenic from the pleistocene aquifers is therefore expected to proceed much faster than from the holocene aquifers . thus , bank infiltration of red river water into the holocene and pleistocene aquifers will likely cause the groundwater arsenic concentration to decrease , but it will take several decades .

recharge of red river water into arsenic - contaminated aquifers below hanoi was investigated . the groundwater age at 40 m depth in the aquifer underlying the river was 1.3 0.8 years , determined by tritium helium dating . this corresponds to a vertical flow rate into the aquifer of 19 m / year . electrical conductivity and partial pressure of co2 ( pco2 ) indicate that water recharged from the river is present in both the sandy holocene and gravelly pleistocene aquifers and is also abstracted by the pumping station . infiltrating river water becomes anoxic in the uppermost aquifer due to the oxidation of dissolved organic carbon . further downward , sedimentary carbon oxidation causes the reduction of as - containing fe - oxides . because the release of arsenic by reduction of fe - oxides is controlled by the reaction rate , arsenic entering the solution becomes highly diluted in the high water flux and contributes little to the groundwater arsenic concentration . instead , the as concentration in the groundwater of up to 1 m is due to equilibrium - controlled desorption of arsenic , adsorbed to the sediment before river water started to infiltrate due to municipal pumping . calculations indicate that it will take several decades of river water infiltration to leach arsenic from the holocene aquifer to below the world health organization limit of 10 g / l .

Introduction Materials and Methods Results and Discussion

PMC3985714

the specificity of interactions is mostly determined by structural and physico - chemical properties of interfaces of interacting proteins . proteins highly similar in sequence can participate in different interactions , and even a small number of amino acid substitutions or insertions / deletions at binding interfaces can lead to different interaction partners or different binding modes . introducing mutations at protein protein interfaces can quite effectively modulate binding affinity or specificity , and site directed mutagenesis has been successfully used in rational protein design and directed evolution to create novel protein complexes . in some cases , the redesign of protein protein interactions may yield new binding partners with differences in specificities of at least 300-fold between the cognate and the noncognate complexes . protein interactions and lead to mendelian diseases or cancer . one possible way to assess the effect of mutation on protein binding affinity is to experimentally measure it . however , although site - directed mutagenesis methods are inexpensive and fast , surface plasmon resonance , isothermal titration calorimetry , fret and other methods used to measure binding affinity can be time - consuming and costly . therefore , the development of reliable computational approaches to predict changes in binding affinity upon mutation is urgently required . it is especially true in light of the rapid development of next generation sequencing technologies , which are producing overwhelming amounts of data on polymorphisms and disease mutations . there have been many studies aiming to develop potentials for protein ligand binding ( see references within ) . according to some studies , the burial of polar groups should not be penalized in the case of hydrophilic protein , the contribution of electrostatic interactions might be more pronounced when switching from protein folding to protein protein interaction potentials . many models have been proposed that try to estimate binding affinities of protein protein complexes , yet they can not reproduce experimental binding data with satisfactory accuracy . this happens mostly due to the difficulties associated with modeling of conformational changes upon binding , estimating the entropy changes and long - range noninterfacial interactions . moreover , most of the approaches are largely tuned to predict the effect of alanine - scanning mutations defined as substitutions of residues into alanine . energy functions used to describe and predict interactions governing protein protein binding linearly combine several energy terms and the coefficients in front of each term can be optimized using datasets of experimentally measured binding affinities or changes in binding affinity due to missense mutations . several approaches have recently been proposed that predict the effects of point mutations on binding energy . they include coarse - grained predictors based on statistical or empirical potentials , molecular mechanics force fields with different solvation models and others . for example , the molecular mechanics poisson boltzmann surface area ( mm - pbsa ) method has been shown to yield good agreement with experimental studies in determining protein stability , binding affinity and ranking docking templates . certain methods specifically investigate the impact of mutations on binding in relation to function or different environmental conditions . for example , recently an in silico mutagenesis method was proposed to calculate the effect of mutation at various ph , whereas another approach predicted the mutations ability to shift the allosteric conformational equilibrium with consequences for functional regulation . although many of these methods report reasonable root - mean - square error ( rmse ) values , their applicability is biased toward very limited sets of proteins and mutations ( on an order of a dozen protein complexes and up to several hundreds of mutations ) used for training and parametrization . despite their limitations , methods that estimate changes in affinity produced by mutations show somewhat better performance compared to computational approaches to predict absolute affinities . it can be partially attributed to counterbalancing of errors , produced by energy calculations or conformational sampling , between the wild - type and mutant structures , under an assumption that structures do not undergo large changes upon mutations . here we introduce a new approach to assess the impact of single and multiple missense mutations on protein binding affinity . it uses a modified mm - pbsa energy function , statistical scoring function and efficient energy minimization protocol . our protocol is parametrized on a large set of several thousands of mutations and their corresponding experimental binding free energy changes assembled from the scientific literature . we find that the choice of water model is very important for the quality of prediction and a simulation protocol with explicit water without restraints on the backbone atoms gives the best results . we show that the conformational sampling by molecular dynamics simulations does not help to achieve better quality predictions for nm set ( see methods ) , and further analyses are needed to prove or disprove this observation for larger sets of mutations . in addition , we describe how prediction accuracy depends on the type of amino acid substitution and its location in the complex . compared to several available computational protocols , our approach achieves very good accuracy and speed . it is benchmarked against a large and independent experimental dataset and shows high correlation coefficients between predicted and experimental values of 0.69 and 0.63 with root - mean - square errors of 1.20 and 1.90 kcal mol for single and multiple mutations , respectively . at the same time , it can efficiently handle the large amount of data generated by modern genomics techniques . we use two datasets of experimentally determined values of changes in standard binding free energy upon mutations ( denoted as g in the paper ) , which are calculated as the difference between binding affinities of mutant ( gmut ) and wild - type ( gwt ) complexes and are derived from the scientific literature for protein protein complexes with experimentally determined wild - type structures . one set is taken from the paper of bockmann et al . and includes 242 single mutations and 123 multiple mutations from nine wild - type protein protein complexes ( we retained only structures of wild - type proteins , eliminated redundant entries and made sure that the skempi set did not overlap with the nm set . we also excluded ten single and eight multiple mutants having initial vmd models ( see next section ) with unrealistically large steric clashes between the substituted and adjacent residues that could not be fixed by the minimization procedure ( table s1 , supporting information ) . as a result , our second experimental set included 1844 single and 574 multiple mutations from 81 wild - type protein protein complexes ( it will be referred to as the skempi set results for our and other methods and main scripts for running the programs are accessible through ftp://ftp.ncbi.nih.gov/pub/panch/mutation_binding . an energy minimization procedure is used to compute the equilibrium configuration and find a minimum of the potential energy of the system . here we use a conjugate gradient algorithm implemented in namd , which finds a nearest minimum to the starting point , repairs distorted geometries and removes steric clashes . the initial crystal structures of wild - type protein protein complexes were obtained from the protein data bank ( pdb ) ; only protein chains were retained in the calculations and missing heavy side chain atoms and hydrogen atoms were added using the vmd ( version 1.9.1 ) program . the procedure for adding atoms in vmd we tested several simulation protocols ( figure s1 , supporting information ) ( 1 ) with and without restrains on the backbone atoms upon minimization using explicit tip3p water model ( 2 ) with an unconstrained molecular dynamics ( md ) simulations with tip3p explicit water model and ( 3 ) with the minimization using the generalized born implicit solvent model for both wild - type and mutant structures . a simulation protocol that used minimization without restraints on the backbone atoms in explicit water showed the best agreement with the experiments ( see results ) . all models were immersed into rectangular boxes of water molecules extending up to 10 from the protein in each direction . to ensure an ionic concentration of 150 mm and zero net charge , the distribution of the system size including water molecules and ions is shown in figure s2 ( supporting information ) . all wild - type protein complexes were minimized using a 40 000-step energy minimization procedure to make all wild - type protein complexes fully optimized for further binding energy calculation and the preparation of the initial mutant structures . this 40 000-step energy minimization procedure included an initial 5000-step minimization that was carried out using harmonic restraints ( with the force constant of 5 kcal mol ) applied on the backbone atoms of all residues , followed by a 35 000-step energy minimization on the whole system . we used the final minimized models of wild - type protein complexes to produce the mutant structures ( figure s1 , supporting information ) ; namely , we introduced an amino acid substitution using the mutator plugin of the vmd and kept the rest of the system invariant including the water box and ions . then we performed an additional 1000-step minimization of the mutant ( altogether 40 000 steps for wild - type and 1000 steps for mutant minimization ) and showed that the agreement with the experimental data increases as the number of steps increases but reaches a plateau after 300500 steps for single mutations . figure s3 ( supporting information ) shows that increasing the number of minimization steps for a mutant above 300500 does not improve the agreement with the experiments . moreover , we showed that even very large complexes did not require more than 300500 minimization steps for mutants ( figure s4 , supporting information ) . the minimized models of mutant protein complexes after 300 minimization steps were used in our further analysis . minimization was done only for the protein complexes , and protein structures of monomers were extracted from the minimized complexes for the following energy calculation . consistent with the previous studies , this approach was more accurate and faster than when minimization was done for complexes and monomers separately . the energy minimization and md simulation were carried out with the namd program version 2.9 using the charmm27 force field . periodic boundary conditions and a 12 cutoff distance for nonbonded interactions were applied to the systems . the particle mesh ewald ( pme ) method was used to calculate the long - range electrostatic interactions . lengths of hydrogen - containing bonds were constrained by the shake algorithm . unlike minimization , md simulation generates atomic trajectories and may be used to generate conformational ensemble and determine macroscopic properties of the system by ensemble averages . in addition , md simulations can in principle explore conformations which are not accessible via energy minimization protocol because of the energy barriers between different conformational states . binding energies were calculated based on the mm - pbsa method that combines the molecular mechanical terms with the poisson boltzmann continuum representation of the solvent calculated using the charmm force field . the total free energy in mm - pbsa is expressed as the following:1here egasmm corresponds to the molecular mechanical energy and is calculated as a sum of the internal energy of the molecule , the van der waals and coulomb electrostatic interactions in the gas - phase . gsolvp is the polar contribution to the solvation free energy of the molecule calculated using the poisson boltzmann ( pb ) equation , which is the difference between the electrostatic energy of solute in the solvent environment and in the reference environment ( gas phase in our study).gsolvnp is the nonpolar solvation energy , which is proportional to the solvent - accessible surface area ( sa ) of a molecule and ts corresponds to the entropy of solute . as entropy calculations are very computationally expensive and might not be very accurate , we did not perform entropy calculations in our analysis . moreover , as was discussed in previous studies , entropy terms may cancel each other later in eq 3 , especially for cases when structures do not undergo large changes upon mutations in the minimization simulation protocol . the binding energy in mm - pbsa approach is usually calculated as a difference between the average energies of the complex and each monomer:2then the change of the binding energy due to a mutation can be calculated as the following:3 we performed a multiple regression fitting procedure to model the linear relationship between the response ( dependent ) variable , which is , in our case , experimental values of changes in binding affinity , and different dependent variables . we tried different types of energy functions composed of different energy components ( see results section ) and estimated the optimal model parameters ( the weight factors for each energy term ) using the nm and skempi experimental sets . we assessed the accuracy of predictions ( tables 2 and 3 ) and found that energy function pred1 had the best agreement with experiments for both datasets and had only four parameters and three energy terms , all of which were derived from charmm:4here evdw is the change of van der waals interaction energy and gsolv is the change of polar solvation energy of solute in water . samut represents a term proportional to the interface area of the mutant complex ( if we choose the wild - type complex interface area , the results are not significantly different from those where mutant interface area is used ) . nonpolar solvation energy and other terms did not contribute significantly to the model quality ( p - value > 0.01 ) and were not used in our model . for the pb calculation dielectric constants = 1 , 2 , 4 and different ion concentrations were tested on single mutations of nm set using the optimized minimization protocol and energy function pred1 ( the testing results are shown in table s2 , supporting information ) . as a result , = 2 for the protein interior , = 80 for the exterior aqueous environment and ion concentration of zero were used for further energy calculations . all pb calculations were performed with the pbeq module ; the van der waals interaction energy was calculated with the energy module , and molecular surface area was obtained with the sasa module of the charmm program . the atomic born radii were previously calibrated and optimized to reproduce the electrostatic free energy of the 20 amino acids in md simulations with explicit water molecules . the foldx method calculates the effect of mutations on protein stability using an empirical force field . it optimizes the side chain configurations without taking into account the backbone conformational movements . we tested two different protocols using foldx : the repairpdb module applied to protein crystal structures and the repairpdb module applied to the minimized structures . we choose the first foldx protocol for comparison as it gave us better correlation with experimental values . to calculate binding energy by foldx , we used eq 2 and calculated the unfolding free energy of the whole complex and each monomer separately . the second method , beatmusic is specifically trained to calculate the change in binding affinity produced by single missense mutations . it uses residue based statistical potentials that were previously optimized to discriminate native from decoy complexes . in addition , the beatmusic energy function has terms accounting for packing defects and for the effect of mutations on the unfolding free energy of the whole complex in cases where unbound monomers could be considered disordered . the third method , cc / pbsa , applies the concoord approach to sample the protein configurational ensemble and the pb approach to calculate the solvent contribution to the binding free energy . we used two main measures to estimate the quality of the agreement between experimental and predicted values . first , we calculated the pearson correlation coefficient and tested a null hypothesis about the equality of the correlation coefficient to zero . all correlation coefficients reported in the paper were significantly different from zero with p - values of less than 0.01 . another measure was the root - mean - square error ( rmse ) , which is the square root of the sum of the differences between values predicted by a model and experimental values divided by the number of cases . five - fold cross validation was done on training sets where the data was randomly partitioned into five subsets of approximately equal size , and one subset was used for testing the model and the remaining four subsets were used as training data . to diminish similarity between training and testing sets and ensure the accuracy of cross - validated correlation coefficients , protein complexes from the same similarity cluster , provided by the skempi database , were used only for training and not for testing . our goal is to construct a computational protocol that would yield good prediction accuracy for a diverse and large set of missense mutations . we obtained a new energy function with weighting factors in front of each terms based on eq 3 ( see methods ) by applying a multiple linear regression procedure . this model was fit to the experimental data of the differences in binding affinities produced by mutations . because several mutations can interact with each other and exhibit cooperativity and positive epistasis , we performed the fitting procedure separately for single and multiple mutations . our results showed that the separate parameter optimization for single and multiple mutations yielded a better agreement with experiments than if we combined them . table 1 ( table s2 , supporting information , has more detailed information ) shows results for different simulation protocols for 242 single mutants from the nm set with the 5-fold cross - validation using the pred1 energy function . as can be seen from table 1 , minimization in explicit water without restrains on the backbone atoms shows the highest correlation which indicates how important the water model is for the quality of prediction . moreover , minimizing the structures of mutants results in a better quality of g estimates than a protocol where the vmd in silico model is used without minimization ( see zero minimization step in figure s3a d , supporting information ) . however , increasing the number of minimization steps after about 300 does not significantly improve the average prediction performance . the exception is the minimization for multiple mutations from the skempi set where the shorter minimization procedure in general produces better results ( figure s3d , supporting information ) . r , the pearson correlation coefficient between experimental and predicted g values , r , the five - fold cross - validated correlation and rmse , the root - mean squared error are each shown for the case of training / testing on single mutations of nm set . this table only shows the results with dielectric constant of two and ion concentration of zero . we executed 1 ns md simulations for 242 single mutants from nine protein complexes of the nm set . no significant conformational changes were observed in most cases in terms of rmsd from the starting mutant complex that was minimized for 1000 steps prior to md simulation . relatively large conformational changes between the wild - type and mutant proteins were observed in a few cases ( rmsd of more than 2.5 ) , although the average backbone rmsd was less than 1.5 ( figure s5 , supporting information ) . we observed even smaller backbone conformational changes during the minimization procedure ( data not shown ) . as can be seen from table 1 and figure s3(e , f ) ( supporting information ) , md simulation over 1 ns yields a maximum correlation of 0.40 between experiments and predictions for cases when md simulations were done for both mutant and wild - type complexes ( figure s3f , supporting information ) . nevertheless , it is considerably lower compared to the protocol when only minimization procedure is used . tables 2 and 3 compare our approach with other methods for single and multiple mutations , respectively . first , we tested two energy functions : pred3 and pred4 with 11 and 13 parameters for single and multiple mutations , respectively ( energy functions are defined in the supporting information ) . pred4 and pred3 were applied to single and multiple mutations of the nm set ( tables 2 and 3 ) and produced very good agreement with the experiment ( cross - validated correlation coefficient of 0.68 and 0.85 for pred4 and pred3 , respectively , applied to single and multiple mutations ) , but they showed rather poor results on the large representative set of skempi mutations with the correlation coefficients of 0.37 and 0.46 for single and multiple mutations , respectively . it should be mentioned that , unlike the correlation coefficient , rmse values are scale dependent and can only be compared for different methods for the same test set , not between different sets . it is known that if parameters are tuned to overminimize mean squared errors and overfit the model , this can lead to decreased generalization performance . r is the pearson correlation coefficient between experimental and predicted g values and r is the five - fold cross - validated correlation coefficient . the last column shows the slope of the regression line between predicted and experimental values . r is the pearson correlation coefficient between experimental and predicted g values and r is the five - fold cross - validated correlation coefficient . the last column shows the slope of the regression line between predicted and experimental values . taking all this into account , we have constructed a model and ensured that it was trained on one large representative set ( skempi ) and yielded a good agreement with experiments when tested on another independent set ( nm ) . our energy function pred1 ( see methods , eq 4 ) had four parameters and three energy terms . energy terms with statistically significant contribution to the quality of multiple regression model are listed in table s3 ( supporting information ) together with their corresponding coefficients / weights . as can be seen from tables 2 and 3 , the pred1 function gives a correlation coefficient of 0.62 and 0.63 and rmse values of 1.27 and 1.90 kcal mol , if trained on skempi and tested on the nm set for single and multiple mutations respectively . interestingly , the energy function pred1 trained on a set of skempi multiple mutations showed better results for multiple mutations compared to using the energy function pred1 trained on skempi single mutations set ( correlation coefficient of 0.55 versus 0.49 ) . we compared our approach with three other independent methods , foldx , beatmusic and cc / pbsa . beatmusic can not be applied to multiple mutations whereas cc / pbsa is not very efficient for large sets like skempi because of its slow computation time . as can be seen from table 2 , beatmusic overall yields better results than foldx whereas the pred1 model gives a better agreement with the experiments compared to both of these methods ( correlation of 0.62 , 0.52 and 0.47 for pred1 , beatmusic and foldx , respectively , for testing on the nm set ) . limited foldx capacity can be attributed to the fact that the foldx empirical energy function was parametrized on the experimental changes of unfolding free energy , not the binding affinity . the comparison of their computational speed will be reported in another section . given the relatively high algorithm efficiency of the foldx and beatmusic methods , we decided to combine their scoring schemes with our pred1 energy function.5the performance of the combined model ( pred2 ) exceeded the performances of the individual pred1 ( p - value < 0.01 , see table s3 , supporting information ) , foldx and beatmusic methods and returned the correlation coefficient of 0.69 with an rmse of 1.20 kcal mol . importantly , the slope of the regression line for pred2 is very close to 1 ( 0.97 ) , which indicates that predicted values are on the same scale as experimental ones . in addition , we used the cc / pbsa server and managed to obtain the results for 66 single mutations randomly selected from the skempi set . after excluding five mutations with very high predicted energies of gcc / pbsa > 100 kcal mol , the correlation between experimental and calculated values for remaining 61 mutations was found to be r = 0.23 ( p - value = 0.07 ) using the cc / pbsa method and r = 0.47 ( p - value < 0.01 ) using the pred2 energy function . we further compared the accuracy separately for mutations that stabilized ( gpred < 0 ) or destabilized ( gpred > 0 ) the protein protein binding . mutations were also subdivided into binding hot spots ( |gpred| 2 kcal mol ) or others ( |gpred| < 2 kcal mol ) . the prediction accuracy was defined as a percentage of correctly identified mutations out of the total number of mutations ( tp + tn)/total , where tp denotes true positives and tn corresponds to true negatives . sensitivity was defined as tp/(tp + fn ) and specificity was calculated as tn/(tn + fp ) ( fn , false negative ; fp , false positive ) . table 4 shows that the pred2 energy function has very high prediction accuracy for destabilizing mutants but low accuracy for stabilizing mutants . stabilizing mutants are not well predicted by any of the methods used in this study and the foldx method provides the highest prediction accuracy for stabilizing mutants . the specificity of predictions of binding hot spots is almost 100% for all methods although the sensitivity is compromised for all of them . we did not have enough data to perform a similar analysis for multiple mutations . figure 1 shows experimental and predicted g for single and multiple mutants for the skempi training set and the nm test set . we can see that only a few stabilizing mutants are predicted as stabilizing whereas the majority of them are predicted as destabilizing . training is performed on skempi single ( a , b ) and multiple ( c , d ) mutation sets . a : testing on skempi single mutation set . beatmusic has the shortest processing time and calculation for one mutation takes less than a second on its webserver ( http://babylone.ulb.ac.be/beatmusic/ ) . foldx is also very fast and takes about 5 min for a protein of about 300 residues when calculations are performed on an intel core duo2 2.8 ghz processor . if we consider the parallel cpu calculation ability , our minimization protocol with namd uses 15 min of cpu time for a protein of 160 residues and 10 water box , altogether of about 23 900 atoms on 16 processors ( with a 2.8 ghz intel emt64 ) . there is no need to redo the long wild - type minimization procedure if several mutations of one protein are analyzed . in our study , we utilized the high computational capabilities of the biowulf linux cluster at the national institutes of health . for the binding free energy calculation using charmm , it takes about 10 min on one processor for about 160 residues protein ( 2.6 ghz amd opteron ) . the cc / pbsa approach uses the conformational sampling method and has long processing time ( 249 min for 149 residue protein on a 3.2 ghz intel xeon processor , as reported in ref ( 22 ) ) . however , the cc / pbsa webserver ( http://ccpbsa.biologie.uni-erlangen.de/ccpbsa/ ) takes much longer to process one mutation and is not practically applicable for large datasets . on the basis of our previously mentioned results , we decided to use skempi as a representative training set for the multiple regression fitting procedure . the resulting coefficients / weights in front of each energy terms and standardized coefficients in front of standardized variables ( variable with the variance equal to one ) are listed in table s3 ( supporting information ) . as can be seen from this table , the polar solvation energy term calculated using poisson boltzmann equation ( gsolv ) and the van der waals ( evdw ) terms have largest contributions to the total change in binding energy gpred1 for single mutants ( standardized coefficients of 0.40 and 0.34 , respectively ) . interestingly , for multiple mutations , gsolv becomes twice as important as for single mutations whereas the role of interface area diminishes . if we use the pred2 model , then the largest contribution also comes from the polar solvation energy term ( standardized coefficient of 0.31 ) . the beatmusic score ( gbm ) has a relatively high impact , whereas foldx and interface area make smaller contributions to the quality of the model ( standardized coefficients of about 0.15 ) . however , all above terms contribute significantly to the agreement between experiments and predictions with p - values of less than 0.01 . in this section , we consider several factors that might influence the quality of the prediction for single mutations . importantly , all of these factors represent features of studied mutation sites that can be derived either from sequences or protein structures a priori . first , we analyzed the effect of the type of residue substitutions on prediction accuracy . figure 2 shows the boxplots of prediction errors for wild - type and mutant residue types , respectively . in each box , the central line is the median , the edges of the box are the 25th and 75th percentiles , the whiskers extend to the most extreme data points and outliers are plotted with circles . the median signed error for most wild - type amino acids is close to zero with two notable exceptions of overprediction , for met and pro . on the other side of the spectrum are glu , tyr and thr , which produce underprediction errors but not as prominently as met and pro . as to the mutant residue type , pro the cyclic structure of proline s side chain introduces constraints on the main - chain dihedral angles and can be structurally important for stability or binding . as can be seen from figure s6 ( supporting information ) , substitutions from / into pro might cause relatively large local conformational changes compared to other substitution types . moreover , substitutions of other amino acids into proline can result in significant destabilization of complexes ( figure s7 , supporting information ) . boxplots of prediction error ( residual ) for different wild - type ( a ) and mutant ( b ) residue types for single mutants from skempi training set . the residual is calculated as a difference between experimental gexp and predicted gpred2 values . as was previously shown , mutations resulting in amino acid substitutions with similar physico - chemical properties may not drastically alter the stability of a protein or a complex . we examined the performance of our model pred2 in relation to the change in charge and volume of side chains produced by mutations ( tables s4 and s5 , supporting information ) . one can see from table s4 ( supporting information ) that the majority of substitutions are neutral - into - neutral , whereas the minority of them change the charge of amino acids . positive - into - negative and neutral - into - negative amino acid substitutions are characterized by notable correlations ( not enough statistics to estimate negative - into - positive charge changing substitutions ) . if we consider changes in the amino acid volume , then the highest correlation is observed for substitutions of small - into - large amino acids ( table s5 , supporting information ) . as can be seen from figure s6 ( supporting information ) , small - into - large substitutions also cause largest conformational changes unlike large - into - small substitutions , which usually do not produce steric clashes . the location of mutations in protein structures is another important feature , and even neighboring mutations might produce very different effects on binding affinity . all mutations can be classified into five types depending on their locations with respect to interface and surface ( see ref ( 61 ) and the supporting information for definition ) . figure 3 shows how mutation location may influence the experimental and predicted ( not shown , but the effect is very similar ) g values . mutations located in the core of the interface ( cor ) produce the largest changes in binding energy followed by the interface mutations partially exposed to the solvent ( rim and sup ) . on the basis of these observations , one might conclude that majority of single mutations from the skempi set do not lead to long - range allosteric effects and only those located directly at the interface regions make the largest contribution to the change in binding energy . nevertheless , the effect of noninterface mutations is not negligible . cor , rim and sup are the core , rim and support regions of the interface and int and sur are the solvent accessible regions . the effect of protein flexibility on binding can not be ignored and in some cases can improve or deteriorate the predictions . here we describe two examples where we account for conformational changes in complex structures when the mutation is introduced . figure 4 compares two structures obtained by minimization and md simulation for mutant alpha - chymotrypsin a / turkey ovomucoid third domain protein complex ( pdb code : 1cho ) ; the leu15glu mutation is introduced into turkey ovomucoid third domain ( chain i ) . if we compare the minimized structure and the complex conformation after md simulation , we can see the changed conformation after md simulation where side chains of ser217 and ser190 from alpha - chymotrypsin a protein flip over to form four extra hydrogen bonds with mutated residue glu15 ; moreover , ser195 moves closer to glu15 to form another hydrogen bond ( figure 4 and table s6 , supporting information ) . this might enhance the interaction between the two partners and results in a smaller destabilizing gpred1 values ( 1.13 kcal mol ) for the md simulation protocol compared to minimization protocol ( 4.32 kcal mol ) and experimental value ( 6.6 kcal mol ) . this example illustrates that although md simulation does allow for larger conformational changes , these transitions might not take place in a native protein . difference between conformations of leu15glu mutant for -chymotrypsin a / turkey ovomucoid third domain protein complex ( pdb code : 1cho ) obtained by minimization ( mm ) and md simulation ( md ) . complex between turkey ovomucoid third domain ( chain i ) and -chymotrypsin a ( chains f and g ) for mm structure are shown in yellow and green , respectively . complex between chains i and f / g for md are shown in red and blue , respectively . black dotted lines correspond to the hydrogen bonds formed between glu15 of chain i and ser217 , ser190 and ser195 of chain g ( ball - and - stick model ) in the md simulated structure . in the case of interleukin-4/receptor chain protein ( pdb code : 1iar ) md simulation protocol made a relatively better prediction compared to the minimization protocol ( gexp = 0.43 kcal mol , gpred1_mm = 4.57 and gpred1_md = 1.89 kcal mol ) . the mutant minimized structure loses seven hydrogen bonds between arg85 and asp67 and asp125 compared to the wild - type minimized structure ( table s6 , supporting information ) whereas the mutant md simulated structure loses only three hydrogen bonds compared to the md simulated wild - type structure ( table s6 , supporting information ) . in this study , we attempt to design an efficient computational protocol in order to estimate the impact of single and multiple missense mutations on protein binding affinity . our analysis showed that the choices of simulation procedure and energy function are both important for achieving accurate predictions . we demonstrated that using an unconstrained minimization procedure in explicit solvent yielded a better agreement with experiments compared to implicit solvent , which is consistent with a previous study on a smaller dataset we used a modified mm - pbsa energy function and optimized its parameters on the experimental data of several thousands of mutations . we showed that even with the 5-fold cross - validation , it is possible to attain a very high correlation coefficient ( r = 0.85 ) with the experimental data using a model with eleven parameters . however , these energy functions do not perform well when applied to an independent set of mutations . it points to the fact that such a model can be overtrained and biased toward certain groups of proteins or mutations . therefore , one should be very cautious in interpreting the results using only one limited dataset for fitting and testing even if cross - validation is done . our approach with an all - atom force field model , explicit water minimization protocol and energy function with only four parameters yielded a reasonable correlation coefficient ( r = 0.62 ) and rmse values ( rmse = 1.27 kcal mol ) between experimental and predicted values after training was done on a representative skempi set and testing was performed on an independent and nonoverlapping set of single mutations . consistent with another study , the mm - pbsa energy function displayed superior performance compared to statistical and empirical potentials . moreover , including statistical scores from foldx and beatmusic into the model produced significantly better agreement with experiments ( p - value < 0.01 ) with a correlation coefficient of 0.69 and rmse of 1.20 kcal mol . we showed that the largest significant contribution that explained the largest proportion of experimental data variation came from the polar solvation energy term . this result once more points to the extreme importance of the solvent model and solvation effects . we also would like to spotlight the drawback of our model that underestimates the contribution of stabilizing mutations . this , in turn , could be the result of an insufficient number of stabilizing amino acid substitutions in the experimental training set . to assess the effect of multiple mutations , we investigated two models trained on single and multiple mutations , respectively . interestingly , we found that the model trained on a set of multiple mutations showed better results for multiple mutations compared to the model trained on single mutations . indeed , nonadditivity of the effect of multiple mutations on binding was observed previously for alanine scanning and disease mutations . it was partially attributed to the modular structure of the binding interface and cooperativity of amino acids within each binding site cluster . it is difficult to assess the conformational changes of monomers occurring upon binding and the effect of flexibility on prediction accuracy . although we did not estimate conformational changes upon binding ( in many cases , no unbound states were available ) , we tried to account for conformational changes in complex structures produced by mutations . as we showed , conformational sampling of mutant structures by molecular dynamics simulations on a 1 ns time scale did not help to achieve better agreement with experiments compared to the protocol when only one minimized mutant complex structure was used . this is consistent with several previous studies demonstrating that averaging over an md - generated ensemble of conformations had a negligible effect on the quality of binding affinity predictions , on estimating the effect of mutations on binding and on homology model refinement . it was previously shown that backbone sampling can do more harm than good when estimating the effects of mutations on protein stability in cases where structural changes are negligible . it implies that either our single minimized conformation dominated a native conformational ensemble or we sampled inaccessible conformations during the unconstrained md simulations . in any case , even without conformational sampling , we were able to achieve similar or better prediction accuracy on a much faster time scale compared to one of the best currently available method ( cc / pbsa ) . this is especially important for high - throughput virtual screening studies because the conformational sampling is very time - consuming . finally , we found that the highest correlation was observed for substitutions that changed either charge or side chain volume . we also observed that missense mutations located directly in the core region of interfaces had the largest effect on binding affinity and long - range effects from noninterface mutations were relatively minor . all these distinctive realistic patterns and the reasonable agreement with experiments validates the use of atomic force fields , statistical potentials or combinatorial scoring schemes for estimating the effects of single and multiple missense mutations on protein

the crucial prerequisite for proper biological function is the protein s ability to establish highly selective interactions with macromolecular partners . a missense mutation that alters the protein binding affinity may cause significant perturbations or complete abolishment of the function , potentially leading to diseases . the availability of computational methods to evaluate the impact of mutations on protein protein binding is critical for a wide range of biomedical applications . here , we report an efficient computational approach for predicting the effect of single and multiple missense mutations on protein protein binding affinity . it is based on a well - tested simulation protocol for structure minimization , modified mm - pbsa and statistical scoring energy functions with parameters optimized on experimental sets of several thousands of mutations . our simulation protocol yields very good agreement between predicted and experimental values with pearson correlation coefficients of 0.69 and 0.63 and root - mean - square errors of 1.20 and 1.90 kcal mol1 for single and multiple mutations , respectively . compared with other available methods , our approach achieves high speed and prediction accuracy and can be applied to large datasets generated by modern genomics initiatives . in addition , we report a crucial role of water model and the polar solvation energy in estimating the changes in binding affinity . our analysis also reveals that prediction accuracy and effect of mutations on binding strongly depends on the type of mutation and its location in a protein complex .

Introduction Methods Results Discussion

PMC4897265

human action recognition has been in great demand in the field of pattern recognition , given its direct relation to video surveillance , human - computer interaction , and ambient - assisted living ( aal ) , among other application scenarios . especially in the latter , human behavior analysis ( hba ) , in which human action recognition plays a fundamental role , can endow smart home services with the required smartness needed to perform fall detection , intelligent safety services ( closing a door or an open tap ) , or activities of daily living ( adl ) recognition . upon these detection stages , aal services may learn subjects ' routines , diets , and even personal hygiene habits , which allow providing useful and proactive services . for this reason , human action recognition techniques are essential in order to develop aal services that support safety at home , health assistance , and aging in place . great advances have been made in vision - based motion capture and analysis , and , at the same time , the society is starting to demand sophisticated and accurate hba systems . this is shown , for example , in the recent rise of interest in devices like the microsoft kinect sensor . current efforts focus on achieving admissible recognition speeds , which is essential for real - time and online systems . another goal is the successful handling of multiview scenarios so as to add robustness to occlusions and improve the quality of the recognition . one of the main drawbacks of multiview techniques is that rich and detailed 3d scene reconstructions are normally incompatible with real - time recognition . on the other hand , the current proposal builds upon earlier work , where we have presented a human action recognition method based on silhouettes and sequences of key poses , which shows to be suitable for real - time scenarios and specially robust to actor variances . in , a study is included comparing different approaches of fusing multiple views using an approach based on a bag of key poses , which is extended in the present contribution . one method stage in which substantial computational cost is added and success in later stages depends upon is feature extraction . in this paper , this specific stage is especially targeted . a low - dimensional radial silhouette - based feature is combined with a simple learning approach based on multiple video streams . working with silhouette contour points , radial bins are computed using the centroid as the origin , and a summary representation is obtained for each bin . this pose representation is used in order to obtain the per - view key poses which are involved in each action performance . therefore , a model fusion of multiple visual sensors is applied . from the obtained bag of key poses , the sequences of key poses of each action class are computed , which are used later on for sequence matching and recognition . experimentation performed on two publicly available datasets ( weizmann and muhavi ) and a self - recorded one shows that the proposed technique not only obtains very high and stable recognition rates but also proves to be suitable for real - time applications . note that by real time we mean that recognition can be performed at video frequency or above , as is common in the field . section 2 summarizes the most recent and relevant works in human action recognition focusing on the type of features used and how multiview scenarios are managed . in section 3 , our proposal is detailed offering a low - dimensional feature based on silhouette contours and a radial scheme . sections 4 and 5 specify the applied multiview learning approach based on a bag of key poses and action recognition through sequence matching . section 6 analyzes the obtained results and compares them with the state of the art in terms of recognition rate and speed , providing also an analysis of the behaviour of the proposed method with respect to its parameters . regarding the feature extraction stage of human action recognition methods based on vision , these can be differentiated by the either static or dynamic nature of the feature . whereas static features consider only the current frame ( extracting diverse types of characteristics based on shape , gradients , key points , etc . ) , dynamic features consider a sequence of several frames and apply techniques like image differencing , optical flow , and spatial - temporal interest points ( stip ) . among the former , we find silhouette - based features which rely either on the whole shape of the silhouette or only on the contour points . in , action primitives are extracted reducing the dimensionality of the binary images with principal component analysis ( pca ) . polar coordinates are considered in , where three radial histograms are defined for the upper part , the lower part , and the whole human body . each polar coordinate system has several bins with different radii and angles , and the concatenated normalized histograms are used to describe the human posture . similarly , in , a log - polar histogram is computed choosing the different radii of the bins based on logarithmic scale . silhouette contours are employed in with the purpose of creating a distance signal based on the pointwise euclidean distances between each contour point and the centroid of the silhouette . conversely , in , the pairwise distances between contour points are computed to build a histogram of distances resulting in a rotation , scale , and translation invariant feature . in , the whole silhouette is used for gait recognition . an angular transform based on the average distance between the silhouette points and the centroid similarly , in , the shape of the silhouette contour is projected on a line based on the transform , which is then made invariant to translation . silhouettes can also be used to obtain stick figures , for instance , by means of skeletonization . chen et al . applied star skeletonization to obtain a five - dimensional vector in star fashion considering the head , the arms , and the legs as local maxima . pointwise distances between contour points and the centroid of the silhouette are used to find the five local maxima . in the work of ikizler and duygulu , a different approach based on a bag - of - rectangles oriented rectangular patches are extracted over the human silhouette , and the human pose is represented with a histogram of circular bins of 15 each . a very popular dynamic feature in pattern recognition based on computer vision is optical flow . fathi and mori rely on low - level features based on optical flow . in their work , weighted combinations of midlevel motion features are built covering small spatiotemporal cuboids from which the low - level features are chosen . in , motion over a sequence of frames is considered defining motion history and energy images . these encode the temporal evolution and the location of the motion , respectively , over a number of frames . this work has been extended by so as to obtain a free - viewpoint representation from multiple views . a similar objective is pursued in , where time is considered as the third dimension building space - time volumes based on sequences of binary silhouettes . action recognition is performed with global space - time features composed of the weighted moments of local space - time saliency and orientation . combine eigenprojections of the width profile of the actor with the centroid of the silhouette and the standard deviation in the x and y axes in a single feature vector . a 3d histogram of oriented gradients ( 3dhog ) is computed for densely distributed regions and combined with temporal embedding to represent an entire video sequence . tran and sorokin merge both silhouette shape and optical flow in a 286-dimensional feature , which also includes the context of 15 surrounding frames reduced by means of pca . this feature has been used successfully in other works as , for instance , recently in . take an interesting approach proposing a novel feature extraction technique , which relies on the surrounding regions of the subjects . these negative spaces present advantages related to robustness to boundary variations caused by partial occlusions , shadows , and nonrigid deformations . rgb - d data , that is , rgb color information along pixel - wise depth measurement , is increasingly being used , since the microsoft kinect device has been released . using the depth data and relying on an intermediate body part recognition process , a markerless body pose estimation in form of 3d skeletal information can be obtained in real time . this kind of data results proficient for gesture and action recognition required by applications , such as gaming and natural user interfaces ( nui ) . in [ 31 , 32 ] , more detailed surveys about these recently appeared depth - based methods can be found . naturally , the usage of static features does not mean that the temporal aspect can not be considered . temporal cues are commonly reflected in the change between successive elements of a sequence of features or in the learning algorithm itself . for further details about the state of the art another relevant area for this work is how human action recognition is handled when dealing with multiple camera views . multiview recognition methods can be classified , for example , by the level at which the fusion of information happens . initially , when dealing with 2d data from multiple sources , these can be used in order to create a 3d representation [ 34 , 35 ] . nevertheless , 3d representations usually imply a higher computational cost as appropriate 3d features need to be obtained . feature fusion places the fusion process one step further by obtaining single - view features for each of the camera views and generating a common representation for all the features afterwards . feature vectors are commonly combined by aggregation functions or concatenation of vectors [ 36 , 37 ] or also more sophisticated techniques as canonical correlation analysis . the appeal of this type of fusion is the resulting simplicity of transition from single- to multiview recognition methods , since multiview data is only handled implicitly . a learning method which in fact learns and extracts information from actions or poses from multiple views requires considerations at the learning scheme . through model fusion , multiple views are learned either as other possible instances of the same class or by explicitly modelling each possible view . these 2d or 3d models may support a limited or unlimited number of points of view ( pov ) . last but not least , information fusion can be applied at the decision level . in this case , for each of the views , a single - view recognition method is used independently , and a decision is taken based on the single - view recognition results . the best view is chosen based on one or multiple criteria like closest distance to the learned pattern , highest score / probability of feature matching , or metrics which try to estimate the quality of the received input pattern . however , the main difficulty is to establish this decision rule because it depends strongly on the type of actions to recognize and on the camera setup . for example , in , a local segment similarity voting scheme is employed to fuse multiple views , and superior results are obtained when compared with feature fusion based on feature concatenation . finally , feature extraction and fusion of multiple views do not necessarily have to be considered two separate processing stages . for instance , in [ 40 , 41 ] , lattice computing is proposed for low - dimensional representation of 2d shapes and data fusion . in our case , model fusion has been chosen because of two reasons : ( 1 ) in comparison with fusion at the decision level , only a single learning process is required in order to perform multiview recognition and ( 2 ) it allows explicit modeling of the poses from each view that are involved in a performance of an action . as a consequence , multiple benefits can be obtained as follows.once the learning process has been finished , further views and action classes can be learned without restarting the whole process . this leads to supporting incremental learning and eliminating the widely accepted limitation of batch - mode training for human action recognition .the camera setups do not need to match between training and testing stages . more camera views may improve the result of the recognition , though it is not required to have all the cameras available.each camera view is processed separately and matched with the corresponding view , without requiring to know specifically at which angle it is installed . once the learning process has been finished , further views and action classes can be learned without restarting the whole process . this leads to supporting incremental learning and eliminating the widely accepted limitation of batch - mode training for human action recognition . more camera views may improve the result of the recognition , though it is not required to have all the cameras available . each camera view is processed separately and matched with the corresponding view , without requiring to know specifically at which angle it is installed . these considerations are important requirements in order to apply the proposed method to the development of aal services . model fusion enabled us to fulfil these constraints , as will be seen in the following sections . as has been previously introduced , our goal is to perform human action recognition in real time and to do so even in scenarios with multiple cameras . human silhouettes contain rich shape information and can be extracted relatively easily , for example , through background subtraction or human body detection . in addition , silhouettes and their contours show certain robustness to lighting changes and small viewpoint variations compared to other techniques , as optical flow . using only the contour points of the silhouette results in a significant dimensionality reduction by getting rid of the redundant interior points . the following variables are used along this section:(1)the number of contour points n;(2)the number of radial bins s;(3)the indices i , j , k , and l , for all i , k , l { 1 , , n } and for all j { 1 , , s}. the number of contour points n ; the number of radial bins s ; the indices i , j , k , and l , for all i , k , l { 1 , , n } and for all j { 1 , , s}. we use the border following algorithm from to extract the n contour points p = { p1 , p2 , , pn } , where pi = ( xi , yi ) . our proposal taking the centroid of the silhouette as the origin , the specific bin of each contour point can be assigned . then , in difference to [ 20 , 21 ] where radial or log - polar histograms are used as spatial descriptors or where star skeletonization is applied , in our approach , an efficient summary representation is obtained for each of the bins , whose concatenation returns the final feature ( figure 1 shows an overview of the process ) . on one hand , it relies on the fact that when using a direct comparison of contours , even after length normalization as in , spatial alignment between feature patterns is still missing . each silhouette has a distinct shape depending on the actor and the action class , and therefore a specific part of the contour can have more or less points in each sample . using an element - wise comparison of the radial bins of different contours , this avoids an element - wise comparison of the contour points , which would imply the erroneous assumption that these are correlated . on the other hand , this radial scheme allows us to apply an even further dimensionality reduction by obtaining a representative summary value for each radial bin . the following steps are taken to compute the feature.(1)the centroid of the contour points c = ( xc , yc ) is calculated as ( 1)xc=i=1nxin , yc=i=1nyin.(2)the pointwise euclidean distances between each contour point and the centroid , d = { d1 , d2 , , dn } , are obtained as in . consider ( 2)di = cmpi , i1, ,n.(3)considering a clockwise order , the corresponding bin si of each contour point pi is assigned as follows ( for the sake of simplicity , i = 0 is considered as i = 360 ) : ( 3)i = arccosyiycdi180,if xi0,180+arccosyiycdi180,otherwise , si = si360 , i{1, ,n}.(4)finally , a summary representation is obtained for the points of each bin . the final feature v results of the concatenation of summary representations . these are normalized to unit sum in order to achieve scale invariance : ( 4)vj = fpk , pk+1, ,plsk, ,sl = jk , l1, ,n , kkkkkkkkkkkkkkkkkkkkkkkkkkkj1, ,s , vj=vj0=1sv0 , j1, ,s , v=v1||v2||||vs. the centroid of the contour points c = ( xc , yc ) is calculated as ( 1)xc=i=1nxin , yc=i=1nyin . the pointwise euclidean distances between each contour point and the centroid , d = { d1 , d2 , , dn } , are obtained as in . consider ( 2)di = cmpi , i1, ,n . considering a clockwise order , the corresponding bin si of each contour point pi is assigned as follows ( for the sake of simplicity , i = 0 is considered as i = 360 ) : ( 3)i = arccosyiycdi180,if xi0,180+arccosyiycdi180,otherwise , si = si360 , i{1, ,n}. finally , a summary representation is obtained for the points of each bin . the final feature v results of the concatenation of summary representations . these are normalized to unit sum in order to achieve scale invariance : ( 4)vj = fpk , pk+1, ,plsk, vj=vj0=1sv0 , j1, ,s , v=v1||v2||||vs. the function f could be any type of function which returns a significant value or property of the input points . we tested three types of summaries ( variance , max value , and range ) , based on the previously obtained distances to the centroid , whose results will be analyzed in section 6 . ,pl=i = kldi2 , where is the average distance of the contour points of each bin . consider ( 6)fmaxpk , pk+1, ,pl = maxdk , dk+1, ,dl , frangepk , pk+1, ,dl mindk , dk+1, ,dl.figure 2 shows an example of the result of the fmax summary function . considering that multiple views of the same field of view are available , our method learns from these views at the model level , relying therefore on model fusion . k - means clustering is used in order to identify the per - view representative instances , the so - called key poses , of each action class . the resulting bag of key poses serves as a dictionary of known poses and can be used to simplify the training sequences of pose representations to sequences of key poses . first , all the training video sequences need to be processed to obtain their pose representations . supposing that m views are available and r action classes need to be learned , k - means clustering with euclidean distance is applied for the pose representations of each combination of view and action class separately . hence , k clusters are obtained for each of the m r groups of data . the center of each cluster is taken as a key pose , and a bag of key poses of k m r class representatives is generated . in this way , an equal representation of each of the action classes and fused views can be assured in the bag of key poses ( figure 3 shows an overview of the process ) . at this point , the training data has been reduced to a representative model of the key poses that are involved in each view of each action class . nevertheless , not all the key poses are equally important . very common poses such as standing still are not able to distinguish between actions , whereas a bend pose can most certainly be only found in its own action class . for this reason , a weight w which indicates the capacity of discrimination of each key pose kp for this purpose , all available pose representations are matched with their nearest neighbor among the bag of key poses ( using euclidean distance ) so as to obtain the ratio of within - class matches wkp = matcheskp / assignmentskp . in this manner , matches is defined as the number of within - class assignments , that is , the number of cases in which a pose representation is matched with a key pose from the same class , whereas assignments denotes the total number of times that key pose got chosen . video recognition presents a clear advantage over image recognition which relies on the temporal dimension . the available training sequences present valuable information about the duration and the temporal evolution of action performances . in order to model the temporal relationship between key poses , the training sequences of pose representations are converted into sequences of key poses . for each sequence , the corresponding sequence of key poses seq = { kp1 , kp2 , , kpt } is obtained by interchanging each pose representation with its nearest neighbor key pose among the bag of key poses . this allows us to capture the long - term temporal evolution of key poses and , at the same time , to significantly improve the quality of the training sequences as noise and outliers are filtered . during the recognition stage , the goal is to assign an action class label to an unknown sequence . for this purpose , ( 1 ) the corresponding pose representation of each video frame is generated and ( 2 ) the pose representations are replaced with the nearest neighbor key poses among the bag of key poses . this way , a sequence of key poses is obtained and recognition can be performed by means of sequence matching . since action performances can nonuniformly vary in speed depending on the actor and his / her condition , sequences need to be aligned properly . dynamic time warping ( dtw ) shows proficiency in temporal alignment of sequences with inconsistent lengths , accelerations , or decelerations . we use dtw in order to find the nearest neighbor training sequence based on the lowest dtw distance . given two sequences seq = { kp1 , kp2 , , kpt } and seq = { kp1 , kp2 , , kpu } , the dtw distance ddtw(seq , seq ) can be obtained as follows : ( 7)ddtwseq , seq'=dtwt , u , dtwi , j = mindtwi1,j , dtwi , j1,dtwi1,j1+dkpi , kpj ' , where the distance between two key poses d(kpi , kpj ) is obtained based on both the euclidean distance between their features and the relevance of the match of key poses . as seen before , not all the key poses are as relevant for the purpose of identifying the corresponding action class . hence , it can be determined how relevant a specific match of key poses is based on their weights wi and wj. in this sense , the distance between key poses is obtained as ( 8)dkpi , kpj'=kpikpj'+zreli , j , reli , j = devi , jwiwj',devi , j = kpikpj'average_distance , where average_distance corresponds to the average distance between key poses computed throughout the training stage . as it can be seen , the relevance rel(i , j ) of the match is determined based on the weights of the key poses , that is , the capacity of discrimination and the deviation of the feature distance . consequently , matches of key poses which are very similar or very different are considered more relevant than those that present an average similarity . table 1 shows the chosen value for each case . in pairings of discriminative key poses which are similar to each other , if the distance among them is higher than average , this indicates that these important key poses do not match well together and therefore the final distance is increased . for ambiguous key poses , that is , key poses with low discriminative value , pairings are not as important for the distance between sequences . on the other hand , a pairing of a discriminative and an ambiguous key pose should be disfavored as these key poses should match with instances with similar weights . otherwise , the operator is based on the sign of dev(i , j ) , which means that low feature distances are favored ( z = 1 ) and high feature distances are penalized ( z = + 1 ) . this way , not only the shape - based similarity between key poses but also the relevance of the specific match is taken into account in sequence matching . once the nearest neighbor sequence of key poses is found , its label is retrieved . the label of the match with the lowest distance is chosen as the final result of the recognition ; that is , the result is based on the best view . this means that only a single view is required in order to perform the recognition , even though better viewing angles may improve the result . note that this process is similar to decision - level fusion , but , in this case , recognition relies on the same multiview learning model , that is , the bag of key poses . in this section , the presented method is tested on three datasets which serve as benchmarks . on this single- and multiview data , our learning algorithm is used with the proposed feature , and the results of the three chosen summary representations ( variance , max value , and range ) are compared . in addition , the distance - signal feature from dedeolu et al . and the silhouette - based feature from boulgouris et al . , which have been summarized in section 2 , are used as a reference so as to make a comparison between features possible . lastly , our approach is compared with the state of the art in terms of recognition rates and speed . it includes video sequences from nine actors performing ten different actions outdoors ( bending , jumping jack , jumping forward , jumping in place , running , galloping sideways , skipping , walking , waving one hand , and waving two hands ) and has been recorded with a static front - side camera providing rgb images of a resolution of 180 144 px . these silhouettes have been obtained automatically through background subtraction techniques ; therefore , they present noise and incompleteness . it is worth to mention that we do include the skip action , which is excluded in several other works because it usually has a negative impact on the overall recognition accuracy . the muhavi dataset targets multiview human action recognition , since it includes 17 different actions recorded from eight views with a resolution of 720 576 px . muhavi - mas provides manually annotated silhouettes for a subset of two views from 14 ( muhavi-14 : collapseleft , collapseright , guardtokick , guardtopunch , kickright , punchright , runlefttoright , runrighttoleft , standupleft , standupright , turnbackleft , turnbackright , walklefttoright , and walkrighttoleft ) or 8 ( muhavi-8 : collapse , guard , kickright , punchright , run , standup , turnback , and walk ) actions performed by two actors . finally , our self - recorded dai rgbd dataset has been acquired using a multiview setup of microsoft kinect devices . this dataset includes 12 actions classes ( bend , carryball , checkwatch , jump , punchleft , punchright , sitdown , standingstill , standup , waveboth , waveleft , and waveright ) , performed by three different actors . using depth - based segmentation , the silhouettes of the so - called users of a resolution of 320 240 px are obtained . in future works , we intend to expand this dataset with more subjects and samples and make it publicly available . we chose two tests to be performed on these datasets as follows.leave-one-sequence-out cross validation ( loso ) . the system is trained with all but one sequence which is used as test sequence . this procedure is repeated for all available sequences and the accuracy scores are averaged . in the case of multiview sequences , each video sequence is considered as the combination of its views.leave-one-actor-out cross validation ( loao ) . this test verifies the robustness to actor - variance . in this sense , the sequences from all but one actor are used for training , while the sequences from the remaining actor , unknown to the system , are used for testing . the system is trained with all but one sequence which is used as test sequence . this procedure is repeated for all available sequences and the accuracy scores are averaged . in the case of multiview sequences , each video sequence is considered as the combination of its views . this test verifies the robustness to actor - variance . in this sense , the sequences from all but one actor are used for training , while the sequences from the remaining actor , unknown to the system , are used for testing . this test is performed for each actor and the obtained accuracy scores are averaged . the feature from boulgouris et al . , which has been originally designed for gait recognition , presents advantages regarding , for instance , robustness to segmentation errors , since it relies on the average distance to the centroid of all the silhouette points of each circular sector . nevertheless , on the tested action recognition datasets , it returned low success rates , which are significantly outperformed by the other four contour - based approaches . both the feature from dedeolu et al . and ours are based on the pointwise distances between the contour points and the centroid of the silhouette . our proposal distinguishes itself in that a radial scheme is applied in order to spatially align contour parts . further dimensionality reduction is also provided by summarizing each radial bin in a single characteristic value . table 2 shows the performance we obtained by applying this existing feature to our learning algorithm . whereas on the weizmann dataset the results are significantly behind the state of the art and the rates obtained on the dai rgbd dataset are rather low , the results for the muhavi dataset are promising . the difference of performance can be explained with the different qualities of the binary silhouettes . the silhouettes from the muhavi - mas subset have been manually annotated in order to separate the problem of silhouette - based human action recognition from the difficulties which arise from the silhouette extraction task . this stands in contrast to the other datasets whose silhouettes have been obtained automatically , respectively , through background subtraction or depth - based segmentation , presenting therefore segmentation errors . this leads us to the conclusion that the visual feature from is strongly dependant on the quality of the silhouettes . table 2 also shows the results that have been obtained with the different summary functions from our proposal . the variance summary representation , which only encodes the local dispersion but not reflects the actual distance to the centroid , achieves an improvement in some tests at the cost of obtaining poor results on the muhavi actor - invariance tests ( loao ) and the dai rgbd dataset . the max value summary representation solves this problem and returns acceptable rates for all tests . finally , with frange , the range summary representation obtains the best overall recognition rates , achieving our highest rates for the weizmann dataset , the muhavi loso tests , and the dai rgbd dataset . in conclusion , the proposed radial silhouette - based feature not only achieves to substantially improve the results obtained with similar features as [ 9 , 10 ] but its low - dimensionality also offers an additional advantage in computational cost ( feature size is reduced from ~300 points in to ~20 radial bins in our approach ) . the presented method uses two parameters which are not given by the constraints of the dataset and the action classes which have to be recognized and therefore have to be established by design . the first one is found at the feature extraction stage , that is , the number of radial bins s. a lower value of s leads to a lower dimensionality which reduces the computational cost and may also improve noise filtering , but , at the same time , it will reduce the amount of characteristic data . the second parameter is the number of key poses per action class and view k. in this case , the appropriate amount of representatives needs to be found to capture the most relevant characteristics of the sample distribution in the feature space , discarding outlier and nonrelevant areas . again , higher values will lead to an increase of the computational cost of the classification . therefore , a compromise needs to be reached between classification time and accuracy . in order to analyse the behavior of the proposed algorithm with respect to these two parameters , a statistic analysis has been performed . due to the nondeterministic behavior of the k - means algorithm , classification rates vary among executions . we executed ten repetitions of each test ( muhavi-8 loao cross validation ) and obtained the median value ( see figure 4 ) . it can be observed that a high value of key poses , that is , feature space representatives , only leads to a good classification rate if the feature dimensionality is not too low ; otherwise , a few key poses are enough to capture the relevant areas of the feature space . note also that a higher feature dimensionality does not necessarily require a higher number of key poses , since it does not imply a broader sample distribution of the feature space . finally , with the purpose of obtaining high and reproducible results , the parameter values have been chosen based on the highest median success rate ( 92.6% ) , which has been obtained with s = 12 and k = 5 in this case . since lower values are preferred for both parameters , the lowest parameter values are used if several combinations reach the same median success rate . comparison between different approaches can be difficult due to the diverse goals human action recognition methods may pursue , the different types of input data , and the chosen evaluation methods . in our case , multiview human action recognition is aimed at an indoor scenario related to aal services . therefore , the system is required to perform in real time as other services will rely on the action recognition output . a comparison of the obtained classification and recognition speed rates for the publicly available weizmann and muhavi - mas datasets is provided in this section . performance has been tested on a standard pc with an intel core 2 duo cpu at 3 ghz and 4 gb of ram with windows 7 64-bit . all tests have been performed using binary silhouette images as input , and no further hardware optimizations have been performed . it can be seen that while perfect recognition has been achieved for the weizmann dataset , our method places itself well in terms of both recognition accuracy and recognition speed when comparing it to methods that target fast human action recognition . on the muhavi-14 and muhavi-8 datasets , our approach achieves to significantly outperform the known recognition rates of the state of the art ( see tables 4 and 5 ) . to the best of our knowledge , this is the first work to report a perfect recognition on the muhavi-8 dataset performing the leave - one - sequence - out cross validation test . the equivalent test on the muhavi-14 dataset returned an improvement of 9.6% in comparison with the work from cheema et al . , which also shows real - time suitability . furthermore , our approach presents very high robustness to actor - variance as the leave - one - actor - out cross validation tests show , and it achieves to perform at over 90 fps with the higher resolution images from the muhavi dataset . it is also worth mentioning that the training stage of the presented approach runs at similar rates between 92 and 221 fps . with these results it is known that silhouette extraction with admissible quality can be performed in real time through background subtraction techniques [ 47 , 48 ] . furthermore , recent advances in depth sensors make it possible to obtain human poses of substantial higher quality by means of real - time depth based segmentation . in addition , depth , infrared , or laser sensors allow preserving privacy as rgb information is not essential for silhouette - based human action recognition . in this work , a low - dimensional radial silhouette - based feature has been proposed , which in combination with a simple , yet effective , multiview learning approach based on a bag of key poses and sequence matching shows to be a very robust and efficient technique for human action recognition in real time . by means of a radial scheme , contour parts are spatially aligned , and , through the summary function , dimensionality is drastically reduced . this proposal achieves to significantly improve recognition accuracy and speed and is proficient with both single- and multiview scenarios . in comparison with the state of the art , our approach presents high results on the weizmann dataset and , to the best of our knowledge , the best rates achieved so far on the muhavi dataset . real - time suitability is confirmed , since performance tests returned results clearly above video frequency . future works include finding an optimal summary representation or the appropriate combination of summary representations based on a multiclassifier system . tests with a greater number of visual sensors need to be performed so as to see how many views can be handled by the learning approach based on model fusion and to which limit multiview data improves the recognition . for this purpose , multiview datasets such as ixmas and i3dpost can be employed . the proposed approach does not require that each viewing angle matches with a specific orientation of the subject because different orientations can be modelled if seen at the training stage . nevertheless , since the method is not explicitly addressing view - invariance , it can not deal with cross - view scenarios .

this paper presents a novel silhouette - based feature for vision - based human action recognition , which relies on the contour of the silhouette and a radial scheme . its low - dimensionality and ease of extraction result in an outstanding proficiency for real - time scenarios . this feature is used in a learning algorithm that by means of model fusion of multiple camera streams builds a bag of key poses , which serves as a dictionary of known poses and allows converting the training sequences into sequences of key poses . these are used in order to perform action recognition by means of a sequence matching algorithm . experimentation on three different datasets returns high and stable recognition rates . to the best of our knowledge , this paper presents the highest results so far on the muhavi - mas dataset . real - time suitability is given , since the method easily performs above video frequency . therefore , the related requirements that applications as ambient - assisted living services impose are successfully fulfilled .

1. Introduction 2. Related Work 3. Pose Representation Feature 4. Multiview Learning Algorithm 5. Action Recognition 6. Experimental Results 7. Conclusion

PMC1160199

the pdb ( ) ( 1 ) currently contains > 30 000 3d structures of bio - molecules ( march 22 , 2005 ) . those who want to look at one of these 3d datasets need additional software to visualize the structure file that is stored in the database . modern web browsers like mozilla , internet explorer or opera normally display only html code and images directly . in order to display other complex data these software components could be java applets , plug - ins or an external helper application . the disadvantage of java applets and plug - ins embedded into the layout of the displayed webpage by the web browser is the low resolution of the images normally < 100 d.p.i . ( dots per inch ) . often screenshots made for publications are not accepted for printing . thus people need to install dedicated programs for rendering molecular pictures , but often these programs are difficult to use and a lot of the functions can only be activated by programming scripts . this is only acceptable for intensive use and not for use from time to time . a pioneering webpage from 1995 called molecules to go ( ) combines a full text within the pdb with a simple structure presentation and the jena image library database ( ) ( 24 ) offers for the generation of mono or stereo pdf images with a fixed representation style either in a default orientation or via a vrml interface in a user - selected orientation , a related option that is based on molscript . at the moment there is no way to get a high quality and high resolution 3d structure image with a user - selected view of a molecule via internet without using additional locally installed software . the alternative concept splits the web interface ( client ) from the rendering program ( server ) . the web browser only gets html and javascript code ( which normally every browser can handle ) and an image of the molecule ( figures 1 and 2 ) . the user has different methods to upload a 3d structure of a molecule in pdb format to the web applications server . the pdb format contains , next to the atom type , the x , y and z co - ordinates of the 3d structure . for manual file in addition to this method a pdb file can be fetched by the four - character pdb i d . unless the files are stored locally they are automatically downloaded from the protein database server . furthermore , aismig ( an interactive server - side molecule image generator ) provides a simple object access protocol ( soap ) interface to be fully integrated with other web applications available on the internet . thus molecules generated on other servers or stored in other databases can be automatically uploaded to the aismig server and directly accessed for the rendering process . see the carbohydrate builder web tool sweet ii ( 5 ) ( ) as an example . at the server side , a script fetches the request from the user and after checking if the file is in pdb format , a script calls the free program pymol ( ) to generate the molecular image . at this first stage the user can manipulate the color and shape of the molecule interactively . the menu structure ( figure 3 ) is quite similar to the rasmol ( 6,7 ) menu panel and provides the standard manipulation functions . the user can access parts of the protein by atom number , atom type , group name , group number and chain . coloring by structure ( coloring the sheets , turns and loops ) is also possible . for the shape of the molecule we provide the following modes : line , sticks , ribbon and cartoon ( only proteins ) , sphere , surface , dots and mesh . static macros like color by b - factor or color by eisenberg hydrophobic scale are the same for all molecules . dynamic macros , in contrary , are generated dynamically at starting time by other service programs . therefore the program needs to analyze the molecule and writes the appropriate data to the dynamic macro . if the user has the molecule in the right perspective , he switches to the second stage where he needs to set the rendering options . at this point pymol produces the molecule as a set of 3d objects that are loaded and parsed by the second stage program part . at this stage , the user can only influence the rendering and scene parameters like image size , additional spotlight or texture elements . the raytracer for generating the high quality and high resolution pictures is the free program povray ( ) . if all settings are established , the user can start the process of rendering the final image . for this process he needs to enter his email address and he gets an email with a link to the result page . until the image is ready , the user can see the progress ( as a percentage ) if the job is running , or the queue position that is displayed . the web server submits the rendering job to a small linux cluster where a modified version of povray runs . an alternative method of internet - based display of 3d structures of molecules has been developed . the aismig web application is an implementation of this alternative concept and the free service is available at . figures 3 and 4 illustrate the object generation at stage 1 , the object rendering at stage 2 and the final image . figure 5 demonstrates the different possibilities of the web application . by uncoupling the object generation from the object rendering it is possible to manipulate the scene and add a second light source or map some textures on the molecule . ( some nice images can be found at the aismig homepage in the art gallery . ) the aismig services can easily be linked to other web applications or databases by using the soap interface in order to upload a 3d pdb file to the webserver . an example of how to connect to another web - application by this mechanism is demonstrated by the sweet ii ( 5 ) carbohydrate builder web tool where the user can create 3d structure files of carbohydrates and can visualize the generated structure by aismig . the concept has advantages and disadvantages , as does every concept . in the following section the alternative concept will be discussed by comparison to other current concepts like java applets , plug - ins and external helper tools . java applets have the advantages that they are nearly independent of browser and operating system . the standard java applets for molecule visualization are jmol ( ) , webmol ( 8) ( ) and pdbjviewer ( 9 ) ( , ) . pdbjviewer is the latest applet and uses the java3d extension for surface generation . at the moment the java virtual machine is a plug - in for web browsers but in former times this was different hence it is listed in the table at this point . the most often used plug - in for visualization of molecule 3d structures is chime ( ) but plug - ins are highly operating system and browser dependent . also , java applets as plug - ins are not able to generate high - resolution images besides they need to be installed on the client system . one advantage of these methods is that the applet or the plug - in are included in the layout of the web page and can interact with other components . the advantage of external programs is that they can render large images and often they have powerful functions for working with small molecules or with proteins . there are lots of programs for displaying 3d structures of molecules and normally every molecular viewing program can be used by this mechanism ( that the webserver calls an external helper program with a file name as parameter ) . the most popular programs are : rasmol ( and ) , swiss - pdb viewer ( 10 ) and pymol . the greatest advantage of the interactive server - side image generation concept ( aismig ) is that no additional software in addition to the web browser is necessary to view and process the molecules , thus everyone can visualize and manipulate the generated molecule structure . furthermore rendering a molecule in high resolution ( e.g. 1200 d.p.i . ) for a publication or poster poses no problem . software modules need only be installed once at the server side and all users of the service can use it . by using pymol for image generation it is possible to generate complex surfaces as pymol uses opengl with all its advantages . however , this server - side image generation method requires for each interaction a request to the server and at every interaction an image is generated and transmitted to the client . the images are small but the sum of all can be quite large . at the moment aismig sends a jpg image with an average size of 15 kb for each request . with one of the menu entries the user can download a png image in a better quality that is 60 to 140 kb . it is slower than client - side running software where real time rotation by mouse movements is possible . an alternative concept similar to the current concepts ( java , plug - ins and external application ) was developed and implemented . aismig will complement the current concepts with its advantages ( no additional software installation and rendering of high - resolution pictures ) , but it was not designed to replace any of the existing methods for molecule visualization . aismig enables the possibility to visualize a molecular 3d structure with only a standard web browser without requiring local installation of any additional structure viewer software . aismig helps even those who are not computer experts to see 3d molecule structures provided by a web service or web - based databases . 1a0s sucrose - specific porin rendered in high quality and high resolution 4650 3300 pixel with the color by rainbow function .

using a web browser without additional software and generating interactive high quality and high resolution images of bio - molecules is no longer a problem . interactive visualization of 3d molecule structures by internet browsers normally is not possible without additional software and the disadvantage of browser - based structure images ( e.g. by a java applet ) is their low resolution . scientists who want to generate 3d molecular images with high quality and high resolution ( e.g. for publications or to render a molecule for a poster ) therefore require separately installed software that is often not easy to use . the alternative concept is an interactive server - side rendering application that can be interfaced with any web browser . thus it combines the advantage of the web application with the high - end rendering of a raytracer . this article addresses users who want to generate high quality images from molecular structures and do not have software installed locally for structure visualization . often people do not have a structure viewer , such as rasmol or chime ( or even java ) installed locally but want to visualize a molecule structure interactively . aismig ( an interactive server - side molecule image generator ) is a web service that provides a visualization of molecule structures in such cases . aismig - url : .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION Figures and Tables

PMC5386443

total maxillary setback ( tms ) osteotomy is an orthognathic surgery technique for correcting skeletal problems of bimaxillary protrusion or severe maxillary excess . in cases of skeletal maxillary excess , maxillary setback can be obtained by using either anterior maxillary segmental osteotomies or tms osteotomy , so that posterior repositioning of the maxilla can be achieved . tms movement can be classified into parallel and rotational setback according to the change of the palatal plane . explained rotational setback as maxillary setback with clockwise rotation of the palatal plane more than 2 degrees and subsequent backward movement of upper central incisors . authors are mainly divided into 2 groups according to the technique used for tms osteotomy ; one suggesting bone removal from posterior maxillary region and the other suggesting fracture of pterygoid plates to avoid any bony interference when maxillary setback is considered . notably , many authors have supported the idea of intentionally fracturing the pterygoid plates to set back the entire maxilla [ 59 ] . the numerical method of obtaining the solution to a complex problem is called the finite element method . the analysis performed with finite element method fea allows the analyzer to evaluate the distribution of stresses and displacements on the simulated model . maxillary advancement surgery offers a highly predictable and widespread treatment for the correction of anteroposterior skeletal deficiency . accuracy and surgical movement pattern of maxillary advancement has been widely investigated in the literature . therefore , the purpose of this study was to evaluate and compare the displacement patterns of the maxilla with regard to the 2 different tms movements described in the literature using fea . in this study , a finite element model of a hemimaxilla was developed from computerized tomography ( ct ) images taken at 0.5 mm intervals of a young man using dicom ( v 3.0 , nema , rosslyn , virginia 22209 usa ) software . sequential ct images were transferred to maxilim ( medicim nv , mechelen , belgium ) software to obtain a three - dimensional image . a tetrahedral finite element mesh model of the image was then constructed using msc marc ( msc software corporation , santa ana , ca 92707 , usa ) software to simulate the maxilla . the horizontal line representing the upper limit of the maxilla model was fixed and zero - displacement boundary condition was imposed on the nodes along this line . the mechanical structures in the model were assumed to be homogenous , isotropic , and linear elastic with the properties shown in table 1 . the periodontal ligament was not simulated . the surgical le fort i osteotomy line was simulated running horizontally along the lateral maxillary wall and extending to the pterygoid plates . two different backward movements were applied to the lower maxillary segment to simulate parallel and rotational setbacks of the maxilla according to the osteotomy line . when the movement was directed in parallel with the osteotomy line , rotational setback movement was applied with a direction of 5 inferior to the osteotomy line to obtain a counterclockwise rotation . actually , the counterclockwise rotation mentioned herein ( on the left lateral aspect of the model ) corresponds to the clockwise rotational setback defined by baek et al . therefore , the use of counterclockwise rotation in this study was because of the left - sided models . in both parallel and rotational setback simulations , 2 and 3 mm of posterior movements were applied to point a and the displacement pattern of the maxillary segment was observed in each simulation . posterior movement amounts of 2 and 3 mm were selected based on the most commonly reported mean setback amounts in the literature [ 5,6,1012 ] . the reference lines assigned to measure the displacement of the maxillary segment were : l1 , the vertical line passing through the apex point of the canine tooth ; and l2 , the vertical line passing through the midpoint between the buccal roots of the second molar . distance change between the upper and lower maxillary segments in l1 was assumed to reflect the displacement pattern of the maxillary segment in the anterior region , while the difference in l2 was assumed to represent the displacement in the posterior region . the created model with the osteotomy line and reference lines is shown in figure 1 . to evaluate the pattern of the tms movement , the amount of displacements ( mm ) was measured on the x - axis ( transverse plane ) and z - axis ( vertical plane ) . displacements on the y - axis ( anteroposterior plane ) were , predictably , either 2 mm or 3 mm based on the amount of posterior movement applied to the maxillary segment . for example , a posterior movement of 2 mm refers to exactly the same amount of posterior displacement of both l1 and l2 reference lines . the amounts of displacements measured in the parallel and rotational setback simulations are shown in tables 2 and 3 , respectively . displacement patterns of the maxilla according to the reference lines l1 and l2 were evaluated in the transverse and vertical planes . positive values indicate lateral displacements of the maxillary segment . in the parallel setback simulation with 2 mm posterior movement , the maxillary segment was displaced laterally , with a larger amount in the anterior part compared to the posterior part ( figure 2 ) . similarly , 3 mm posterior movement revealed lateral displacement of the maxillary segment with increased amounts in both anterior and posterior parts ( figure 3 ) . in the rotational setback simulation , maxillary segment showed the same movement pattern as that of parallel setback , with slightly increased displacement amounts . it was obvious that maxillary segment showed a tendency toward lateral displacement in all simulated movements . lateral displacements did not exceed 1 mm in the posterior region as opposed to the anterior region . the largest lateral displacement was 1.84 mm , which occurred with the rotational setback of 3 mm . negative values indicate inferior displacements of the maxillary segment . in the parallel setback simulation , the model produced smaller inferior displacements in the anterior region than in the posterior region . during the rotational setback movement , figure 4 shows the displacement pattern of maxillary segment with a rotational setback of 2 mm . particularly , a marked increase was observed in the anterior region where the largest inferior displacement ( 3.0 mm ) was measured . l2 displacement amounts in the rotational setback movement seemed to decrease slightly compared to parallel setback values ; nevertheless , the values were still very close to each other . when comparing the parallel and rotational setback movements , the posterior part of the maxillary segment showed a tendency to move more inferiorly in parallel setback , whereas the anterior part noticeably showed a greater inferior displacement with the application of rotational setback ( figure 5 ) . positive values indicate lateral displacements of the maxillary segment . in the parallel setback simulation with 2 mm posterior movement , the maxillary segment was displaced laterally , with a larger amount in the anterior part compared to the posterior part ( figure 2 ) . similarly , 3 mm posterior movement revealed lateral displacement of the maxillary segment with increased amounts in both anterior and posterior parts ( figure 3 ) . in the rotational setback simulation , maxillary segment showed the same movement pattern as that of parallel setback , with slightly increased displacement amounts . it was obvious that maxillary segment showed a tendency toward lateral displacement in all simulated movements . lateral displacements did not exceed 1 mm in the posterior region as opposed to the anterior region . the largest lateral displacement was 1.84 mm , which occurred with the rotational setback of 3 mm . negative values indicate inferior displacements of the maxillary segment . in the parallel setback simulation , the model produced smaller inferior displacements in the anterior region than in the posterior region . during the rotational setback movement , vertical displacement in the inferior direction tended to increase . figure 4 shows the displacement pattern of maxillary segment with a rotational setback of 2 mm . particularly , a marked increase was observed in the anterior region where the largest inferior displacement ( 3.0 mm ) was measured . l2 displacement amounts in the rotational setback movement seemed to decrease slightly compared to parallel setback values ; nevertheless , the values were still very close to each other . when comparing the parallel and rotational setback movements , the posterior part of the maxillary segment showed a tendency to move more inferiorly in parallel setback , whereas the anterior part noticeably showed a greater inferior displacement with the application of rotational setback ( figure 5 ) . understanding the maxillary displacement characteristics under parallel and rotational setback movements is of great importance to achieve an accurate result following the surgical operation . thus , the purpose of the analysis was to observe the characteristics of maxillary setback when different posterior movements in parallel and rotational directions were applied . a limited number of publications are available in the literature that focus on the accuracy of tms osteotomy . studies examining maxillary displacement with fea are also limited and generally concentrated on rapid palatal expansion ( rpe ) [ 1316 ] . other than rpe studies , one study evaluated the maxillary displacement under different headgear forces and another under protraction forces . the present study is the first fea that addresses the displacement pattern of the maxillary segment during tms surgery . many authors emphasized that tms osteotomy could be more advantageous than segmental setback procedures , because it does not rely on tooth extraction and it overcomes the concerns about vascularization of segments [ 24,19 ] . most of the articles reported that tmso plays a significant role in the treatment of maxillary excess or bimaxillary protrusion [ 13,2022 ] . as more information about tmso has become available , a need has arisen to determine the pattern of maxillary setback movement . some authors suggested that fracture of the pterygoid plates is a safe and effective technique to set back entire maxilla ; however , possible outcomes of setback movement carried out with the fracture of pterygoid plates are unclear and the displacement pattern of the maxilla is unpredictable . the main reason for simulating such a technique was to avoid any interference between the maxillary tuberosity and pterygoid plates . furthermore , the technique of pterygoid plate fracture is a matter of great interest since there exist many studies addressing this technique for maxillary setback movement [ 59 ] . in the current study , parallel setback was simulated using a posterior movement parallel to the osteotomy line , while rotational setback was represented with a posterior movement directed 5 degrees inferior to the osteotomy line . reported that two - thirds of their study sample received rotational setback in clockwise direction with a mean angulation of 1.3 ( sd 3.3 ) . similarly , baek et al . performed clockwise rotational setback with an angulation more than 2 , but the amounts of exact angular change were not specified . in another 2 studies , clockwise rotational setback was mentioned to be the type of setback movement ; however , again no information on its angulation was presented . selected setback movement amounts for assessment in this simulation were 2 and 3 mm based on the commonly reported magnitudes of tms [ 5,6,1012 ] . as the lower maxillary segment was completely separated from the upper segment with the conventional le fort i osteotomy , the lower segment moved freely , so forces applied at point a were accepted as negligible . reference line l1 was used to observe the displacement pattern of the maxillary segment in the canine area , so the results were interpreted as representing the displacement of anterior maxilla as well as anterior dentition . the l2 line was assigned in the second molar area to represent the posterior maxillary displacement pattern , especially at the end of the dental arch . this result supports the findings of gautam et al . that the maxillary structures were displaced laterally with the different types of headgear retractions . all lateral displacements in the anterior region were found to be more than 1 mm . lateral displacements in the posterior region were relatively smaller , with the maximum amount being 0.90 mm in the 3 mm rotational setback simulation . this observation is consistent with the findings of hirose et al . , who reported that the maxilla moved inferiorly in general . inferior displacement amounts as small as 0.15 to 0.23 mm were detected with 2 mm setback movements . however , a sudden increase from 0.2 mm to 3.0 mm occurred in the anterior maxilla when the rotational setback amount increased to 3 mm . in contrast , the inferior displacements tended to significantly increase in the anterior maxilla with the rotational setbacks . to help develop a better understanding of tms , a fea was conducted to evaluate the displacement pattern of the maxilla under different circumstances . the fea results indicated that the inferior displacement of the maxilla occurred even in the parallel type of setback movement . not surprisingly , the anterior part of the maxillary segment moved in the inferior direction with the applied counterclockwise direction . another interesting observation was that the maxillary segment was displaced laterally with relatively larger amounts in the anterior maxilla . these results may provide insight into how the maxilla tends to move during tms surgery .

backgroundthe purpose of this analysis was to evaluate the displacement patterns of the maxilla under parallel and rotational setbacks using the finite element method ( fem).material / methodsa three - dimensional ( 3d ) finite element model of a hemimaxilla was constructed . through a conventional le fort i osteotomy , 2 and 3 mm of posterior movement in a parallel and rotational manner were simulated and the displacement pattern of the maxilla in each movement type was evaluated.resultsboth parallel and rotational setbacks resulted in lateral and inferior displacement of the maxillary segment . the largest inferior displacement was 3.0 mm and the largest lateral displacement was 1.84 mm . all lateral displacements in the anterior region were found to be more than 1 mm.conclusionsthe results of this study may provide insight into how the maxilla tends to move during total maxillary setback surgery .

Background Material and Methods Results Displacement in the transverse plane (x-axis) Displacement in the vertical plane (z-axis) Discussion Conclusions

PMC5430265

it also gives rise to long - term handicap giving negative influences on activities of daily living in stroke patients1 , 2 . there are a lot of stroke sequelae which have bad effects on quality of life in stroke patients . among these aftereffects , hemiparesis defined as motor impairments of the limb contralateral to unilateral brain damage3 , 4 . the increased activation in unaffected hemisphere could be a possible cause of a pathological inhibition in affected hemisphere as well as motor impairments in affected side of the body5 , 6 . a number of researches on a physical activity reported that there is a close relationship between a performance of exercise and an improvement of brain plasticity7 . skilled reaching can be considered as task - specific training via its purposeful characteristics , so it is used as a training program to rehabilitate stroke patients with a motor disability in their upper limb8,9,10 . treadmill exercise has been studied as one of conventional treatments for a long time . in general , treadmill exercise is commonly applied to stroke patients thanks to its beneficial effect on neuroprotection11 , 12 . to date , the only therapy approved by fda has been thrombolytic tissue plasminogen activator ( tpa ) although approximately 80% of human strokes are classified into ischemic stroke13,14,15 . it is suggested that continuous researches via experimental ischemic stroke models should be performed to develop novel , proper , and helpful solutions for patients with ischemic stroke . in this regards , the present study investigated effects of skilled reach training with affected forelimb and treadmill exercise on the expression of neurotrophic factor following ischemia - induced brain injury in rats . thirty male sprague - dawley rats were divided into 3 groups randomly : namely , the control sacrified 2 weeks after surgery ( con ) , skilled reach training with forepaw contralateral to brain injury for 2 weeks ( sc ) , and treadmill exercise for 2 weeks ( te ) . all the experiments were performed in accordance with protocols approved by the animal experiment committee in daegu university , based on the nih guidelines for the care and use of laboratory animals . transient focal cerebral ischemia was induced by intraluminal occlusion of the left middle cerebral artery ( mca ) . briefly , right common carotid artery ( cca ) was exposed and the external carotid artery ( eca ) and the cca were ligated . middle cerebral artery is occluded by a 40 nylon monofilament coated with a silicone tip , the monofilament was inserted into the internal carotid artery from the external carotid artery until mild resistance was felt . reperfusion was established by completely withdrawing the nylon monofilament after 120 min of occlusion . skilled reach training was conducted in a plexiglas chamber ( 45 cm in height , 15 cm in width , 40 cm in length ) containing of a 1 cm by 10 cm window in front wall allowed for one of the rat s paw to reach via for a pellet . animals were acclimated to the chamber with 4.5 mg of sugar - flavored food pellets ( research diets , new brunswich , nj , usa ) placed on the shelf placed in a small indentation 3 cm from the inside wall of the chamber and trained in 30 minutes sessions administrated 6 days per week for 2 weeks based on previous reports . when a pellet was removed by a rat , the shelf was refilled for a time period of 30 minutes continuously . a motor - driven treadmill was used for the treadmill exercise . before treadmill exercise , rats in te experienced 1 days of adaptive running exercise at a speed of 69 m / min for 5 min . the brains of each group were collected , washed twice in pbs , and then homogenized and lysed with buffer ( 137 mm nacl , 8.1 mm na2hpo4 , 2.7 mm kcl , 1.5 mm kh2po4 , 2.5 mm edta , 1 mm dithiothreitol , 0.1 mm pmsf , 10 g / ml leupeptin [ ph 7.5 ] ) for 30 min on ice . the lysates were centrifuged for 10 min at 15,000 rpm at 4 c , and the protein concentration was determined as described previously . equal amounts of protein ( 40 g ) were resolved via 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) and transferred to nitrocellulose membranes . the blots were washed with tbst ( 10 mm tris hcl [ ph 7.6 ] , 150 mm nacl , 0.05% tween 20 ) , blocked with 5% skim milk for 1 hour , then incubated with the appropriate primary antibodies at the dilutions recommended by the suppliers . the membranes were washed , and the primary antibodies were detected using horseradish peroxidase - conjugated goat anti - rabbit igg or goat - anti mouse igg . the bands were then visualized via enhanced chemiluminescence ( amersham pharmacia biotech , piscataway , nj , usa ) . the expression of brain - derived neurotrophic factor ( bdnf ) and nerve growth factor ( ngf ) in con , sc , and te were also investigated to identify the effect of skilled reach training with contralesional forelimb and treadmill exercise on bdnf expression . there were significant differences in bdnf and ngf expression between con and sc , con and te ( p<0.05 ) . there were no significant differences in bdnf and ngf expression between sc and te ( p>0.05 ) . motor function in upper - extremity is closely related to quality of life in stroke patients via having influences on activity and participation , and this is the reason to focus on the recovery of upper limb motor function in stroke patients16,17,18 . skilled reach training , one of task - specific exercises , has frequently been mentioned as a treatment strategy for stroke patients in many researches19 , 20 . in many studies , skilled reach training was applied to animal models in order to investigate its effect on neurological diseases . pagnussat et al . displayed an effect of skilled reaching with affected forelimb on functional recovery in their research21 . above this , several studies also reported that recovery of motor function can be induced by applying skilled reach training with contralesional forelimb9 , 22 . in addition , effects of treadmill exercise have been studied in many researches for a long time . it has several beneficial effects such as motor function recovery and neuroprotection12 . in many studies investigating recovery of a damaged brain , plasticity is often mentioned23 . particularly , it is reported that the activation of neurotrophic factors is able to facilitate plasticity via inducing generation and differentiation of neuronal progenitor cell24,25,26 . park et al . reported that neurotrophic factors have an influence on neuroprotective function after brain injury27 . for these reasons , the present study investigated the expression of bdnf and ngf via western blot analysis to examine the effect of skilled reach training and treadmill exercise on neurotrophic factor expression . from results , there were significant differences in bdnf and ngf expression between con and sc , con and te ( p<0.05 ) . although expression of bdnf and ngf in sc slightly increased compared with in te , the difference was not statistically significant . ( p>0.05 ) ( table 1table 1.expression of bdnf and ngf in each group ( unit : pixel)consctebdnf2,046.5 108.74,240.1 72.5 * 4,173.3 65.1*ngf2,211.3 87.93,881.6 91.4 * 3,830.8 88.7*values are reported as the mean sd.*p<0.05 vs. con ) . these results imply that both skilled reach training with affected forelimb and treadmill exercise can induce expression of neurotrophic factors . it is suggested that skilled reach training may have more influence on them than treadmill exercise because of its repetitive and task - specific characteristics . values are reported as the mean sd . there is the limitation of the present study . since the results of the study only showed expression of bdnf and ngf , it is not enough to explain the relationship between expression of neurotrophic factors and recovery of function . further study applying tests for evaluating motor function is necessary to make the results of the present study more obvious . in consequence , especially , skilled reach training with affected forelimb showed more beneficial effects on neurotrophic factor expression than treadmill exercise with a moderate intensity .

[ purpose ] the aim of the present study was to investigate effects of skilled reach training with affected forelimb and treadmill exercise on the expression of neurotrophic factor following ischemia - induced brain injury in rats . [ subjects and methods ] thirty male sprague - dawley rats were divided into 3 groups randomly : namely , the control sacrified 2 weeks after surgery , skilled reach training with forepaw contralateral to brain injury for 2 weeks , and treadmill exercise for 2 weeks . transient focal cerebral ischemia was induced by intraluminal occlusion of the left middle cerebral artery . after that , skilled reach training and treadmill exercise were conducted . western blot analysis was performed to investigate expressions of neurotrophic factors . [ results ] there were significant differences in brain - derived neurotrophic factor and nerve growth factor expression between the control group and the experimental group . there were no significant differences in brain - derived neurotrophic factor and nerve growth factor expression between the skilled reach training group and the treadmill exercise group . [ conclusion ] skilled reach training and treadmill exercise can affect the expression of neurotrophic factors .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4974191

rotator cuff lesions are the most common causes of painful shoulder in the elderly , accounting for up to 70% of cases ( 1 ) . although magnetic resonance ( mr ) arthrography is reported to be the most sensitive and specific technique for diagnosing rotator cuff tears , ultrasound ( us ) is widely used since it is a cost - effective and easily assessable tool , providing dynamic imaging studies ( 234 ) . ultrasound is reported to be accurate , with a sensitivity range from 92.4% to 96% and a specificity range from 93% to 94.4% for diagnosing full thickness tear and a sensitivity range from 66.7% to 84% and a specificity range from 89% to 93.5% for partial thickness tear ( 25 ) . measuring the tendon thickness by ultrasound is proven to be a valid method in diagnosing enthesopathy of plantar fascia , distal achilles tendon , patellar ligament , distal quadriceps and brachial triceps tendon in spondyloarthropathy patients ( 6 ) . in addition , ultrasound dimension of achilles tendon thickness can be used as one of the diagnostic criteria in achilles tendon pathology ( 7 ) . in diagnosing rotator cuff lesions , tendon thickness should be compared with normal dimensions of the rotator cuff . there have been researches suggesting diagnostic cut off values of rotator cuff tear or supraspinatus tendinopathy ( 58 ) . however , there is no definite reference providing normal ultrasound dimensions of the shoulder with a wide range of age groups , especially in the korean population . in this study , the ultrasound dimensions of the rotator cuff , long head of biceps tendon , deltoid muscle , subacromial subdeltoid bursa and acromioclavicular joint in healthy korean adults were measured and the possible variability among different sex , dominant hands , and ages were assessed to provide accurate references for rotator cuff measurements . all of the subjects enrolled in the study were recruited from volunteers who visited our institution , from april 2014 to february 2015 . healthy adults aged between 20 and 70 years who had no shoulder problems were included . exclusion criteria were the following : subjects with 1 ) shoulder pain , 2 ) history of shoulder instability or dislocation , 3 ) shoulder pathology such as rotator cuff injury , impingement syndrome , biceps tendinopathy , adhesive capsulitis , subacromial bursitis , or acromioclavicular joint injury , 4 ) history of surgery involving rotator cuff injury , 5 ) shoulder weakness due to underlying pathology such as suprascapular neuropathy , brachial plexopathy , cervical root disorder , cervical myelopathy and stroke 6 ) diabetes mellitus , 7 ) rheumatic disorders or systemic diseases ( renal , hepatic , cardiac , etc . ) . to exclude preexisting shoulder pathology , physical examinations including shoulder range of motion and palpation were performed . in addition , provocative tests to evaluate glenohumeral instability , labral pathology , rotator cuff injury , impingement , acromioclavicular joint pathology , bicep tendon injury were performed . patients who have pain , tenderness , limited range of motion , or positive findings in at least one of those physical examinations were excluded . ultrasound examination was performed by a single physiatrist , who is a formal member of korean academy of rehabilitation medicine , with experience in musculoskeletal ultrasound scanning for more than 10 years . acuson sequoia 512 ( siemens , germany ) ultrasound scanner with an 8 - 15 mhz linear array probe was used . ultrasonographic scanning was performed according to the protocol recommended by the european society of musculoskeletal radiology . the thickness of long head of biceps tendon the thickness of subscapularis tendon the thickness of supraspinatus tendon the thickness of subacromial subdeltoid ( sasd ) bursa the interval of acromioclavicular joint ( ac joint ) the thickness of infraspinatus tendon the thickness of deltoid muscle the tendon of long head of biceps was measured in the transverse view at the highest point of the groove with the subject having neutral shoulder and elbow flexed 90 ' with palm up position . then , with the probe fixed in the same position , the subject was instructed to externally rotate the arm , fixing the elbow on the lateral chest . when the subscapularis tendon emerged inferior to the coracoid process , the probe was slightly moved to find the site of insertion to the lesser tuberosity and the thickness of subscapularis tendon was measured at just medial to the insertion site ( fig . the thickness of supraspinatus tendon was measured on the coronal view at the sulcus located between greater tuberosity and articular cartilage with the modified crass position ( fig . the modified crass position means placing the subjects ' arm posteriorly and the palmar side of the hand on the superior aspect of the iliac wing with the elbow flexed , directed posteriorly . with this position , the reason why we chose the modified crass position over the crass position is that the majority of patients with rotator cuff pathology experience less pain and are able to position closer to the instruction in the former than the latter . the probe was moved anteriorly and posteriorly to precisely observe the insertion of supraspinatus tendon located anteriorly to the running of the biceps tendon . the thickness of the subacromial subdeltoid bursa and supraspinatus tendon were measured on the coronal view in the same plane . acromioclavicular joint interval was measured over the top of the shoulder in a coronal plane . infraspinatus tendon thickness was measured at the level of the posterior border of the acromion with the hand placing on the opposite shoulder ( fig . deltoid muscle thickness was measured at the anterolateral edge of acromion with the same position as the infraspinatus tendon thickness . ( a ) the thickness of subscapularis tendon was measured at just medial to the site of insertion to the lesser tuberosity . ( b ) the thickness of supraspinatus tendon was measured on the coronal view at the sulcus located between greater tuberosity and articular cartilage . ( c ) the thickness of infraspinatus tendon was measured at the level of the posterior border of the acromion . considering previous researches of measuring ultrasound dimensions in asymptomatic adults , our sample size was decided ( 89 ) . in our study , we assumed ultrasonographic dimensions of shoulders would be values of standard deviation of 0.7 and a difference of 0.5 mm in thickness of rotator cuff between males and females would be significant statistically with p value of 5% . the sample size of 43 in each sex group was assumed to be adequate to compare with each other with power of 80% . the rate of falling out was assumed to be 15% , we planned to enroll 55 patients for each group . unpaired t - test ( or wilcoxon rank sum test for nonparametric test ) was used to determine differences in measurements between males and females . paired t - test ( or wilcoxon signed rank test for nonparametric test ) was used to compare measurements of dominant and non - dominant shoulders . differences in values according to the age were determined by analysis of variance ( anova or kruskal - wallis for nonparametric tests ) . data analyses were performed with spss ver . 21.0 for windows ( spss inc . , chicago , il , usa ) . all of the subjects enrolled in the study were recruited from volunteers who visited our institution , from april 2014 to february 2015 . healthy adults aged between 20 and 70 years who had no shoulder problems were included . exclusion criteria were the following : subjects with 1 ) shoulder pain , 2 ) history of shoulder instability or dislocation , 3 ) shoulder pathology such as rotator cuff injury , impingement syndrome , biceps tendinopathy , adhesive capsulitis , subacromial bursitis , or acromioclavicular joint injury , 4 ) history of surgery involving rotator cuff injury , 5 ) shoulder weakness due to underlying pathology such as suprascapular neuropathy , brachial plexopathy , cervical root disorder , cervical myelopathy and stroke 6 ) diabetes mellitus , 7 ) rheumatic disorders or systemic diseases ( renal , hepatic , cardiac , etc . ) . to exclude preexisting shoulder pathology , physical examinations including shoulder range of motion and palpation were performed . in addition , provocative tests to evaluate glenohumeral instability , labral pathology , rotator cuff injury , impingement , acromioclavicular joint pathology , bicep tendon injury were performed . patients who have pain , tenderness , limited range of motion , or positive findings in at least one of those physical examinations were excluded . ultrasound examination was performed by a single physiatrist , who is a formal member of korean academy of rehabilitation medicine , with experience in musculoskeletal ultrasound scanning for more than 10 years . acuson sequoia 512 ( siemens , germany ) ultrasound scanner with an 8 - 15 mhz linear array probe was used . ultrasonographic scanning was performed according to the protocol recommended by the european society of musculoskeletal radiology . the thickness of long head of biceps tendon the thickness of subscapularis tendon the thickness of supraspinatus tendon the thickness of subacromial subdeltoid ( sasd ) bursa the interval of acromioclavicular joint ( ac joint ) the thickness of infraspinatus tendon the thickness of deltoid muscle the tendon of long head of biceps was measured in the transverse view at the highest point of the groove with the subject having neutral shoulder and elbow flexed 90 ' with palm up position . then , with the probe fixed in the same position , the subject was instructed to externally rotate the arm , fixing the elbow on the lateral chest . when the subscapularis tendon emerged inferior to the coracoid process , the probe was slightly moved to find the site of insertion to the lesser tuberosity and the thickness of subscapularis tendon was measured at just medial to the insertion site ( fig . the thickness of supraspinatus tendon was measured on the coronal view at the sulcus located between greater tuberosity and articular cartilage with the modified crass position ( fig . the modified crass position means placing the subjects ' arm posteriorly and the palmar side of the hand on the superior aspect of the iliac wing with the elbow flexed , directed posteriorly . with this position , the reason why we chose the modified crass position over the crass position is that the majority of patients with rotator cuff pathology experience less pain and are able to position closer to the instruction in the former than the latter . the probe was moved anteriorly and posteriorly to precisely observe the insertion of supraspinatus tendon located anteriorly to the running of the biceps tendon . the thickness of the subacromial subdeltoid bursa and supraspinatus tendon were measured on the coronal view in the same plane . acromioclavicular joint interval was measured over the top of the shoulder in a coronal plane . infraspinatus tendon thickness was measured at the level of the posterior border of the acromion with the hand placing on the opposite shoulder ( fig . deltoid muscle thickness was measured at the anterolateral edge of acromion with the same position as the infraspinatus tendon thickness . ( a ) the thickness of subscapularis tendon was measured at just medial to the site of insertion to the lesser tuberosity . ( b ) the thickness of supraspinatus tendon was measured on the coronal view at the sulcus located between greater tuberosity and articular cartilage . ( c ) the thickness of infraspinatus tendon was measured at the level of the posterior border of the acromion . considering previous researches of measuring ultrasound dimensions in asymptomatic adults , our sample size was decided ( 89 ) . in our study , we assumed ultrasonographic dimensions of shoulders would be values of standard deviation of 0.7 and a difference of 0.5 mm in thickness of rotator cuff between males and females would be significant statistically with p value of 5% . the sample size of 43 in each sex group was assumed to be adequate to compare with each other with power of 80% . the rate of falling out was assumed to be 15% , we planned to enroll 55 patients for each group . unpaired t - test ( or wilcoxon rank sum test for nonparametric test ) was used to determine differences in measurements between males and females . paired t - test ( or wilcoxon signed rank test for nonparametric test ) differences in values according to the age were determined by analysis of variance ( anova or kruskal - wallis for nonparametric tests ) . data analyses were performed with spss ver . 21.0 for windows ( spss inc . , chicago , il , usa ) . out of 109 volunteers , 9 subjects were excluded because of positive findings in physical examinations and experiences of shoulder injection to relieve pain . a total of 200 shoulders from 100 subjects ( 50 males and 50 females , 95 right handed and 5 left handed , age 21 to 69 years ) were scanned . baseline demographic features of the participants are presented on table 1 . in male subjects , the mean thickness of supraspinatus , infraspinatus and subscapularis tendon was 5.1 0.8 mm , 4.7 0.6 mm , 4.6 0.8 mm in dominant arm and 5.0 0.7 mm , 4.8 0.7 mm , 4.7 0.7 mm in non - dominant arm , respectively . in female subjects , the mean thickness of supraspinatus , infraspinatus and subscapularis tendon was 4.6 0.9 mm , 4.0 0.7 mm , 4.1 0.7 mm in dominant arm and 4.4 0.8 mm , 4.1 0.6 mm , 4.2 0.6 mm in non - dominant arm , respectively ( table 2 ) . the supraspinatus , infraspinatus , subscapularis tendon and deltoid muscle thickness were significantly different between males and females for dominant and non - dominant arms . the measurements of sasd bursa thickness were significantly different between males and females for non - dominant arms . p < 0.05 ; unpaired t - test ; wilcoxon rank sum test . the differences in measurements of rotator cuff tendons between the dominant and non - dominant arm among males and females showed no statistical significance ( table 3 ) . only deltoid muscle thickness and ac joint interval in males and deltoid muscle thickness in females p < 0.05 ; unpaired t - test ; wilcoxon rank sum test . when subjects were stratified by the age groups , divided by ten years , the measurements of supraspinatus tendon thickness showed tendency of increase with the age , whereas the ac joint interval showed decreasing tendency ( table 4 ) . in the other measurements , no significant difference among age groups was found . this study suggests normative reference data of rotator cuff tendon thickness and acromioclavicular joint interval among korean population . to make ultrasonographic diagnosis of rotator cuff pathology , especially rotator cuff tear or tendinopathy , although there has been a report suggesting diagnostic criteria of supraspinatus tendinopathy demonstrating maximal thickness of supraspinatus tendon based on comparison between symptomatic patients and asymptomatic controls , there is no report suggesting normal reference values of rotator cuff dimension ( 10 ) . this study is in great value for the fact that it is the first report providing normative ultrasound dimensions of the rotator cuff in healthy korean adults with varying age . the results of our study possess certain degree of validity since the results show the correlation with prior studies ( 911 ) . in our study , the supraspinatus , infraspinatus , subscapularis tendon and deltoid muscle thickness were significantly different between males and females for dominant and non - dominant arms . the increasing tendency of rotator cuff thickness in male subjects in our research is assumed to be related to larger strength of the shoulder in males than females . there has been research demonstrating significant correlations between supraspinatus thickness and external rotation strength , infraspinatus thickness and internal rotation thickness , subscapularis thickness and internal rotation strength ( 8) . in addition , in other prior research on the rotator cuff dimensions of young adults ( aged 18 - 40 years ) , the rotator cuff dimensions between males and females were significantly different ( 9 ) . the dimensions between dominant and non - dominant arms were not significantly different in all of the thickness of rotator cuff tendon , biceps tendon and subacromial subdeltoid bursa with the exception of ac joint interval and the thickness of deltoid muscle . our study showed similar results with previous researches , which means the asymptomatic contralateral shoulder can be used to estimate the expected dimension ( 912 ) . however , in the male group , the ac joint interval of dominant arms was significantly lower than non - dominant arms . this result could be associated with arthritic change as a consequence of more usage of dominant arm in males . further researches regarding association of activities of upper extremities with ac joint interval are needed . when the measurements were stratified by age , the measurements of supraspinatus tendon thickness revealed increasing tendency with increasing age groups . this tendency could be related to asymptomatic rotator cuff tendinopathy which shows frequent incidence rate with aging . the prevalence of rotator cuff pathology is reported to increase by natural aging process ( 13 ) . in a research of ultrasonographic findings in asymptomatic shoulders , supraspinatus tendon was significantly thicker and demonstrated a lower echogenicity ratio in elderly patients aged more than 60 years and the thickness showed positive correlation with age . this study suggested the increase in thickness of the supraspinatus tendon might be due to chronic tendinopathy by age - related degeneration . in a research study of ultrasonographic findings of asymptomatic shoulders , supraspinatus tendinosis was the third most common abnormal finding , accounting for 39% , followed by subacromial subdeltoid bursal thickening and acromioclavicular joint osteoarthritis ( 14 ) . further studies for clarifying correlations between sonographic findings and pathology are needed . in our data , this tendency may be the result of natural degeneration by aging process and similar results are documented in the other studies . ( 15 ) reported more advanced arthritic changes in acromioclavicular joint were detected in mri in the over 30 age group . nicholson et al . ( 16 ) reported significant increase in degenerative changes at the acromial facet of the acromioclavicular joint occurred with advancing age . first , the ultrasound scanning was performed once by a single physiatrist , which means intraobserver and interobserver agreement was not assessed . the previous study regarding rotator cuff dimension in young healthy adults showed good intraobserver and interobserver agreement ( 9 ) . in the study of sonographic evaluation of the painful shoulder , the examiners were in very good agreement for full - thickness rotator cuff tears , supraspinatus tendinosis , abnormalities of the long head of biceps tendon , subacromial bursa abnormalities , acromioclavicular osteoarthritis and moderate agreement for partial thickness tear and intratendinous tears ( 17 ) . the other study revealed good interobserver reliability in grading fatty degeneration of rotator cuff muscles ( 18 ) . although operator dependence has been considered a limitation of ultrasonography , good interobserver and intraobserver reliability was reported by previous studies . although our study could not assess the intraobserver and interobserver reliability , ultrasonography measurements were performed on both arms for each participant and the results of each measurement showed congruence , indirectly increasing the reliability of the single physiatrist 's measurement . second , the number of subjects was not large enough to objectively assess differences among age . however , this is the first report that evaluated the normal dimensions of rotator cuff tendons where at least 10 subjects were recruited within each subgroup of stratified age , having a range of 20 to 70 years . third , all of the subjects were chosen from one institution and there could be selection bias . however , participants in our study possess diversity in age and gender , which could represent general korean population . fourth , subjects ' anthropometric factors that can possibly affect the measurements such as height , weight and body mass index ( bmi ) were not assessed . as the previous study showed there were no significant correlation between the height or weight of the subjects and the rotator cuff tendon measurements , we assumed weight and height could be omitted in measuring normal rotator cuff tendon ( 9 ) . however , because there has been a study demonstrating obesity is related to increased risk for rotator cuff tendinitis and rotator cuff related surgery , further studies assessing relationship between body mass index and cuff tendon thickness under adjustment of age and gender are needed ( 19 ) . fifth , echogenicity that could be useful in diagnosing rotator cuff tendinopathy was not measured in our research . further studies assessing echogenicity as well as rotator cuff tendon thickness in healthy adults would be needed to suggest more accurate normal reference data . further studies with larger groups of subjects assessing normal rotator cuff dimensions and defining influencing factors including a wide range of age would be needed to further validate our normative reference values . furthermore , studies regarding comparison between measurements of normal healthy adults and patients with rotator cuff lesion and suggesting the cut - off value of rotator cuff lesions in korea would be needed .

in evaluating patients complaining of shoulder pain , ultrasonography is an emerging imaging tool due to convenience , low cost , high sensitivity and specificity . however , normative values of ultrasound dimensions of the shoulder to be compared with pathologic findings in korean adults are not provided yet . we evaluated the ultrasound dimensions of the rotator cuff , long head of biceps tendon , deltoid muscle and acromioclavicular joint in korean healthy adults . shoulder ultrasonography was performed on 200 shoulders from 100 healthy adults . the dimensions of the thickness of rotator cuff ( supraspinatus , infraspinatus , subscapularis tendon ) , deltoid muscle , long head of biceps tendon , subacromial subdeltoid bursa , and acromioclavicular joint interval were measured in a standardized manner . differences in measurements among sex , age , and dominant arms were compared.the thickness of rotator cuff tendons ( supraspinatus , infraspinatus , subscapularis ) and deltoid muscle were significantly different between men and women . the thickness of subacromial subdeltoid bursa was significantly different between men and women for non - dominant side . in rotator cuff tendon measurements , the differences between dominant and non - dominant shoulders were not significant , which means the asymptomatic contralateral shoulder can be used to estimate the normal reference values . when stratified by age divided by 10 years , the measurements of supraspinatus , subscapularis and deltoid thickness showed tendency of increase with the age . the acromioclavicular joint interval , on the other hand , revealed decreasing tendency . this report suggests normative values of ultrasound dimensions of healthy korean population with varying age , and can be useful as reference values in evaluating shoulder pathology , especially in rotator cuff tendon pathology .

INTRODUCTION MATERIALS AND METHODS Subjects Data collection Statistical analysis Ethics statement RESULTS DISCUSSION

PMC3982812

specimens : five species of adult salamanders representing five families were examined in this study ( table 1table 1.specimens used in this studyspecieshabitatsvl * ( mm)amphiuma tridactylumaquatic408434471necturus maculosusaquatic175172168cynops pyrrhogastersemi - aquatic534845hynobius nigrescensterrestrial707273ambystoma tigrinumterrestrial11110386*snout - vent length . ) . aquatic species included amphiuma tridactylum ( n=3 ) and necturus maculosus ( n=3 ) , which have elongated bodies and shortened limbs ( a. tridactylum more so than n. maculosus ) . semi - aquatic species were represented by cynops pyrrhogaster ( n=3 ) , which has a short body and relatively robust limbs . terrestrial species included hynobius nigrescens ( n=3 ) and ambystoma tigrinum ( n=3 ) , which have short trunks and robust limbs . specimens were fixed in straight body position using 10% formalin and preserved in 70% ethanol solution . m. dorsalis trunci makes up most of the epaxial muscle mass and m. interspinalis , m. intertransversarius and m. subvertebralis are perivertebral muscles . m. obliquus externus , m. obliquus externus superficialis , m. obliquus externus profundus , m. obliquus internus and m. transversus abdominis are grouped into lateral hypaxial muscles , and m. rectus lateralis is found along the trunk on the lateral hypaxial muscles . the following trunk muscles were examined in this study : m. dorsalis trunci , m. interspinalis , m. intertransversarius , m. subvertebralis , m. obliquus externus , m. obliquus externus superficialis , m. obliquus externus profundus , m. obliquus internus , m. transversus abdominis , m. rectus profundus , m. rectus lateralis and m. rectus abdominis . each trunk muscle on the left side was dissected using tweezers in the five species , and specimens were observed from the lateral view ( fig . 1.ambystoma tigrinum ( a ) : lateral view of trunk musculature ( b ) : dorsal view of trunk musculature after removal of m. dorsalis trunci . subsequently , cross - sections were obtained from the middle of the trunk between the pectoral and pelvic girdles on the right side of the body . images of the lateral view and cross - sections were observed using a microscope with a single - lens reflex camera . ambystoma tigrinum ( a ) : lateral view of trunk musculature ( b ) : dorsal view of trunk musculature after removal of m. dorsalis trunci . , specimens were moistened with water to avoid drying , which may cause measurement error . each trunk muscle was weighed to an accuracy of 0.01 mg using an electronic balance shimadzu auw220d ( simadzu , kyoto , japan ) . the ratio of the weight of each muscle to the weight of all trunk muscles was calculated as a percentage . in this study , to calculate these ratios , the weight of all trunk muscles was treated except that of m. rectus lateralis , because of its small size . for distinction among the five species , values for some muscles were grouped according to anatomical position , running direction and function : m. obliquus externus superficialis and m. obliquus externus profundus ; m. obliquus internus and m. transversus abdominis ; m. rectus profundus and m. rectus abdominis . these tests were conducted using statistical processing software ( jmp pro 9 , sas institute japan , tokyo , japan ) . homogeneity of variance and means between species were confirmed by analysis of variance ( anova ) . when significant differences were identified by anova , differences between species were estimated using tukey s test . francis , naylor and maurer [ 12 , 13 ] were used as references for the following muscle descriptions . two superficial lateral hypaxial layers consist of m. obliquus externus superficialis and m. obliquus externus profundus . these muscle fibers run from the craniodorsal region caudoventrally towards the pubic bone . m. obliquus internus and m. transversus abdominis make up the inner lateral hypaxial layers . these muscle fibers run from the cranioventral trunk region to the dorsal parts of the hip girdle . some species have m. obliquus externus instead of m. obliquus externus superficialis and m. obliquus externus profundus . observations of muscle morphology : cross - sectional areas and lateral views of the trunk muscles for the five species of salamanders are shown in figs . 2 and 3fig . 2.cross-sections through the midtrunk of ( a ) : amphiuma tridactylum , ( b ) : necturus maculosus , ( c ) : cynops pyrrhogaster , ( d ) : hynobius nigrescens , ( e ) : ambystoma tigrinum . scale bar=5 mm.fig . 3.lateral view of ( a ) : amphiuma tridactylum , ( b ) : necturus maculosus , ( c ) : cynops pyrrhogaster , ( d ) : hynobius nigrescens , ( e ) : ambystoma tigrinum . the number of muscle layers and specialization of each muscle differed by species . in a. tridactylum , an aquatic species , four layers ( m. obliquus externus superficialis , m. obliquus externus profundus , m. obliquus internus and m. transversus abdominis ) composed the lateral hypaxial musculature . m. rectus abdominis in a. tridactylum could not be distinguished from m. obliquus externus profundus and m. obliquus internus . the lateral hypaxial muscles of a. tridactylum were relatively thick and covered the m. dorsalis trunci . in n. maculosus , an aquatic species , three layers ( m. obliquus externus , m. obliquus internus and m. transversus abdominis ) made up the lateral hypaxial muscles , which were relatively thick . the m. rectus abdominis of n. maculosus was not separated from m. obliquus externus and m. obliquus internus . the lateral hypaxial muscles of c. pyrrhogaster , a semi - aquatic species , consisted of four layers : m. obliquus externus superficialis , m. obliquus externus profundus , m. obliquus internus and m. transversus abdominis . m. rectus abdominis was easily separated from m. obliquus externus profundus and m. obliquus internus . nigrescens , a terrestrial species , two layers were evident in the lateral hypaxial muscles : m. obliquus externus and m. transversus abdominis . h. nigrescens possessed m. rectus profundus and an isolated m. rectus abdominis . in a. tigrinum , a terrestrial species , four layers ( m. obliquus externus superficialis , m. obliquus externus profundus , m. obliquus internus and m. transversus abdominis ) made up the lateral hypaxial muscles , which were relatively thin . m. rectus abdominis of a. tigrinum separated from other muscular structures . cross - sections through the midtrunk of ( a ) : amphiuma tridactylum , ( b ) : necturus maculosus , ( c ) : cynops pyrrhogaster , ( d ) : hynobius nigrescens , ( e ) : ambystoma tigrinum . . lateral view of ( a ) : amphiuma tridactylum , ( b ) : necturus maculosus , ( c ) : cynops pyrrhogaster , ( d ) : hynobius nigrescens , ( e ) : ambystoma tigrinum . scale bar=5 mm . muscle weight : weight ratios of trunk muscles to overall muscle weight are represented in table 2table 2.muscle weight ratios ( % ) measured at midtrunk ( mean sem)speciesm . rectus abdominis , m. rectus profundus + m. rectus abdominisamphiuma tridactylum33.3 0.83.2 0.42.1 0.39.5 1.323.2 1.225.0 1.93.7 0.4necturus maculosus34.0 1.83.0 0.52.0 0.310.4 0.620.6 0.825.8 2.94.0 1.814.5 1.99.4 0.4hynobius nigrescens46.4 2.35.0 0.33.4 0.514.4 0.510.4 2.67.3 1.213.1 1.5ambystoma tigrinum45.7 3.86.8 1.23.6 0.216.9 2.16.0 0.47.7 2.612.3 0.2different superscript letters indicate significant differences ( anova and tukey s test , p<0.05 ) . and 4.muscle weight ratios of each trunk muscle in a. tridactylum , n. maculosus , c. pyrrhogaster , h. nigrescens and a. tigrinum . different superscript letters indicate significant differences ( anova and tukey s test , p<0.05 ) . black bar : aquatic species , gray bar : semi - aquatic species , white bar : terrestrial species .. considerable variations were observed between these salamanders . significant differences were shown among species in m. dorsalis trunci weight ratios [ f ( 4 , 10)=22.79 , p<0.01 ] ( f explains f - value of anova , and numbers in bracket mean degrees of freedom ) . for m. dorsalis trunci , weight ratios were smaller in aquatic species ( a. tridactylum of 33.3% and n. maculosus of 34.0% ) and significantly smaller than those of terrestrial species ( p<0.05 ) . for the terrestrial species , h. nigrescens and a. tigrinum , m. dorsalis trunci accounted for more than 46% of muscle weight . among the more aquatic species , m. interspinalis occupied a smaller percentage of total muscle weight than that of more terrestrial species , and significant interspecific differences were demonstrated [ f ( 4 , 10)=17.05 , p<0.01 ] . in a. tigrinum , which is a terrestrial species , m. interspinalis made up less than 7% of total muscle weight , but the value for this muscle was significantly larger than that of aquatic species . significant differences were exhibited among species [ f ( 4 , 10)=15.82 , p<0.01 ] in m. intertransversarius weight ratios . in more aquatic species , the weight ratio of m. intertransversarius in h. nigrescens and a. tigrinum was significantly larger than in aquatic species ( p<0.05 ) . for m. subvertebralis , significant differences in weight ratios among species were shown [ f ( 4 , 10)=19.87 , p<0.01 ] . in aquatic species , the weight ratio for m. subvertebralis was significantly smaller than that of terrestrial species ( p<0.05 ) . different superscript letters indicate significant differences ( anova and tukey s test , p<0.05 ) . muscle weight ratios of each trunk muscle in a. tridactylum , n. maculosus , c. pyrrhogaster , h. nigrescens and a. tigrinum . different superscript letters indicate significant differences ( anova and tukey s test , p<0.05 ) . black bar : aquatic species , gray bar : semi - aquatic species , white bar : terrestrial species . significant interspecific differences in weight ratio of m. obliquus externus were seen among species [ f ( 4 , 10)=61.20 , p<0.01 ] . weight ratios for m. obliquus externus were significantly larger in more aquatic species ( p<0.05 ) . m. obliquus externus weight ratios were significantly larger than 20% in aquatic species ( a. tridactylum and n. maculosus ) . significant differences in weight ratios of m. transversus abdominis were observed among species [ f ( 4 , 10)=51.68 , p<0.01 ] . similarly , in more aquatic species , weight ratios for m. transversus abdominis were larger . in a. tridactylum and n. maculosus , weight ratios for m. transversus abdominis were significantly larger than in other species ( p<0.05 ) . in terrestrial species ( h. nigrescens and a. tigrinum ) , m. transversus abdominis occupied less than 8% of total muscle weight , a significantly smaller percentage than in other species . finally , significant differences among species were exhibited in weight ratio for m. rectus abdominis [ f ( 4 , 10)=90.82 , p<0.01 ] ; it was smaller in more aquatic species . in aquatic species ( a. tridactylum and n. maculosus ) , m. rectus abdominis accounted for less than 5% of total muscle weight , which was significantly smaller than that in other species ( p<0.05 ) . terrestrial species ( h. nigrescens and a. tigrinum ) were equipped with significantly larger m. rectus abdominis . morphological trends : a comparison of trunk muscles on cross sections through the midtrunk region showed interspecific variations in relative muscle thickness ( fig . thicker lateral hypaxial muscles were found in more aquatic species , whereas thinner lateral hypaxial muscles were found in more terrestrial species . this finding supports the results of a previous study by simons and brainerd comparing s. lacertina , a. tridactylum , cryptobranchus alleganiensis and a. tigrinum . in the more developed limbs of the terrestrial species , the locomotive function of the lateral hypaxial muscles shifts , and their thickness decreases . m. obliquus internus , one of the lateral hypaxial muscles , is used during both swimming and walking . in swimming , m. obliquus internus mainly produces lateral bending and produces or resists long - axis torsion and produces sagittal flexion . in terrestrial locomotion , m. obliquus internus plays a role in counteracting long - axis torsion of the trunk . while lateral hypaxial muscles are used on the ground , more aquatic species may need larger lateral hypaxial muscles for lateral bending , because of water viscosity and the resulting drag . especially in a. tridactylum , although both a. tridactylum and n. maculosus are fully aquatic species , a. tridactylum possesses smaller limbs than n. maculosus . therefore , a. tridactylum may depend more than n. maculosus on dorsally enlarged lateral hypaxial muscles for locomotion . m. rectus lateralis was recognized in a. tigrinum , which is a terrestrial species , and c. pyrrhogaster , which is a semi - aquatic species , and m. rectus profundus was observed in h. nigrescens , which is a terrestrial species . although differences were observed in the number of layers of lateral hypaxial muscles among species , no significant trend was found related to habitat or predominant mode of locomotion . these results are consistent with those of simons and brainerd for other taxa . the same authors did not find a close association between ecology and predominant mode of locomotion of salamanders with the number of hypaxial muscle layers in their analysis of ten families of urodela . m. rectus abdominis was independent of lateral hypaxial muscles in semi - aquatic species ( c. pyrrhogaster ) and terrestrial species ( h. nigrescens and a. tigrinum ) . however , m. rectus abdominis in aquatic species ( a. tridactylum and n. maculosus ) was continuous with the lateral hypaxial musculature . therefore , it is reasonable to assume a more specific function of m. rectus abdominis in more terrestrial species . m. rectus abdominis plays a role in counteracting sagittal extension of the trunk produced by the action of the epaxial muscles [ 5 , 20 ] and by the gravity with the elongate trunk in salamanders . therefore , we suggest that the terrestrial species examined in this study use their relatively greater and clearly isolated m. rectus abdominis in resisting sagittal extension of the trunk and that this adaptation is related to the amount of time they spend on land . muscle weights : we found m. dorsalis trunci was larger in more terrestrial species ( fig . 4 ) . during both swimming and walking , m. dorsalis trunci produces lateral bending [ 5 , 6 , 9 ] . in addition , most lateral strands of the autochthonous m. dorsalis trunci may be in a position to exert ventral flexing of the trunk ( fig . therefore , in order to counteract the effect of gravitational forces , m. dorsalis trunci was greater in more terrestrial species . m. interspinalis may also contribute to flexing the trunk ventrally to counteract the influence of the gravity by its position ( fig . 2 ) . thus , m. interspinalis was larger in more terrestrial species , which employ m. interspinalis on land although the function of m. intertransversarius has not been clarified , it seems to play a role in ensuring spinal integrity and ventral flexing similar to m. interspinalis , because of its location between vertebrae . 4 ) . functional subunits that can stabilize and mobilize the trunk and adjust body stiffness compose m. subvertebralis . in addition because of its role in trunk stabilization , m. subvertebralis is more often utilized in terrestrial environments . thus , heavier m. subvertebralis is characteristic of more terrestrial species . lateral hypaxial muscles ( m. obliquus externus superficialis , m. obliquus externus profundus , m. obliquus internus and m. transversus abdominis ) were larger in more aquatic species ( fig . lateral hypaxial muscles function in torsion control and maintaining stability [ 3 , 5 ] . as mentioned earlier , lateral hypaxial muscles are used in both swimming and walking . m. obliquus internus has functions of mainly producing lateral bending and producing or resisting long - axis torsion of the trunk as well as some sagittal flexion in swimming . in terrestrial locomotion , m. obliquus internus plays a role in counteracting long - axis torsion of the trunk . trunk muscles in species with diminutive limbs produce lateral bending actively during aquatic locomotion , whereas lateral bending is produced passively by the muscle action of extrinsic limbs . aquatic species rely more on lateral bending of the trunk to move forward ; therefore , weights of lateral hypaxial muscles are relatively greater in more aquatic species . though all lateral hypaxial muscles play a role in stabilization and torsion control on ground , muscle ratios of lateral hypaxial muscles may be affected by differences in lateral bending in different environments . since muscular forces along the ventral contour line are needed in animals with lengthy trunks that are supported by limbs , the m. rectus abdominis , which is the most ventral muscle , must be heavier in more terrestrial species [ 5 , 17 ] . it is suggested that the function of m. rectus abdominis is to prevent sagittal trunk extension caused by the action of the epaxial muscles and by the gravity with the elongate trunk in salamanders . since larger weight ratios for m. dorsalis trunci 4 ) and influence of gravity occurs , dorsally concave curvature of the trunk by dorsal muscles may also be larger than in more aquatic species . to resist greater dorsally concave curvature of the trunk , larger the aquatic species were larger than the terrestrial species , and lateral hypaxial muscles were thicker and heavier in the aquatic forms . it is considerable that the stronger development of the lateral hypaxial musculature in the aquatic species may be enforced by the factor by size . in this study , interspecific variation in trunk musculature among urodela reflects their main mode of locomotion in different ecological habitats .

abstracttrunk musculature in urodela species varies by habitat . in this study , trunk musculature was examined in five species of adult salamanders representing three different habitats : aquatic species , amphiuma tridactylum and necturus maculosus ; semi - aquatic species , cynops pyrrhogaster ; terrestrial species , hynobius nigrescens and ambystoma tigrinum . more terrestrial species have heavier dorsal and ventral trunk muscles than more aquatic forms . by contrast , the lateral hypaxial musculature was stronger in more aquatic species . the number of layers of lateral hypaxial musculature varied among urodela species and did not clearly correlate with their habitats . the m. rectus abdominis was separated from the lateral hypaxial musculature in both terrestrial and semi - aquatic species . in aquatic species , m. rectus abdominis was not separated from lateral hypaxial musculature . lateral hypaxial musculature differed in thickness among species and was relatively thinner in terrestrial species . in more terrestrial species , dorsal muscles may be used for stabilization and ventral flexing against gravity . ventral muscle may be used in preventing dorsally concave curvature of the trunk by dorsal muscles and by weight . the lengthy trunk supported by limbs needs muscular forces along the ventral contour line in more terrestrial species . and , the locomotion on well - developed limbs seems to lead to a decrease of the lateral hypaxial musculature .

MATERIALS AND METHODS RESULTS DISCUSSION

PMC3062964

the nutritional benefits of linum usitatissium seed , more commonly known as flaxseed , are of significant interest as flax is seen as healthy . oil is the main constituent of flaxseed , with more than 70% of the oil composed of polyunsaturated fat . flaxseed oil is the richest source of the essential omega-3 fatty acid , alpha - linolenic acid . biotechnology and biomedicine around flax is developing its nutraceutical potential and thus contributes to the value added of the seed and oil [ 24 ] . flaxseed is rich in nutrients , such as lignans which have been shown in laboratory studies to help protect against certain types of cancers [ 58 ] . flax also contains an abundance of hydrophobic cyclolinopeptides ( clps ) , identified as cycloinopeptides a - i , to the extent that 500 mg of clps are very easily extracted from 1000 ml of pressed oil . cyclolinopeptide a ( clp - a ) , a nonapeptide with a primary sequence of pro - pro - phe - phe - leu - ile - ile - leu - val shown in figure 1 , was first isolated in 1959 from flaxseed oil precipitates . the role of clp - a and other clps in flax is unclear but in laboratory studies these peptides exhibit potent immunosuppressive activity , as well as inhibitory activity towards calcium - dependent activation of t - lymphocyte cell division , as well as show antimalarial activity . depending on the peptide sequence , clps may have an abundance of c = o , nh , and sulfur containing side chains , which could be used for linking drug delivery vessels . for instance , clps were synthesized with an even number of d and l amino acids , and were found to stack through intermolecular hydrogen bonding forming nanotubes with an antiparallel structure . the amino acid residues are located on the exterior of the nanotube , which allows the outer surface properties to be optimized to its environment while the interior of the tube is uniform and adjustable by the number of amino acid residues inserted in the peptide . in another example , human serum albumin ( hsa ) was modified with the cyclic - rgd peptide and a peg linker was used to link a previously hsa is a globular protein and is the most abundant protein present in the blood plasma commonly occurring at concentrations of approximately 40 mg ml . it is a transport protein due to its interactions with many organic / inorganic compounds including fatty acids , giving it an important function of regulating intercellular fluxes . hsa is composed of three structurally similar domains ( i , ii , and iii ) , each contain 2 subdomains ( a and b ) which are stabilized by 17 disulfide bridges . in addition to the 7 binding sites for fatty acids and a high affinity binding site on the n - terminus , there are two hydrophobic pockets ( generally referred to as site i and site ii ) located in subdomains iia and iiia . investigation of the thermodynamics and kinetics of clp - a / hsa - binding and release is necessary when clps are considered as a potential drug / nutraceutical ingredient , or used as part of a potential nanodelivery device used for delivering materials to a targeted environment in biomedical applications . here , we report the binding interaction of clp - a and hsa using surface plasmon resonance ( spr ) . quantitative association ( ka ) and dissociation ( kd ) constants were obtained at different temperatures , which also allowed the determination of the thermodynamic parameters ( h , g and s ) . circular dichroism ( cd ) in both the near - uv and far - uv was used to evaluate any conformational changes of hsa upon binding with the clp - a . amine coupling reagents ( n - ethyl - n-(dimethylaminopropyl)-carbodiimide ( edc ) , n - hydroxysuccinimide ( nhs ) , ethanolamine ( ea ) ) , hbs - ep ( 10 mm hepes , 150 mm nacl , 0.005% surfactant p20 and 3.4 mm edta at ph 7.4 ) and cm5 carboxylated dextran sensor chips were obtained from biacore ( ge healthcare , montreal , qu ) . hsa ( fatty acid free ) was obtained from sigma ( oakville , on ) . bethune ) was extracted using a continuous oilseed screw press ( komet , type ca59 c ; ibg monforts oekotec gmbh & co. germany ) . to release the flaxseed oil . flaxseed oil was diluted with 5% ethyl acetate in hexane , loaded on a silica - gel column , and clp 's were separated from the oil by successive elutions of 10% ( v / v ) ethyl acetate in hexane , 50% ethyl acetate in hexane , 100% ethyl acetate , 10% methanol in dichloromethane , and 20% methanol in dichloromethane . the resulting clps extracted from the flaxseed oil were further separated by reverse - phase hplc using a gradient mixture of acetonitrile : water ( 30 : 70 ( v / v ) ) , and increasing the acetonitrile to 65% over 120 min . approximately 180 mg of clp - a was isolated per 1000 ml of flaxseed oil . the extracted clp - a was recrystallized in methanol prior to the spr and cd experiments detailed below . running buffer for all experiments was prepared by diluting a 10x phosphate - buffered saline ( pbs ) stock solution ( 100 mm phosphate buffer , 20.7 mm kcl , 1370 mm nacl , ph 6.7 ) to a 1x pbs , ph 7.4 solution containing 5% dmso . analyte solutions containing the clp - a peptides were prepared by diluting the stock solution ( 10 mm in dmso ) into 1.05x pbs , giving a final 1x pbs , ph 7.4 buffer containing 5% dmso ( same as the running buffer ) . all spr measurements were performed at different temperatures using a biacorex instrument equipped with a research - grade cm5 carboxylated - dextran sensor chip ( biacore ) . the fluidic unit of the instrument was cleaned prior to experiments using the desorb method ( injections of 0.5% sodium dodecyl sulphate , followed by 50 mm glycine , ph 9.5 ) equipped in the instrument 's software . on some occasions a super - clean method was also used ( injections of 1% ch3cooh , 1 m nahco3 , 10 mm hcl , and 6 m gndhcl ) . the carboxylated - dextran sensor chips were preconditioned prior to hsa immobilization by priming the instrument 3 times with hbs - ep buffer , followed by 3 successive 1 min injections of 50 mm naoh at 50 l min . hsa was covalently immobilized onto a cm5 sensor surface at 25c using standard amine - coupling chemistry . flow cell 1 ( fc1 ) and flow cell 2 ( fc2 ) were activated by a 7 min injection of a 1 : 1 mixture of 0.1 m nhs and 0.4 m edc using a flow rate of 5 l min . immediately after activation , hsa ( 30 g ml , 10 mm acetate buffer ph 5.3 ) a 7 min injection of 1 m ea ( ph 8.5 ) was conducted over fc1 and fc2 to deactivate the surfaces . clp - a solutions ( 50200 m ) were injected in triplicate and flowed over reference and hsa surfaces for 2 min at a flow rate of 30 m min , followed by a dissociation phase of 3 min for all experimental temperatures . at the end of the clp - a injection , the flowing solution was switched to the running buffer until the response returned to the original baseline . instances where the baseline was not readily achieved , a short pulse of 50% ( v / v ) dmso solution was injected at high flow rate to remove any remaining clp - a . data were collected at a rate of 5 hz and processed using the biaevaluation v4.2 software . the equilibrium constant , ka , was determined using the rectangular hyperbola equation : ( 1)req = rmax ka[clp - a]1+ka[clp - a ] , where req , rmax , [ clp - a ] are the measured response values , the binding site saturation value , and the concentration of clp - a , respectively . curve - fitting was done with microcal origin v7.1 using a global rmax value of 2000 . cd measurements of hsa ( 18 m ) , hsa / clp - a ( 18 m , 50 m ) in 10 mm pbs , 5% dmso , ph = 7.4 buffer were made with a pistar-180 cd spectrometer ( applied photophysics ltd . , the far - uv region was scanned from 260180 nm in 0.5 nm steps at a scan rate of 10 nm min and a bandwidth of 6 nm using a 0.010 cm optical pathlength quartz cuvette . the near - uv region was scanned from 350250 nm in 0.5 nm steps at a scan rate of 10 nm min , and a bandwidth of 2 nm using a 10 mm optical pathlength cuvette . typical spr response sensorgrams of clp - a - binding interaction with hsa which was covalently immobilized onto a carboxylated dextran gold - coated sensor surface via amine coupling are shown in figure 2 . the response of clp - a for concentrations ranging between 50 m and 200 m are shown at 20c , 25c , 30c , and 37c in figures 2(a ) , 2(b ) , 2(c ) and 2(d ) respectively . the gradual increase of response to a steady state during clp - a injection indicates very slow recognition kinetics of the clp - a to the hsa - binding site . quantitative kinetic data were initially extracted using the kinetics profiling within the biacore evaluation software . increased concentration of clp - a resulted in an increased response suggesting that the saturation point of hsa was not reached within the concentration range of clp - a used . the slow recognition and possible rate limiting step is due to cyclic peptide becoming available for interaction with hsa . the clp - a has a large hydration sphere which dissociates before interaction with hsa . this process requires energy and also increases the entropy hsa has seven known fatty - acid - binding sites , each of which is known to bind other types of small molecules . two of the more common binding sites for small molecule drugs are commonly known as sudlow 's site i ( i.e. the warfarin - binding site ) , and sudlow 's site ii ( the benzodiazapene site ) . entropy - driven site i bound drug molecules , which are structurally bulky , heterocyclic compounds with centrally localized negative polar / charges and hydrophobic substitutions , have been suggested to bind through hydrophobic interactions and/or must be bound in a hydrophobic crevice within the binding cavity . clp - a concentration was limited to below 200 m due to the solubility of the peptide in the buffer . the affinity of interaction was estimated by first assuming that hsa - binding sites were equally available and second that only high affinity sites were occupied in the working concentration range . the data sets shown in figure 3 were fit to ( 1 ) , and the ka and kd values are summarized in table 1 . the shape of the sensorgram as the temperature increases shows that in addition to the increase in affinity with temperature there is also a change in recognition before steady state is reached . the kinetic information shows that there is slower association and faster dissociation at 25c compared to the values at 37c . the faster release at the lower temperature suggests that thermodynamic parameters would also be important in understanding this binding . the fast dissociation at 25c and the slower dissociation at 37c show that 12c change contribute significantly to reaching the activation energy . the larger binding at higher temperatures indicates that the activation energy is relatively low . to further elucidate the hsa / clp - a interaction , thermodynamic parameters are extracted using the van't hoff equation ( 2)ln kd=hr(1t)sr , where r = 8.314 j mol k. a plot of ln kd versus t shown in figure 4 is linear . this yields h and s for the interaction to be 204 kj mol and 746 j mol k respectively . this indicates an association with the hydrophobic pockets of hsa and the opening of binding sites . therefore , the entire solvation complex consisting of clp - a , dmso and h2o requires energy to expose the binding site . this energy plus the binding energy for interaction in the hydrophobic pockets of hsa result in an overall endothermic process . the large entropy of the endothermic process clearly indicate that binding is entropy - driven . this is due to contribution from breaking of the solvation complex and hydrophobic binding . it was found that the site ii bound drugs formed enthalpy driven complexes ( large-h , large-s ) whereas site i bound drugs produced more entropy - driven complexes ( small-h , minimal negative or positive s ) . interestingly , the g value for all drug molecules was approximately constant ( 30 kj mol at 37c ) . for the clp - a hsa interaction , the calculated g is approximately 27 kj mol ( at 37c ) , which is slightly lower compared to the anionic drugs . however , this g value is comparable to values reported for short - medium chain ( c4c8 ) fatty acid - binding to hsa ( 26 to 28 kj mol at 37c ) . these fatty acids were found to bind in only one class of binding sites of hsa at a ratio of 2 : 1 . the binding process formed enthalpy driven complexes , reflecting van der waals and/or hydrogen binding in low dielectric media presently , it is unclear as to the role of individual amino acids or possible sequence that play a primary role for clp - a 's binding with hsa . research involving other clps ( clp - b , clp - e ) and some of their modified counterparts is currently ongoing . previous studies involving the biological activity of this peptide has shown that the pro - phe - phe- tripeptide block played an important role in its ability to inhibit cholate uptake into hepatocytes . other studies have found that the clp - a has immunosuppressive activity , and many structural analogues have been synthesized to study the peptide sequence 's role . it was concluded that the flexibility of the peptide structure plays an important role in its biological function . other work has focused on increasing the solubility of clp - a without suppressing its biological activity . it was determined that substitution of the phe residues with their sulfonated derivates for clp - a and its linear and cyclic derivatives maintained their immunosuppresor activity . for the current study , it could be speculated that increasing the solubility of clp - a would reduce the overall solvation complex , which would reduce the thermodynamic parameters of the clp - a / hsa interaction . another interesting feature observed from the spr data was that the measured req for a given concentration of clp - a also increased with temperature . given that the hsa surface concentration was approximately 11 kru , one can estimate that the theoretical rmax for this binding interaction is approximately 160 ru using the relation : ( 3)rmax theoretical = mwamwlrlsm , where mwa , mwl are the molecular weights of the analyte and ligand , respectively , rl is the surface density of ligand , and sm is the stoichiometry of the interaction . from ( 3 ) , the increase in the given response could be due to peptide aggregation , or additional binding to other active binding sites on the hsa . it should be noted that spr is measuring a change in the refractive index at the sensor surface , and this will be affected by temperature change , although it should not be significant in this narrow temperature range . if any conformational changes were present upon clp - a complexation with hsa , we compared the far - uv and near - uv cd spectra of hsa and hsa / clp - a in the running buffer used in the biacore experiments shown in figure 5 . cd spectra taken in the far - uv spectral region provide insight to the secondary structure of a protein . in figure 5(a ) , we observe that the cd spectrum for the hsa / clp - a complex has a slightly different lineshape compared to the native hsa cd spectrum , suggesting that there are slight changes to the secondary structure of hsa upon clp - a - binding . there are also some significant changes in the near - uv cd spectra shown in figure 5(b ) . this region of the spectra corresponds to signals from the phenylalanine , tyrosine , and tryptophan residues on the protein and it provides insight to changes in the tertiary structure of the protein . the observed change in cd signal indicates a tertiary conformational change upon clp - a - binding , shown in figure 5(c ) . the clp - a / hsa interaction investigated by spr shows the complex is formed by a strong endothermic and entropy - driven reaction . this entropy - driven complexation indicates that the interaction occurs through a hydrophobic interaction of the clp - a and possibly sudlow site i of the hsa . complex formation is through slow recognition kinetics and is relative stable at physiological temperatures . a slight change in both secondary and tertiary structure of hsa are observed upon the binding of clp - a . although there are still many potential questions that remain regarding the binding of clp - a to hsa , the fact that quantitative thermodynamic and binding constants has been demonstrated can help lay the foundation for the future development of any potential biomedical applications involving clp - a . this would add to understanding the nutraceutical potential of clp - a and clp in general .

the kinetics , energetics , and structure of cyclolinopeptide a binding with human serum albumin were investigated with surface plasmon resonance and circular dichroism . the complex is formed through slow recognition kinetics that is temperature sensitive in the range of 20c37c . the overall reaction was observed to be endothermic ( h = 204 kj mol1 ) and entropy driven ( s = 746 j mol1k1 ) with overall small changes to the tertiary structure .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusions

PMC3136699

acl ruptures are a common injury in orthopaedic practice . the acl is frequently replaced by a tendon autograft or allograft to restore normal knee laxity and to prevent the development of early osteoarthritis induced by persistent abnormal laxity . reconstruction techniques were improved over the last 10 years , but graft failure is not uncommon : 0.710% . even though substantial research efforts have been presented on various aspects of acl reconstruction [ 2 , 3 , 10 , 13 , 15 , 27 , 28 , 3234 ] , little current studies evaluating human biopsies pose inherent limits : often sample size was small and only a limited number of time points were evaluated postoperatively [ 6 , 11 , 18 , 19 , 21 , 35 ] . furthermore , use of different types of grafts limits comparability [ 6 , 11 ] . studies in humans describe a prolonged remodelling process compared with animal studies [ 6 , 11 , 18 , 19 , 21 , 35 ] . most animal studies found completion of the remodelling process between 24 weeks and one year . the graft undergoes a transition from its initial biological and biomechanical properties to properties resembling the intact acl . however , a complete restoration of intact acl properties has not been found , but an adaptation , which has been termed the ligamentization process [ 1 , 4 , 8 , 9 , 14 , 17 , 2123 , 25 , 31 ] . knowledge about the duration and the remodelling process itself in humans can potentially influence and improve outcome and rehabilitation protocols following acl reconstruction [ 11 , 21 ] . aim of this study was to analyse the remodelling process of human hamstring tendon grafts after standardized acl reconstruction with accelerated brace - free rehabilitation . the hypothesis of the study is that hamstring tendon grafts undergo a remodelling process that will adapt its histological appearance to the morphology of the intact acl and that this process is completed by one year . sixty - seven patients underwent retrieval of biopsies between 6 and 117 months after replacement of the acl from 2001 up to 2007 . the biopsies were carried out during second - look arthroscopies , which were not associated with the initial acl reconstruction . previous acl reconstruction by one senior orthopaedic surgeon ( hs ) using quadruple hamstring autograft with bone mulch screw fixation in femur and washerloc fixation on tibia ( arthrotec , warsaw , in , usa ) ; 2 . no signs of abnormal laxity on clinical examination nor at examination under anaesthesia at the time of second - look arthroscopy . a knee with normal laxity is defined as a knee without giving way sensation , kt 1000 < 3 mm differences on 133 n side - to - side testing ( medmetric co. , san diego , ca , usa ) and a negative pivot shift test ; 3 . informed consent to participate in the study . exclusion criteria were the following : 1 . unwillingness to participate in the study ; 2 . previous acl reconstruction by another orthopaedic surgeon or different method of fixation ; 3 . abnormal laxity on clinical examination or found in examination under anaesthesia at the time of second - look arthroscopy . abnormal laxity is defined as a knee with giving way sensation , kt-1000 > 3 mm differences on 133 n side - to - side testing and/or positive pivot shift test ; 4 . cyclops lesion , extension deficit or other reasons related to possible acl graft problems ; 5 . the study was approved by the medical ethics board of the mxima medical center eindhoven - veldhoven , the netherlands . the surgical technique for acl reconstruction was identical in all patients : a trans - tibial technique with bone mulch screw fixation on the femur and washerloc fixation on the tibia ( surgical technique by s.m . tension on the hamstring autograft at the time of fixation was 90100 n , with the knee in full extension . in addition to active flexion and extension exercises , the knee was flexed to 90 degrees by means of continuous passive motion machine ( orthorehab , oakville , ontario , canada ) . after discharge , physiotherapy was continued 23 times a week according to a standardized , brace - free , protocol . unrestricted return to heavy work activities was allowed after 3 months and competitive contact sports after 46 months . a single tissue sample was collected from one of the four strands of the quadruple hamstring autograft . the synovial layer was cleared from the middle section of the graft bundle . a shutt mini - tip straight forceps ( 2.75 mm diameter , linvatec , fl , usa ) were used through the anteromedial portal to take a superficial mid - substance biopsy of the hamstring tendon graft bundle . size of the biopsies was 23 mm . in order to create a timeline of acl autograft remodelling belonging to different individuals , samples were allocated to one of the following groups depending on the time point of their retrieval after acl reconstruction : group 1 = 612 months , group 2 = 1324 months and group 3 = greater than 24 months . two control groups were made : native hamstring tendon ( ht ) and native acl . the ht control group biopsies were taken in patients ( non - related to groups 13 ) at the end of acl reconstructive surgery . a biopsy was taken from the excess of the hamstring autograft exiting the tibia tunnel after tensioning and fixation of the graft . the acl control biopsies were taken from patients ( non - related to groups 13 ) who underwent acl reconstruction after an acute femoral acl tear ( not later than 8 weeks after injury ) . therefore , it was possible to obtain tissue samples from non - injured mid - substance areas . all patients gave written consent that tissue samples were allowed to be obtained and to be processed histologically . after the biopsies were taken , the remaining tissue was removed to continue with the acl reconstruction procedure . directly after retrieval , samples were fixed in formalin for 2 to 3 days , automatically dehydrated for 3 days and embedded in paraffin . serial cuts ( 4 m ) were prepared and mounted on slides with 3% silane ( sigma chemical , st . for descriptive and quantitative cell analysis , haematoxylin eosin ( he ) and masson - goldner - trichrom ( mg ) staining were used following standard histological protocols . vascular density was evaluated by immunostaining sections with a polyclonal rabbit anti - human von willebrandt factor ( factor viii ) antibody ( cat .- no . this antibody binds on the endothelial surface of blood vessels . for the detection of myofibroblasts , tissue sections were immunostained with a mouse anti - human asma monoclonal antibody ( cat . m0851 , dako , glostrup , denmark ) , which binds to -smooth muscle actin ( sma ) especially present in myofibroblast . all tissue samples were hydrated and pre - treated with 0,1% pronase for 10 min at 37c for factor - viii analysis . for both analyses , 10% normal horse serum ( vector laboratories inc . , burlington , ca , usa ) was used for 20 min at room temperature to block non - specific binding sites . the antibody was diluted 1:200 for factor - viii and 1:100 for -sma and added to the tissue samples overnight in a humidity chamber at 4c . after rinsing the samples with tris - buffered saline , they were incubated with the biotinylated horse anti - mouse immunoglobulin g secondary antibody for 30 min . this was followed by incubation with an avidin biotin complex ( abc kit , vectors laboratories inc . , burlington , ca , usa ) linked with alkaline phosphatase as a reporter enzyme for 50 min . tissues were counterstained with mayers haematoxylin , dehydrated and mounted in a xylol - soluble mount ( vitroclud , r langenbrinck , emme both endingen , germany ) . for all assessments ( cellular density , vessel density and myofibroblast density ) , sections were automatically digitized and saved using a digital video analysis system ( ks 400 3.0 , carl zeiss ag , gttingen , germany ) . ten regions of interest ( roi ) of different sizes , depending on the sample size , were placed on the sample at random . these cells vary by their cell shape , the proximity to vessels and show a different distribution between matrix fibres . for descriptive analysis , cell distribution pattern ( uniform / not uniform ) , morphology ( oblong / spindled / rounded , ovoid ) and the evidence of inflammatory reactions were analysed at 50 , 100 , 200 and 400 power . a shapiro wilks test was used to evaluate the normal distribution of all parameters of interest . the pearsons chi - square test was used for comparison of gender . finally , the mann whitney u test was used to compare cellular density , vascular density and myofibroblast density pairwise between the three groups , native ht and native acl . a single tissue sample was collected from one of the four strands of the quadruple hamstring autograft . the synovial layer was cleared from the middle section of the graft bundle . a shutt mini - tip straight forceps ( 2.75 mm diameter , linvatec , fl , usa ) were used through the anteromedial portal to take a superficial mid - substance biopsy of the hamstring tendon graft bundle . size of the biopsies was 23 mm . in order to create a timeline of acl autograft remodelling belonging to different individuals , samples were allocated to one of the following groups depending on the time point of their retrieval after acl reconstruction : group 1 = 612 months , group 2 = 1324 months and group 3 = greater than 24 months . two control groups were made : native hamstring tendon ( ht ) and native acl . the ht control group biopsies were taken in patients ( non - related to groups 13 ) at the end of acl reconstructive surgery . a biopsy was taken from the excess of the hamstring autograft exiting the tibia tunnel after tensioning and fixation of the graft . the acl control biopsies were taken from patients ( non - related to groups 13 ) who underwent acl reconstruction after an acute femoral acl tear ( not later than 8 weeks after injury ) . therefore , it was possible to obtain tissue samples from non - injured mid - substance areas . all patients gave written consent that tissue samples were allowed to be obtained and to be processed histologically . after the biopsies were taken , the remaining tissue was removed to continue with the acl reconstruction procedure . directly after retrieval , samples were fixed in formalin for 2 to 3 days , automatically dehydrated for 3 days and embedded in paraffin . serial cuts ( 4 m ) were prepared and mounted on slides with 3% silane ( sigma chemical , st . haematoxylin eosin ( he ) and masson - goldner - trichrom ( mg ) staining were used following standard histological protocols . vascular density was evaluated by immunostaining sections with a polyclonal rabbit anti - human von willebrandt factor ( factor viii ) antibody ( cat .- no . this antibody binds on the endothelial surface of blood vessels . for the detection of myofibroblasts , tissue sections were immunostained with a mouse anti - human asma monoclonal antibody ( cat . m0851 , dako , glostrup , denmark ) , which binds to -smooth muscle actin ( sma ) especially present in myofibroblast . all tissue samples were hydrated and pre - treated with 0,1% pronase for 10 min at 37c for factor - viii analysis . for both analyses , 10% normal horse serum ( vector laboratories inc . , burlington , ca , usa ) was used for 20 min at room temperature to block non - specific binding sites . the antibody was diluted 1:200 for factor - viii and 1:100 for -sma and added to the tissue samples overnight in a humidity chamber at 4c . after rinsing the samples with tris - buffered saline , they were incubated with the biotinylated horse anti - mouse immunoglobulin g secondary antibody for 30 min . this was followed by incubation with an avidin biotin complex ( abc kit , vectors laboratories inc . , burlington , ca , usa ) linked with alkaline phosphatase as a reporter enzyme for 50 min . tissues were counterstained with mayers haematoxylin , dehydrated and mounted in a xylol - soluble mount ( vitroclud , r langenbrinck , emme both endingen , germany ) . for all assessments ( cellular density , vessel density and myofibroblast density ) , sections were automatically digitized and saved using a digital video analysis system ( ks 400 3.0 , carl zeiss ag , gttingen , germany ) . ten regions of interest ( roi ) of different sizes , depending on the sample size , were placed on the sample at random . these cells vary by their cell shape , the proximity to vessels and show a different distribution between matrix fibres . for descriptive analysis , cell distribution pattern ( uniform / not uniform ) , morphology ( oblong / spindled / rounded , ovoid ) and the evidence of inflammatory reactions were analysed at 50 , 100 , 200 and 400 power . a shapiro wilks test was used to evaluate the normal distribution of all parameters of interest . the pearsons chi - square test was used for comparison of gender . finally , the mann whitney u test was used to compare cellular density , vascular density and myofibroblast density pairwise between the three groups , native ht and native acl . total number of biopsies ( one per patient ) was 67 : 15 biopsies in group 1 ( 612 months after acl reconstruction ) , 16 in group 2 ( 1324 months ) , 11 in group 3 ( > 24 months ) , 17 ht control and 8 acl control . groups 13 were statistically comparable for age , gender , activity level and results of the kt 1000 measurements ( table 1 ) . table 2 shows the detailed results for cell , vessel and myofibroblast density for all biopsy groups.table 1demographics ( age , gender ) of all study groups and kt 1000 results ( 133 n difference between injured and non - injured leg)agegender ( m / f)kt 1000 133 nmedian ( range)inj . non-inj.mean ( sd)mean ( sd)group 128.0 ( 1954)p = 0.4619/6p = 0.9320.8 ( 0.8)<12 months ( n = 15)31.1 ( 11.8)group 225.5 ( 1746)10/60.6 ( 0.7)1324 months ( n = 16)27.7 ( 8.7)group 329.0 ( 2137)7/101.0 ( 0.8)>24 months ( n = 11)29.0 ( 6.3)table 2results of all biopsy groups for cellular density , vessel density and myofibroblast densitygroup 1group 2group 3ht controlacl controlcells / mm mean736.3789.5718.2327.6535.8 range1550.71243.51173.2663.31545.9 median482.0850.9595.6330.4371.9 sd454.1371.1386.9201.6489.4vessels / mm mean17.96.18.27.411.9 range181.841.922.667.731.5 median0.03.43.63.110.1 sd47.310.38.515.99.3myofibroblast / mm mean35.6224.1207.93.45.7 range144.7820.6815.014.625.7 median11.5199.697.71.91.4 sd47.3234.4275.34.48.9 demographics ( age , gender ) of all study groups and kt 1000 results ( 133 n difference between injured and non - injured leg ) results of all biopsy groups for cellular density , vessel density and myofibroblast density compared with native ht , total cell number was significantly increased in groups 13 ( group 1 : p = 0.036 ; group 2 : p = 0.005 ; and group 3 : p = 0.043 ) . the highest value was found at 1324 months ( fig . 1 ) . total cell number decreased from group 2 to group 3 without reaching the cell density level of the native acl ( n.s.).fig . significant differences between groups 13 and native hamstring tendon are illustrated with * results of the median cellular density for all biopsy groups . significant differences between groups 13 and native hamstring tendon are illustrated with * figure 2 illustrates the results for vessel density . vessel density showed the lowest value in group 1 . consecutively , vessel density increased up to the level of native ht in group 2 and higher values in group 3 , without reaching the vessel density of the native acl at any time point ( n.s . ) . comparing the controls 2results of median vessel density for all biopsy groups results of median vessel density for all biopsy groups the results for myofibroblast density are illustrated in fig . myofibroblast density was significantly higher in group 2 compared with native ht ( p = 0.014 ) . myofibroblast density increased from group 1 to group 2 ( n.s . ) . from group 2 to group 3 , myofibroblast density decreased but was still increased compared with both controls ( n.s . ) . myofibroblast density was significantly higher in group 2 ( 1324 months ) compared with native ht control ( marked with * ) fig . 4alpha - smooth staining : group 1 ( top left ) showed a moderate number of myofibroblasts compared with groups 2 and 3 ( right and down ) . note an increased number of myofibroblast and vessels in groups 2 and 3 results for median myofibroblast density for all biopsy groups . myofibroblast density was significantly higher in group 2 ( 1324 months ) compared with native ht control ( marked with * ) alpha - smooth staining : group 1 ( top left ) showed a moderate number of myofibroblasts compared with groups 2 and 3 ( right and down ) . note an increased number of myofibroblast and vessels in groups 2 and 3 necrosis was absent in the biopsies . thereafter , collagen orientation became more regular , adapting to , but not fully restoring , the appearance of the intact acl . ensuing cell morphology changed to spindle shaped in group 2 and predominantly narrow long cells over 24 months ( fig . 5he staining : hamstring tendon ( top left ) with regular collagen orientation , moderate cellular density and little fibroblast activity . group 1 ( top right ) : irregular collagen alignment , increased cellular density , ovoid cell morphology . group 2 . further increased cellular and vessel density with an irregular collagen orientation ( below left ) . group 3 ( below right ) : improved regular collagen arrangement he staining : hamstring tendon ( top left ) with regular collagen orientation , moderate cellular density and little fibroblast activity . group 1 ( top right ) : irregular collagen alignment , increased cellular density , ovoid cell morphology . group 2 . further increased cellular and vessel density with an irregular collagen orientation ( below left ) . the most important finding of the present study was that human hamstring autografts showed typical stages of graft remodelling , which was not complete up to 2 years after acl reconstruction . animal studies have analysed tendon remodelling in bone tunnels in relation to the type of fixation after acl reconstruction [ 7 , 16 , 24 , 29 , 30 ] . weiler et al . found that biodegradable interference fit fixation of a soft tissue graft may alter the mechanical properties in the early remodelling stage because of tissue compromise at the screw insertion site . singhatat et al . examined early strength and stiffness of soft tissue fixation comparing biodegradable interference screw versus washerloc ( arthrotec , warsaw , in , usa ) fixation at 4 weeks . the strength and stiffness of the complex deteriorated after 4 weeks of implantation with the interference screw but was either maintained or improved with the use of the washerloc device . they postulated that aggressive rehabilitation after acl reconstruction should only be allowed after acl reconstruction with a fixation device that maintains strength and stiffness in the first few weeks of implantation . in the present study , other animal studies have analysed the intra - articular remodelling process of different graft types after acl replacement . return of overall graft integrity and histological appearance was completed 612 months after reconstruction without full restoration of the mechanical strength of the intact acl [ 4 , 8 , 25 , 26 , 31 ] . grafts approached only 5060% of the intact acl failure strength [ 1 , 13 , 20 , 31 ] . human biopsy studies have been performed with various techniques to analyse the remodelling of autografts after acl reconstruction [ 6 , 11 , 18 , 19 , 35 ] . performed a biochemical study to analyse the remodelling of 30 patellar tendon and 20 hamstring autografts after acl reconstruction . native acl , patellar tendon , gracilis and semitendinosus biopsies served as controls ( fresh cadavers ) . they concluded that , based on amount of collagen crosslinks and their architecture , remodelling occurs within one year after acl reconstruction . they hypothesized that a low - aggressive rehabilitation programme might seem more desirable , although this was not specifically examined in their study . their method of tissue biopsy was comparable with the technique of the present study . both studies examined mid - substance biopsies for the analysis of remodelling of hamstring autografts in clinically successful acl reconstructions . in contrast to the study by marumo et al . the present study used a standardized surgical technique with autograft hamstring tendons and a standardized accelerated rehabilitation protocol . analysis of cellular density , vascular density and myofibroblast density indicated a prolonged remodelling process compared with their study . zaffagnini et al . took 10 mid - substance biopsies of patellar tendon autografts from 6 months to 10 years after acl reconstruction . they performed a qualitative and quantitative histological evaluation of collagen fibrils by means of transmission electron microscopy . the patellar tendon autograft underwent a transformation period up to 2 years without reaching the mean diameter and bimodality of a native acl . in the present study , hamstring tendon autograft biopsies were examined by immunohistochemical evaluation of cellular density , vascular density and myofibroblast density . despite the differences in methodology , we agree with their findings that human remodelling was prolonged compared with animal studies . they found four stages of ligamentization , but duration and evolution of ligamentization was different from animal studies . in the first phase ( 12 months ) the excessive graft necrosis observed in animal studies could not be confirmed in humans , where necrosis never involved more than 30% of the graft s biopsies . during the second phase ( 210 months ) , increase in metabolic active fibroblasts , vessels and irregular collagen fibres occurred . from 1 to 3 years , fibroblast and vessel density decreased . grafts showed histology similar to a ligament without reaching normal ligament cell or vessel density levels . at 3 years this might explain the absence of necrosis since it would be expected in an earlier stage of remodelling . analysed 48 biopsies from human patella and hamstring tendons , 3120 months after acl replacement . they concluded that remodelling was completed 12 months after acl reconstruction because no significant differences for vascularity and fibre pattern were evident . they postulated that their results may support early postoperative return to full activity and an accelerated rehabilitation programme . the present study found an increasing cell and vessel density up to 24 months . especially the strong increase in myofibroblast density , from 13 up to 24 months , indicated an active remodelling process from 1 to 2 years . furthermore , vessel density increased over 24 months , whereas cell and myofibroblast density decreased but stayed higher compared with native controls . recent work by snchez et al . focused on ligamentization of hamstring autografts treated with platelet - rich plasma preparation rich in growth factors ( prgf ) after acl reconstruction . the prgf - treated grafts showed more remodelling compared with untreated grafts . at 1418 months , the uniform linear collagen orientation and the spindle - shaped morphology of the native ht differed from the remodelled collagen and the ovoid cells present after ligamentization . specific information on the duration of the ligamentization process was not described . in the present study , in contrast to the aforementioned human biopsy studies , special immunohistochemical staining techniques were performed to detect vessel and myofibroblast density . unterhauser et al . showed that the factor - viii staining can improve vessel detection possibility . the increasing myofibroblast density between 13 and 24 months in the present study indicated that the remodelling process in humans was prolonged compared with the results obtained in several animal studies . in the light of the current insights into acl surgery , the trans - tibial technique in the present study was a non - anatomic acl reconstruction . as a result , knee joint motion will not provide the same mechanical stimulus to the healing acl graft as to the intact acl [ 3 , 10 , 28 , 3234 ] . scheffler et al . presented the importance of restoring knee joint mechanics for graft remodelling after acl reconstruction in order to replicate the loading conditions of the intact acl . it was not possible to study acl remodelling by subsequent hamstring autograft biopsies in the same individuals . therefore , it was not possible to verify whether changes occurring in grafts from one time point to another may partially be attributed to amongst - subject variation . the present study was limited to small biopsies of the periphery of the acl graft . finally , the crimp pattern could not be assessed due to the small biopsies compared with animal study models . knowledge about the duration of remodelling process of acl grafts may influence and improve outcome as well as rehabilitation protocols [ 11 , 21 ] . this is the first study to document remodelling of human hamstring autografts in relation to a standardized surgical technique and standardized accelerated rehabilitation . the clinical relevance of the study is that , based on the prolonged remodelling of human hamstring grafts , it may be questionable whether an accelerated rehabilitation protocol after non - anatomic acl reconstruction is to be recommended . future research should focus on the remodelling process of hamstring grafts in an anatomic acl reconstruction . human hamstring grafts showed typical stages of graft remodelling , which was not complete up to 2 years after acl reconstruction . the remodelling process in humans was prolonged compared with the results obtained in several animal studies .

purposehistological analysis of the remodelling process of human hamstring tendon ( ht ) grafts after standardized anterior cruciate ligament reconstruction ( aclr ) with an accelerated rehabilitation protocol.methodssixty-seven patients underwent retrieval of mid - substance biopsies after clinically successful hamstring autograft aclr . samples were allocated to one of three groups depending on the time point of retrieval : group 1 ( 612 months ; n = 15 ) , group 2 ( 1324 months ; n = 16 ) and group 3 ( > 24 months ; n = 11).biopsies from native ht ( n = 17 ) and acl ( n = 8) served as controls . cellular density , vascular density and myofibroblast density and collagen fibril alignment were analysed by haematoxylin eosin , masson - goldner - trichrom and immunohistochemical staining protocols.resultscompared with native ht ( 330.4/mm ) , total cell number was increased in groups 1 - 3 ( group 1 = 482.0/mm ( p = 0.036 ) ; group 2 = 850.9/mm ( p = 0.005 ) ; and group 3 = 595.6/mm ( p = 0.043 ) . there were no significant differences between the groups for vessel density . myofibroblast density was higher in group 2 ( 199.6/mm ) compared with native ht ( 1.9/mm , p = 0.014 ) . collagen orientation was irregular up to 12 months . thereafter , collagen orientation became more regular , adapting to , but not fully restoring , the appearance of the intact acl . for the first 12 months , cells were predominantly ovoid . ensuing cell morphology changed to spindle shaped in group 2 and predominantly narrow long cells over 24 months.conclusionhuman hamstring grafts showed typical stages of graft remodelling , which was not complete up to 2 years after aclr . the remodelling process in humans was prolonged compared with the results obtained in several animal studies . level of evidencecase control study , level iii .

Level of evidence Introduction Materials and methods Histological examination Statistical analysis Results Discussion Conclusion

PMC3143319

the most common human diseases have a complex aetiology involving both genetic and environmental factors . genome - wide association studies ( gwas ) applying a selected set of single nucleotide polymorphism ( snp ) covering the entire genome have recently become popular and quite successful methods in search for genetic determinants of common diseases . however , the genetic variants discovered so far account for only a small portion of common disease heritability , indicating that there still are many genes or loci with small effects to be discovered . the possibility exists that a gene s effect is more easily detected within a framework that accommodates gene - environment and gene gene interactions . for example , a gene s overall effect might be too small to detect with any reasonable sample size , or a genetic effect is entirely dependent on an environmental exposure and may show a substantial effect when a specific environmental factor or another genetic variant is present . results from many association studies have not been replicated , possibly due in part to the omission of incorporating interactions among disease associated loci in the analysis where allele frequencies for interacting genes or environmental exposures may have differed in the investigated populations . in fact , gene - environment interactions and gene gene interactions are increasingly recognized phenomena in the field of human genetics [ 14 ] . this pinpoints the needs for the development of a computation tool for detecting gene - environment and gene gene interactions in a genome - wide fashion , i.e. a tool that allows the analysis of interaction between a specific environmental or genetic factor and a large number of snps . in statistics , the term interaction is used to refer to departure from the underlying form of a statistical model . because there are various statistical models , interaction does not have a consistent meaning . interaction is often assessed by entering a product term into the linear or multiplicative regression model . for example , in a logistic regression model a product term is estimated as departure from multiplicativity of odds ratios . interaction in epidemiology commonly refers to the situation where the joint effect of two risk factors on disease risk differs from the sum of their independent effects . this concept of interaction has a consistent meaning and is particularly applicable in the context of the sufficient cause model as described by rothman [ 5 , 6 ] . a sufficient cause is defined as minimal sets of actions , events , or states of nature that together initiated a process that inevitably resulted in the outcome . for a particular outcome there it can be shown that in the absence of interaction in this sense , disease rates are additive [ 5 , 7 , 8 ] . it can also be shown that when the joint effect of two contributory causes of a disease exceeds the sum of their independent effects ( on the additative scale ) then sufficient cause interaction is present ( but not vice versa ) . rothman has argued that interaction as departure from additivity better reflects biologic interaction [ 5 , 7 ] . he presents three measures of biologic interaction : reri , the relative excess risk due to interaction ; ap , the attributable proportion due to interaction ; and s , the synergy index [ 57 , 912 ] . these measures are defined as follows:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{reri } } = { \text{rr}}_{11 } -{\text{rr}}_{10 } -{\text{rr}}_{01 } + 1 , $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{ap } } = { \text{reri}}/{\text{rr}}_{11 } , $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{s } } = \left [ { { \text{rr}}_{11 } -1 } \right]/\left [ { \left ( { { \text{rr}}_{10 } -1 } \right ) + \left ( { { \text{rr}}_{01 } -1 } \right ) } \right ] $ $ \end{document}where rr11 is the relative risk in the exposure category when both risk factors are present , rr10 is the relative risk for the first risk factor in the absence of the second and rr01 is the relative risk for the second risk factor in the absence of the first . those who are unexposed to both the first and the second risk factor are used for reference category , i.e. , rr00 = 1 . if there is no interaction , reri and ap equals 0 and s equals 1 . for the calculation of confidence interval there are a few options [ 13 , 14 ] . in the current version of geira program , we use hosmer and lemeshow s delta method , which is a straight forward taylor expansion of the variances and covariances based on wald - type statistics using approximate variance estimators . the calculation will be faster in terms of computation time compared with the re - sampling based bootstrap method in the context of genome - wide scale . the limited number of snps that come out from the screening can then easily be re - calculated with confidence limits based on e.g. assmann s method . in the literature on gene - environment and gene gene interactions , there is no definite agreement on which computational methods for calculations of interactions are most appropriate in which contexts [ 1517 ] . irrespective of what choice of method that is preferred , there is a definite need for better computational methods for quantification of interactions . the primary goal of present article is to provide tools for large scale interaction calculation for several of the most utilized methods for detection of interactions . we have chosen to use the term additive interaction referring to rothman s biologic interaction and multiplicative interaction referring to the product term in a multiplicative model . in the program , multiplicative interaction is estimated by inclusion of an interaction term in a logistic regression model while the estimation of additive interaction is based on the three measures , i.e. , reri , ap and s as described above . the program analyzes genome - wide snp data or any list of bi - allelic genetic markers or dichotomous environmental factors in a case control study setting using toward the single risk factor of choice ( major risk factor ) . it includes simple data processing steps , which makes it easy and user - friendly and enables users to modify and extend the program . after user defined single factor of choice and other variables are determined , the program will run automatically . all relevant measures for both additive interaction and multiplicative interaction will be included in the output in format of a table . users can easily generate a ranked top list of snps based on certain user defined criteria . 1a pictorial representation of the geira algorithm a pictorial representation of the geira algorithm step 1 ( data importing ) here we assume that some other software package has previously been used to quality control genome - wide genotype data , including dataset filtering on the basis of snp genotype call rate , minor allele frequency , hardy weinberg equilibrium , individual missing genotype , outliers , etc . the program will read in transposed plink format data files , i.e. , tped ( containing snp and genotype information where one row is a snp ) and tfam ( containing individual and family information where one row is an individual ) ( see plink documentation for details . http://pngu.mgh.harvard.edu/~purcell/plink/pdf.shtml ) . in addition to the tped and tfam files , a covariate file containing covariate information ( including an environmental variable or a major genetic variable of choice ) is needed . step 2 ( risk allele assigning ) a minor allele is determined using all subjects ( both cases and controls ) . a risk allele is determined by comparing the minor allele frequency ( maf ) in cases and controls . if the maf in cases is greater than or equal to that in controls , the minor allele is assigned to the risk allele . if the maf in cases is less than that in controls , the major allele is assigned to the risk allele ( fig . 2 and user s manual for details).fig . example 1 illustrates a situation where a major allele is assigned to a risk allele . example 2 illustrates a situation where a minor allele is assigned to a risk allele . c_gen1_f genotype count of wide type homozygote ( homozygote 1 ) in controls , c_het_f genotype count of heterozygote in controls , c_gen2_f genotype count of homozygote 2 in controls , d_gen1_f genotype count of wide type homozygote ( homozygote 1 ) in cases , c_het_f genotype count of heterozygote in cases , c_gen2_f genotype count of homozygote 2 in cases . c_p : control minor allele frequency ; d_p : case minor allele frequency ; wid wide type , het heterozygote , hom homozygote , ma minor allele , mj major allele detailed algorithm for step 2 ( risk allele assignment ) . example 1 illustrates a situation where a major allele is assigned to a risk allele . example 2 illustrates a situation where a minor allele is assigned to a risk allele . c_gen1_f genotype count of wide type homozygote ( homozygote 1 ) in controls , c_het_f genotype count of heterozygote in controls , c_gen2_f genotype count of homozygote 2 in controls , d_gen1_f genotype count of wide type homozygote ( homozygote 1 ) in cases , c_het_f genotype count of heterozygote in cases , c_gen2_f genotype count of homozygote 2 in cases . c_p : control minor allele frequency ; d_p : case minor allele frequency ; wid wide type , het heterozygote , hom homozygote , ma minor allele , mj major allele step 3 ( data converting ) the raw genotype dataset will then be converted into a dataset based on a dominant , recessive or co - dominant model , according to the following coding : assuming c is the minor allele and also the risk alleledominant model coding : a_a 0 , a_c 1 , c_c 1.recessive model coding : a_a 0 , a_c 0 , c_c 1co - dominant model coding : a_a 0 , a_c 1 , c_c 2 assuming c is the minor allele and also the risk allele dominant model coding : a_a 0 , a_c 1 , recessive model coding : a_a 0 , a_c 0 , c_c 1 co - dominant model coding : a_a 0 , a_c 1 , c_c 2 step 4 ( interaction calculation ) users can choose one of these models , i.e. , a dominant , recessive or a co - dominant model . calculate estimates for both additive and multiplicative interactions , incorporating all estimates into one output table . we therefore created a supervising module that will pass the correct parameters to each step in order . step 6 ( adjustment for multiple testing ) p - value adjustments using bonferroni , sidak ( a technique slightly less conservative than bonferroni ) and false discovery rate ( fdr ) are corrected for total tests performed and are calculated using simple functions of the raw p - values . the program analyzes genome - wide snp data or any list of bi - allelic genetic markers or dichotomous environmental factors in a case control study setting using toward the single risk factor of choice ( major risk factor ) . it includes simple data processing steps , which makes it easy and user - friendly and enables users to modify and extend the program . after user defined single factor of choice and other variables are determined , the program will run automatically . all relevant measures for both additive interaction and multiplicative interaction will be included in the output in format of a table . users can easily generate a ranked top list of snps based on certain user defined criteria . 1a pictorial representation of the geira algorithm a pictorial representation of the geira algorithm step 1 ( data importing ) here we assume that some other software package has previously been used to quality control genome - wide genotype data , including dataset filtering on the basis of snp genotype call rate , minor allele frequency , hardy weinberg equilibrium , individual missing genotype , outliers , etc . the program will read in transposed plink format data files , i.e. , tped ( containing snp and genotype information where one row is a snp ) and tfam ( containing individual and family information where one row is an individual ) ( see plink documentation for details . http://pngu.mgh.harvard.edu/~purcell/plink/pdf.shtml ) . in addition to the tped and tfam files , a covariate file containing covariate information ( including an environmental variable or a major genetic variable of choice ) is needed . step 2 ( risk allele assigning ) a minor allele is determined using all subjects ( both cases and controls ) . a risk allele is determined by comparing the minor allele frequency ( maf ) in cases and controls . if the maf in cases is greater than or equal to that in controls , the minor allele is assigned to the risk allele . if the maf in cases is less than that in controls , the major allele is assigned to the risk allele ( fig . 2 and user s manual for details).fig . example 1 illustrates a situation where a major allele is assigned to a risk allele . example 2 illustrates a situation where a minor allele is assigned to a risk allele . c_gen1_f genotype count of wide type homozygote ( homozygote 1 ) in controls , c_het_f genotype count of heterozygote in controls , c_gen2_f genotype count of homozygote 2 in controls , d_gen1_f genotype count of wide type homozygote ( homozygote 1 ) in cases , c_het_f genotype count of heterozygote in cases , c_gen2_f genotype count of homozygote 2 in cases . c_p : control minor allele frequency ; d_p : case minor allele frequency ; wid wide type , het heterozygote , hom homozygote , ma minor allele , mj major allele detailed algorithm for step 2 ( risk allele assignment ) . example 1 illustrates a situation where a major allele is assigned to a risk allele . example 2 illustrates a situation where a minor allele is assigned to a risk allele . c_gen1_f genotype count of wide type homozygote ( homozygote 1 ) in controls , c_het_f genotype count of heterozygote in controls , c_gen2_f genotype count of homozygote 2 in controls , d_gen1_f genotype count of wide type homozygote ( homozygote 1 ) in cases , c_het_f genotype count of heterozygote in cases , c_gen2_f genotype count of homozygote 2 in cases . c_p : control minor allele frequency ; d_p : case minor allele frequency ; wid wide type , het heterozygote , hom homozygote , ma minor allele , mj major allele step 3 ( data converting ) the raw genotype dataset will then be converted into a dataset based on a dominant , recessive or co - dominant model , according to the following coding : assuming c is the minor allele and also the risk alleledominant model coding : a_a 0 , a_c 1 , c_c 1.recessive model coding : a_a 0 , a_c 0 , c_c 1co - dominant model coding : a_a 0 , a_c 1 , c_c 2 assuming c is the minor allele and also the risk allele dominant model coding : a_a 0 , a_c 1 , recessive model coding : a_a 0 , a_c 0 , c_c 1 co - dominant model coding : a_a 0 , a_c 1 , c_c 2 step 4 ( interaction calculation ) users can choose one of these models , i.e. , a dominant , recessive or a co - dominant model . calculate estimates for both additive and multiplicative interactions , incorporating all estimates into one output table . we therefore created a supervising module that will pass the correct parameters to each step in order . step 6 ( adjustment for multiple testing ) p - value adjustments using bonferroni , sidak ( a technique slightly less conservative than bonferroni ) and false discovery rate ( fdr ) are corrected for total tests performed and are calculated using simple functions of the raw p - values . the program analyzes genome - wide snp data or any list of bi - allelic genetic markers or dichotomous environmental factors in a case control study setting using toward the single risk factor of choice ( major risk factor ) . it includes simple data processing steps , which makes it easy and user - friendly and enables users to modify and extend the program . after user defined single factor of choice and other variables are determined , the program will run automatically . all relevant measures for both additive interaction and multiplicative interaction will be included in the output in format of a table . users can easily generate a ranked top list of snps based on certain user defined criteria . 1a pictorial representation of the geira algorithm a pictorial representation of the geira algorithm step 1 ( data importing ) here we assume that some other software package has previously been used to quality control genome - wide genotype data , including dataset filtering on the basis of snp genotype call rate , minor allele frequency , hardy weinberg equilibrium , individual missing genotype , outliers , etc . the program will read in transposed plink format data files , i.e. , tped ( containing snp and genotype information where one row is a snp ) and tfam ( containing individual and family information where one row is an individual ) ( see plink documentation for details . http://pngu.mgh.harvard.edu/~purcell/plink/pdf.shtml ) . in addition to the tped and tfam files , a covariate file containing covariate information ( including an environmental variable or a major genetic variable of choice ) is needed . step 2 ( risk allele assigning ) a minor allele is determined using all subjects ( both cases and controls ) . a risk allele is determined by comparing the minor allele frequency ( maf ) in cases and controls . if the maf in cases is greater than or equal to that in controls , the minor allele is assigned to the risk allele . if the maf in cases is less than that in controls , the major allele is assigned to the risk allele ( fig . 2 and user s manual for details).fig . example 1 illustrates a situation where a major allele is assigned to a risk allele . example 2 illustrates a situation where a minor allele is assigned to a risk allele . c_gen1_f genotype count of wide type homozygote ( homozygote 1 ) in controls , c_het_f genotype count of heterozygote in controls , c_gen2_f genotype count of homozygote 2 in controls , d_gen1_f genotype count of wide type homozygote ( homozygote 1 ) in cases , c_het_f genotype count of heterozygote in cases , c_gen2_f genotype count of homozygote 2 in cases . c_p : control minor allele frequency ; d_p : case minor allele frequency ; wid wide type , het heterozygote , hom homozygote , ma minor allele , mj major allele detailed algorithm for step 2 ( risk allele assignment ) . example 1 illustrates a situation where a major allele is assigned to a risk allele . example 2 illustrates a situation where a minor allele is assigned to a risk allele . c_gen1_f genotype count of wide type homozygote ( homozygote 1 ) in controls , c_het_f genotype count of heterozygote in controls , c_gen2_f genotype count of homozygote 2 in controls , d_gen1_f genotype count of wide type homozygote ( homozygote 1 ) in cases , c_het_f genotype count of heterozygote in cases , c_gen2_f genotype count of homozygote 2 in cases . c_p : control minor allele frequency ; d_p : case minor allele frequency ; wid wide type , het heterozygote , hom homozygote , ma minor allele , mj major allele step 3 ( data converting ) the raw genotype dataset will then be converted into a dataset based on a dominant , recessive or co - dominant model , according to the following coding : assuming c is the minor allele and also the risk alleledominant model coding : a_a 0 , a_c 1 , c_c 1.recessive model coding : a_a 0 , a_c 0 , c_c 1co - dominant model coding : a_a 0 , a_c 1 , c_c 2 assuming c is the minor allele and also the risk allele dominant model coding : a_a 0 , a_c 1 , recessive model coding : a_a 0 , a_c 0 , c_c 1 co - dominant model coding : a_a 0 , a_c 1 , c_c 2 step 4 ( interaction calculation ) users can choose one of these models , i.e. , a dominant , recessive or a co - dominant model . calculate estimates for both additive and multiplicative interactions , incorporating all estimates into one output table . we therefore created a supervising module that will pass the correct parameters to each step in order . step 6 ( adjustment for multiple testing ) p - value adjustments using bonferroni , sidak ( a technique slightly less conservative than bonferroni ) and false discovery rate ( fdr ) are corrected for total tests performed and are calculated using simple functions of the raw p - values . rheumatoid arthritis ( ra ) is a complex autoimmune disorder with both genetic and environmental influences on the disease pathogenesis . family aggregation and twin studies have estimated a genetic component of approximately 50% [ 19 , 20 ] . we applied geira to the swedish epidemiologic investigation of rheumatoid arthritis ( eira ) gwas data ( illumina 300 k ) . a total of 1,147 autoantibody to citrullinated protein antigens ( acpa)-positive rheumatoid arthritis cases and 1,079 age , gender and residence area matched controls were used for calculation . in order to demonstrate the use of the geira program , we chose to investigate interactions between a large number of genetic variants available in cases ( acpa - positive ra ) and controls as well as data on the environmental exposure smoking in the same cases and controls . we used already published gwas data on these cases and controls as our basic genetic information [ 22 , 24 ] . we conducted an extra quality control procedure in plink , thereafter 301,238 autosomal snps remained for analysis and were used as an input in the geira program . the environmental factor , smoking status , was coded as 1 for ever smoker and 0 for never smoker . from the analysis , a total of 15 snps were found to be statistically significantly interacting with smoking on the additive scale . of these 15 snps , two are from hla region ( hla - dqa2 and hla - dra ) . the genome - wide significance defined as a p for ap of 1.66 10 this application took about 7 h to complete on a desktop with two intel xeon processors running at 3.2 ghz and a ram of 32 g . the further results of these experiments together with biological interpretation of the findings are submitted separately for publication ( submitted ) . the geira program aims at detecting gene - environment as well as gene gene interactions , and in this first report , we describe both the generic algorithms , and their application in a specific case of gene - environment interaction . the main effect of each snp can be estimated using other whole - genome association toolsets , such as plink . the results from the geira program can be considered as a first screening step for selecting potential candidate gene - environment ( or gene snps on the top of the list can be furthered examined by follow - up studies , such as replication in another independent population , dense mapping , and so on . adjustment for confounding factors can be easily done by assigning the macro variables , covar_cat or covar_cont , to the confounding variables ( see user s manual for details ) . for adjustment of multiple testing , an additional step ( step 6 ) for correction for multiple testing was included in the program . notwithstanding the fact that the step 2 is developed to ensure that the odds ratio ( or ) for the risk allele is greater than 1 , the or associated with the genetic factor might still be less than 1 among subject unexposed to the environmental factor . in this case ap is an estimate of the proportion of disease , which is due to interaction among persons with both exposures . if rr11 < [ ( rr10 + rr01)1 ] , ap will be negative . in this case the joint effect from the simultaneous presence of the two factors is less than what is expected by summing their independent effects . we advise users to read the user s manual of the program for more details . displays in the output window are suppressed and contents in the log window are saved in an external directory so that users can trace the program running process if there are some unexpected results in the outputs . to analyze only certain chromosomes , users only need assign a chromosome number of interest to the macro variable chr and run the program . it takes only 735 min , depending on which chromosome selected , to complete the analyses for a single chromosome . this program can be easily modified to estimate genome - wide gene gene interactions . in this case , the only parameter needed to be changed is the environmental variable ( envir ) . the environmental variable should be replaced by a dichotomous genetic variable ( snp , cnv , haplotype etc . ) coded as 0 ( unexposed ) and 1 ( exposed ) with the remaining parameters unchanged . when applying to the very large gwas data , parallel computing on a multiple cpus / clusters server , we will extend the interaction estimation to one continuous and one dichotomous variable , and further to two continuous variables . the algorithm and the program geira presented in this article offers a powerful , user - friendly tool for performing interaction analyses with whole - genome data .

the geira ( gene - environment and gene gene interaction research application ) algorithm and subsequent program is dedicated to genome - wide gene - environment and gene gene interaction analysis . it implements concepts of both additive and multiplicative interaction as well as calculations based on dominant , recessive and co - dominant genetic models , respectively . estimates of interactions are incorporated in a single table to make the output easily read . the algorithm is coded in both sas and r. geira is freely available to non - commercial users at http://www.epinet.se . additional information , including user s manual and example datasets is available online at http://www.epinet.se .

Introduction Methods SAS version 9.2 for windows and R version 2.6.2 were used to develop the GEIRA program. Features Application to real genome-wide data Discussion

PMC4075871

although patients with interstitial pulmonary fibrosis or pulmonary arterial hypertension can have normal spo2 at rest , some will show oxygen desaturation after submaximal exercise . ( , ) end - exercise pao2 decreases after maximal exercise , and submaximal steady - state exercise was found to be an important measure of disease severity in interstitial pulmonary fibrosis . . demonstrated that patients with usual interstitial pneumonia who developed oxygen desaturation during and after a six - minute walk test ( 6mwt)a 4% decrease in oxygen saturation from baseline ( sat 4%)were more than four times more likely to die during follow - up . ( ) the abovementioned findings led us to hypothesize that a decrease in oxygen saturation during self - paced walking ( a submaximal exercise test ) is a meaningful measure of disease status in patients with scleroderma . our results showed that , in the multiple logistic regression analysis , the variable sat 4% was significantly associated with age , dyspnea , and two other variables related to pulmonary involvement , i.e. , fvc < 80% of the predicted value ( as assessed by spirometry ) and positivity for the scl-70 antibody , which is a marker of pulmonary disease in scleroderma . however , the statistical model applied to the data did not indicate which of the dependent variables analyzed ( sat 4% or the distance walked ) was better at predicting pulmonary disease . nevertheless , sat 4% appeared to be able to provide more information on this issue than did the distance walked . ( ) another study conducted by our research group and involving patients with systemic lupus erythematosus showed that those with post-6mwt sat 4% ( as assessed by pulse oximetry ) showed a significant reduction in the six - minute walk distance ( 6mwd ) , which was 443 m in the group of patients who developed oxygen desaturation and 497 m in that of those who did not ( p = 0.0291 ) . however , both 6mwds were well above the lower limit of the normal range . in addition , when compared with the patients who did not develop oxygen desaturation , those who did had a higher post-6mwt hr ( p = 0.0170 ) , a lower mep ( p = 0.0282 ) , a lower mip ( p = 0.0504 ) , and a restrictive pattern of lung disease ( as determined by spirometry ) . these findings suggest that oxygen desaturation is more sensitive in detecting the presence of respiratory abnormalities in patients with systemic lupus erythematosus than is the 6mwd ( unpublished data ) . therefore , oxygen desaturation during submaximal exercise seems to be a more sensitive endpoint to detect subtle respiratory abnormalities in pulmonary diseases and in systemic diseases that affect respiration . maximal exercise tests , such as cardiopulmonary exercise tests , can provide important information on the integration between the cardiovascular and respiratory systems during exercise . although cardiopulmonary exercise tests have been extensively used in order to investigate various lung diseases , they are complex tests that require expensive equipment . the incremental shuttle walk test ( iswt ) is a maximal exercise test that provides data that correlate well with measurements made during incremental cardiopulmonary exercise testing , being simpler and less expensive than the latter . the fact that even patients with few symptoms and mild pulmonary involvement can present with a decrease in oxygen saturation in a submaximal test led to us inquire how oxygen saturation would behave during a maximal walk test , such as the iswt . could it improve the sensitivity of the 6mwt in detecting respiratory impairment ? with the objective of exploring this hypothesis , we decided to perform the iswt in healthy individuals in order to determine the probability of oxygen desaturation at the end of the test . this was a cross - sectional study involving healthy subjects who attended a private fitness center in the city of campinas , brazil . all of the enrolled subjects attended the fitness center in order to keep physically active and in good health , and none were professional athletes . they were invited to participate in the study and agreed to perform the tests included in the protocol . the study was approved by the research ethics committee of the universidade estadual de campinas ( unicamp , state university at campinas ) hospital de clnicas , located in the city of campinas , brazil , and all participants gave written informed consent . subjects were considered ineligible for the iswt if they had a history of lung or heart disease or any other health condition that might preclude the performance of the iswt . a standard questionnaire was applied to all participants to ensure that they did not meet any of the exclusion criteria . a digital peak flow meter ( koko peak pro 6 ; ferraris cardiorespiratory pds healthcare products inc . , louisville , co , usa ) was used in order to determine pre - iswt fev1 and fev6 . the fev1/fev6 ratio was calculated by the device and was displayed on the screen . each subject performed at least three forced expiratory maneuvers after maximal inspiratory maneuvers , the best maneuver being automatically chosen by the peak flow meter . pre - iswt hr and oxygen saturation were determined with a pulse oximeter equipped with a finger probe ( nonin medical , inc . , fingernail polish , if worn by the subject , was removed before testing . in order to guarantee an accurate assessment of oxygen saturation , the principal investigator checked that the pulse oximeter showed an acceptable pulse signal and that the oximeter light was green and pulsing in synchrony with the hr before the beginning of the tests . the protocol used for the iswt was the 12-level version proposed by singh et al . it was delimited by two cones inset 0.5 m from either end to avoid the need for abrupt changes in direction . the speed at which subjects walked was dictated by an audio signal played on a portable microcomputer . each participant received a standardized explanation ( " you should walk at a steady pace , aiming to turn around whenever you hear the signal ; you should continue to walk until you feel that you are unable to maintain the required speed " ) . at the first level of the test , the calculated walking speed was 0.5 m / s and the number of expected shuttles was 3 ; at the twelfth level , walking speed was 2.37 m / s and 14 shuttles were expected . all subjects were carefully observed during the test so that they would not exceed their exercise limit . for the purpose of data analysis , oxygen desaturation was defined as a 4% decrease in oxygen saturation ( spo2 ) from baseline ( sat = oxygen saturation at rest oxygen saturation immediately after the iswt ) . this 4% decrease in oxygen saturation was validated in studies of exercise - induced hypoxemia during maximal exercise tests in athletes . ( ) maximum incremental shuttle walk distance ( iswd ) was defined as the maximum distance that the subjects were able to walk during the iswt . a 4% decrease in oxygen saturation the anderson - darling test was applied to the measured variables and the demographic characteristics of the two groups to determine their distribution . categorical data were compared by the chi - square test or fisher 's exact test . the statistical analysis was performed with the sas software , version 8 ( sas institute , inc . , eighty - three individuals who attended a fitness center in the city of campinas , brazil , were invited and agreed to participate in the study . the mean age was 35.05 12.53 years , the median being 32 years . the mean resting hr was 75.12 12.48 bpm , the median being 73 bpm . the mean pre - iswt spo2 was 97.96 1.02% , the median being 98% . the mean fev1 was 3.75 0.81 l , the median being 3.65 l. the mean fev6 was 4.45 0.87 l , the median being 4.38 l. the mean fev1/fev6 ratio was 0.83 0.08 , the median being 0.82 . in all enrolled subjects , measured fev6 was above the lower limit of the predicted fvc ( as determined by the equations devised for the brazilian population ) , and all subjects had a fev1/fev6 ratio 0.8 . these findings allow the assumption that none of the participants had restrictive or obstructive lung disease ( table 1 ) . table 1 comparison between the two groups ( with and without oxygen desaturation).a the mean iswd was 958.30 146.32 m , the median being 1,020 m. the mean post - iswt hr was 162.41 18.24 bpm , the median being 166 bpm . the mean post - iswt spo2 was 96.27 2.21% . in 11 subjects , post - iswt oxygen saturation values were higher than pre - iswt oxygen saturation values . in 17 subjects , oxygen saturation had decreased by 4% by the end of the test . in 2 subjects , table 2 proportions of individuals who reached more than 85% or 85% or less of the predicted maximal hr in the two groups.a the study population was divided into two groups on the basis of the presence of a post - iswt oxygen desaturation 4% . no differences were found between the two groups regarding age , gender , fev1 , fev6 , fev1/fev6 , initial oxygen saturation ( pre - iswt spo2 ) , iswd , pre - iswt hr , post - iswt hr , or percentage of maximal hr ( table 1 ) . the bmi was significantly higher in those who developed oxygen desaturation ( p = 0.01 ) , and post - iswt spo2 was significantly different between the two groups ( p < 0.0001 ) . in 66 subjects , post - iswt oxygen saturation values were quite similar to pre - iswt oxygen saturation values , a finding that was expected because of the intensity of the exercise performed . in 11 subjects , post - iswt spo2 values were higher than pre - iswt spo2 values . this finding is not unusual , given that physical activity improves ventilation and alveolar recruitment . however , 17 ( 20.7% ) of the 83 individuals in the study sample showed a significant drop in oxygen saturation during the iswt ( sat 4% ) . this was an unexpected finding , and there is little information in the literature regarding what happens with oxygen saturation after the iswt in healthy subjects . exercise - induced hypoxemia in athletes is arbitrarily defined as a decrease in pao2 of approximately 7.5 mmhg , ( ) an sao2 below 95% , or both ; extreme cases will show an sao2 of less than 88% . ( ) oxygen uptake increases during exercise in order to meet the needs imposed by an increased metabolic rate and correlate with work intensity until all subjects achieve maximal oxygen uptake . ( ) each step of oxygen transport from ambient air to the cells can limit whole - body oxygen uptake , and circulation has been considered the most important factor limiting maximal oxygen uptake during large muscle mass exercise . ( ) in such subjects , cardiac output can exceed 30 l / min ; under such circumstances , the ability to renew alveolar air and maintain high oxygen partial pressures , the diffusion resistance to oxygen at the alveolar - capillary membrane , the reduction in red blood cell transit time in the pulmonary capillary , and the increased probability of ventilation / perfusion mismatch are critically important to oxygen uptake . ( ) the prevalence of exercise - induced hypoxemia seems to be as high as 50% . ( ) oxygen desaturation is also more pronounced during whole - body exercise , such as rowing or running , than during leg exercise , and leg exercise is more capable of inducing hypoxemia than is arm exercise . ( ) this suggests that the amount of muscle mass involved in the exercise influences the development of oxygen desaturation . in 1984 , dempsey et al . ( ) studied the incidence of exercise - induced arterial hypoxemia in 16 highly trained healthy runners who were capable of achieving and sustaining very high metabolic rates , including a maximal oxygen uptake of 72 2 ml / kg . arterial blood gases and acid - base status were determined at each load of a progressive short - term exercise test and repeatedly determined during constant - load treadmill running . three types of response were encountered and were quite reproducible within subjects : four runners maintained pao2 within 10 mmhg of resting values ; another four showed a decrease of 10 - 15 mmhg in pao2 ; and the remaining eight runners showed remarkably decreased pao2 . the decrease in pao2 ranged from 21 mmhg to 35 mmhg , pao2 having decreased to less than 75 mmhg in all cases and to less than 60 mmhg in two cases . during constant - load exercise , pao2 was often maintained during the initial 30 s , when hyperventilation was greatest ; subsequently , hypoxemia occurred , the severity of hypoxemia having either remained the same or worsened over the ensuing 3 - 4 min in most of the cases . the most severe hypoxemia during heavy exercise was associated with an ( estimated ) alveolar oxygen tension to a ( measured ) pao2 difference in excess of 40 mmhg . the analysis of all of the variables measured during the investigation led the authors to hypothesize that the observed hypoxemia was attributable to a diffusion limitation secondary to very short red cell transit times in at least a portion of the pulmonary circulation ; such short transit times can occur at high metabolic rates . tidal breathing during heavy exercise can frequently exceed the maximal flow - volume curve , and hyperventilation can be limited by the mechanical load on the chest wall secondary to increased pulmonary impedance , a situation that can also impair the renewal of alveolar air . although it has been described as an incremental field walking test that produces a symptom - limited maximal performance , the iswt can not be compared with the maximal exercise tests cited above . nevertheless , 17 healthy subjects showed oxygen desaturation after the iswt in the present study . the only significant difference between the individuals who developed oxygen desaturation and those who did not was that the bmi was higher in the former . this finding is consistent with the hypothesis that the lungs , during physical activities that cause the hr to get closest to the maximal hr expected for a given subject , are unable to arterialize the fast flowing blood , especially in those subjects whose needs are amplified because of a higher body mass . although durand et al . ( ) found no differences in height , weight , or lung volume between athletes who developed oxygen desaturation and those who did not , it can be argued that the proportions of those variables are more important than their absolute values . our findings and the data from the literature lead to a worrisome possibility : oxygen desaturation during aerobic activity is probably more common than previously thought and can pose a threat to high - performance athletes that has been systematically overlooked . although the occurrence of oxygen desaturation is acknowledged in studies that date back to the second half of the last century , none of those studies elaborated on the potential harmful effects of such periods of intermittent hypoxemia . we are unaware of any evaluation protocol for high - performance aerobic training that includes the determination of exercise - induced oxygen desaturation . given that sudden death is relatively common in athletes , screening for oxygen desaturation seems justified . sudden cardiac death ( scd ) is considered the leading cause of death in young athletes . the studies reporting the highest incidence estimated that up to 110 deaths occur each year in young athletes , which is equivalent to 1 death every 3 days in the united states . ( ) the available evidence points to a structural cardiac abnormality as the underlying cause of scd . hypertrophic cardiomyopathy and coronary artery anomalies account for approximately 25% and 14% , respectively , of all scds in the united states . ( ) arrhythmogenic right ventricular cardiomyopathy / dysplasia is a cardiac disease characterized by myocardial necrosis followed by fibrofatty replacement . these altered myocardial areas constitute the anatomical substrate for reentry circuits that propitiate the onset of ventricular arrhythmias . ( ) this last condition can be particularly significant in the context of the present study . oxygen desaturation during intense physical activity can cause repeated episodes of hypoxic pulmonary vascular constriction and pulmonary hypertension . the walls of the right ventricle can suffer during these episodes , to the point of myocardial necrosis , fibrofatty replacement being the expected consequence of this kind of stress . another common cause of scd is brugada syndrome , ( ) which is characterized by an st - segment elevation in the right precordial electrocardiogram leads followed by a negative t wave . the worldwide prevalence of brugada syndrome is estimated at 1 - 5 per 10,000 population , although it is higher in southeast asia . ( ) brugada syndrome is traditionally thought of as a primary electrical cardiac disease arising in myocardium that is otherwise structurally normal . however , magnetic resonance imaging , positron emission tomography , and pathological evaluation of biopsy specimens have identified structural abnormalities in many patients with a diagnosis of brugada syndrome , including fibrofatty replacement of the right ventricular free wall and fibrotic disruption of the right bundle branch . it is established in the literature that brugada syndrome is the result of an autosomal dominant mutation in the scn5a gene on chromosome 3 , resulting in a loss of function sodium channel abnormality . ( ) it has become increasingly clear that ion channel gene expression is highly dynamic and can respond to many environmental stimuli . therefore , a genetic predisposition to cardiac arrhythmia does not preclude the superimposition of hypoxemia causing the sudden deaths of young people or athletes . the data in the present study and the accumulated knowledge regarding oxygen desaturation during physical activity raise the hypothesis that hypoxemia during exercise can be dangerous and suggest that it is advisable to include a screening test for oxygen desaturation in the evaluation protocols for endurance athletes . in conclusion , because of the possibility of oxygen desaturation in healthy individuals undergoing the iswt , the use of the iswt to predict the presence of subtle respiratory abnormalities undetected by submaximal tests such as the 6mwt can be misleading . the finding that oxygen desaturation is common in healthy subjects undergoing the iswt adds to the knowledge that oxygen desaturation during intense physical activity is quite common and can have deleterious effects . determinar a probabilidade de dessaturao arterial em indivduos saudveis submetidos ao incremental shuttle walk test ( iswt ) . foram estudados 83 indivduos saudveis , dos quais 55 eram homens ( 1 deles fumante ) e 28 eram mulheres . foram determinados vef1 e vef6 antes da realizao do iswt , assim como fc e spo2 antes e depois do iswt . as mdias gerais foram as seguintes : idade , 35,05 12,53 anos ; ndice de massa corporal , 24,30 3,47 kg / m ; fc em repouso , 75,12 12,48 bpm ; spo2 em repouso , 97,96 1,02% ; vef1 , 3,75 0,81 l ; relao vef1/vef6 , 0,83 0,08 ( sem restrio ou obstruo ) ; distncia percorrida no iswt , 958,30 146,32 m ; fc ps - iswt , 162,41 18,24 bpm e spo2 ps - iswt , 96,27 2,21% em 11 indivduos , houve um aumento da spo2 aps o iswt , ao passo que em 17 houve uma queda de 4% . no houve diferena estatstica entre os grupos com e sem dessaturao aps o iswt no tocante s variveis idade , gnero , vef1 , vef6 , vef1/vef6 , spo2 basal , distncia percorrida no iswt , fc e porcentagem da fc mxima . nos indivduos que apresentaram dessaturao , o ndice de massa corporal foi maior ( p = 0,01 ) e a spo2 ps - iswt foi menor ( p = 0,0001 ) . o uso do iswt para prever a presena de problemas respiratrios sutis pode ser enganador . em indivduos saudveis , a dessaturao um evento comum aps o iswt , assim como o durante a atividade fsica intensa . embora pacientes com fibrose pulmonar intersticial ou hipertenso arterial pulmonar possam apresentar spo2 normal em repouso , alguns apresentam dessaturao de oxignio aps o exerccio submximo . ( , ) a pao2 ao final do exerccio diminui aps o exerccio mximo , e o exerccio de estado estacionrio submximo mostrou - se uma importante medida da gravidade da doena na fibrose pulmonar intersticial . demonstraram que pacientes com pneumonia intersticial usual que apresentaram dessaturao de oxignio durante e aps um teste de caminhada de seis minutos ( tc6 ) uma diminuio 4% da saturao de oxignio em relao saturao basal ( "sat 4% ) eram mais de quatro vezes mais propensos a morrer durante o seguimento . ( ) os achados mencionados acima nos levaram hiptese de que uma diminuio da saturao de oxignio durante uma caminhada autocadenciada ( um teste de esforo submximo ) seria uma medida significativa do estado da doena em pacientes com esclerodermia . nossos resultados mostraram que , na anlise de regresso logstica mltipla , a varivel "sat 4% associou - se significativamente a idade , dispneia e duas outras variveis relacionadas ao envolvimento pulmonar , ou seja , cvf < 80% do previsto ( avaliada por meio de espirometria ) e positividade para o anticorpo scl-70 , que um marcador de doena pulmonar na esclerodermia . no entanto , o modelo estatstico aplicado aos dados no indicou qual das variveis dependentes analisadas ( "sat 4% ou a distncia percorrida ) era melhor em prever doena pulmonar . no obstante , "sat 4% pareceu ser capaz de fornecer mais informaes sobre essa questo do que a distncia percorrida . ( ) outro estudo realizado por nosso grupo de pesquisa , com pacientes com lpus eritematoso sistmico , mostrou que aqueles com "sat 4% aps o tc6 ( avaliada por oximetria de pulso ) apresentaram reduo significativa da distncia percorrida no tc6 ( dtc6 ) , que foi 443 m nos pacientes que apresentaram dessaturao de oxignio e 497 m naqueles que no o fizeram ( p = 0,0291 ) . no entanto , ambas as dtc6 situaram - se bem acima do limite inferior da normalidade . alm disso , quando comparados aos pacientes que no apresentaram dessaturao de oxignio , aqueles que o fizeram apresentaram maior fc ps - tc6 ( p = 0,0170 ) , menor pemx ( p = 0,0282 ) , menor pimx ( p = 0,0504 ) e um padro de doena pulmonar restritiva ( determinado por meio de espirometria ) . esses resultados sugerem que a dessaturao de oxignio mais sensvel que a dtc6 para detectar a presena de alteraes respiratrias em pacientes com lpus eritematoso sistmico ( dados no publicados ) . portanto , a dessaturao de oxignio durante o exerccio submximo parece ser um desfecho mais sensvel para detectar alteraes respiratrias sutis em doenas pulmonares e em doenas sistmicas que afetam a respirao . testes de esforo mximo , como os testes de exerccio cardiopulmonar , podem fornecer informaes importantes sobre a integrao entre os sistemas cardiovascular e respiratrio durante o exerccio . embora os testes de exerccio cardiopulmonar tenham sido amplamente usados para investigar vrias doenas pulmonares , so testes complexos que exigem equipamentos caros . o incremental shuttle walk test ( iswt , teste de caminhada com carga progressiva ) um teste de esforo mximo que fornece dados que se correlacionam bem com as medies feitas durante testes de exerccio cardiopulmonar incremental e mais simples e menos caro do que estes . o fato de que mesmo pacientes com poucos sintomas e envolvimento pulmonar leve podem apresentar diminuio da saturao de oxignio em um teste submximo nos levou a perguntar como a saturao de oxignio se comportaria durante um teste mximo de caminhada , como o iswt . com o objetivo de explorar essa hiptese , decidimos realizar o iswt em indivduos saudveis , a fim de determinar a probabilidade de dessaturao de oxignio ao final do teste . trata - se de um estudo transversal com indivduos saudveis que frequentavam uma academia particular de ginstica na cidade de campinas ( sp ) . todos os participantes frequentavam a academia a fim de se manter fisicamente ativos e saudveis , e nenhum deles era atleta profissional . foram convidados a participar do estudo e concordaram em realizar os testes includos no protocolo . o estudo foi aprovado pelo comit de tica em pesquisa do hospital de clnicas da universidade estadual de campinas , em campinas ( sp ) , e todos os participantes assinaram um termo de consentimento livre e esclarecido . os indivduos eram considerados inaptos para o iswt se apresentassem histria de doena pulmonar ou cardaca ou qualquer outro problema de sade que pudesse impedi - los de realizar o iswt . um questionrio padronizado foi aplicado a todos os participantes para garantir que no preenchiam nenhum dos critrios de excluso . um medidor de pico de fluxo digital ( koko peak pro 6 ; ferraris cardiorespiratory pds healthcare products inc . , louisville , co , eua ) cada participante realizou pelo menos trs manobras expiratrias foradas aps manobras inspiratrias mximas , e a melhor manobra foi escolhida automaticamente pelo medidor de pico de fluxo . a fc e saturao de oxignio em repouso foram determinadas com um oxmetro de pulso equipado com um sensor de dedo ( nonin medical , inc . , plymouth , mn , eua ) . caso o participante a ser testado estivesse usando esmalte nas unhas , o esmalte era removido antes do teste . a fim de garantir a preciso da medida da saturao de oxignio , o investigador principal certificou - se de que o oxmetro de pulso mostrasse um sinal de pulso aceitvel e que a luz do oxmetro estivesse verde e pulsando em sincronia com a fc antes do incio dos testes . o protocolo usado para o iswt foi a verso de 12 nveis proposta por singh et al . ( ) para a avaliao da capacidade funcional em pacientes com dpoc . todos os participantes foram testados em condies padronizadas , pelo mesmo investigador . a pista de caminhada era plana e tinha 10 m de comprimento . foi delimitada por dois cones posicionados dentro da pista , 0,5 m antes de cada extremidade , para evitar a necessidade de mudanas bruscas de direo . a velocidade com que os participantes caminharam foi ditada por um sinal de udio reproduzido em um microcomputador porttil . cada participante recebeu uma explicao padronizada ( " voc deve caminhar em ritmo constante , com o objetivo de virar sempre que ouvir o sinal ; voc deve continuar a caminhar at que sinta que no mais capaz de manter a velocidade exigida " ) . no primeiro nvel do teste , a velocidade de caminhada calculada foi 0,5 m / s , e o nmero de shuttles esperados eram 3 ; no dcimo segundo nvel , a velocidade de caminhada foi 2,37 m / s e esperavam - se 14 shuttles . todos os indivduos foram cuidadosamente observados durante o teste para que no excedessem seu limite de exerccio . a saturao de oxignio tambm foi medida imediatamente aps o final do iswt . para a anlise dos dados , considerou - se dessaturao de oxignio a diminuio 4% da saturao de oxignio ( spo2 ) em relao ao valor basal ( "sat = saturao de oxignio em repouso saturao de oxignio imediatamente aps o iswt ) . essa diminuio de 4% na saturao de oxignio foi validada em estudos de hipoxemia induzida pelo exerccio durante testes de esforo mximo em atletas . ( ) considerou - se incremental shuttle walk distance ( iswd ) mxima a distncia mxima que os participantes foram capazes de percorre durante o iswt . o teste de anderson - darling foi aplicado s variveis medidas e s caractersticas demogrficas dos dois grupos para determinar sua distribuio . as variveis cuja distribuio foi normal foram analisadas com o teste t de student . as variveis cuja distribuio no foi os dados categricos foram comparados por meio do teste do qui - quadrado ou do teste exato de fisher . a anlise estatstica foi realizada com o programa sas , verso 8 ( sas institute , inc . , cary , nc , eua ) . as diferenas oitenta e trs indivduos que frequentavam uma academia de ginstica em campinas ( sp ) foram convidados a participar do estudo e aceitaram o convite . dos 83 participantes , 55 eram do sexo masculino e 28 eram do sexo feminino . a mdia de idade foi de 35,05 12,53 anos , e a mediana foi 32 anos . a mdia do ndice de massa corporal ( imc ) foi de 24,30 3,47 a mdia da fc em repouso foi de 75,12 12,48 bpm , e a mediana foi 73 bpm . a mdia da spo2 em repouso foi de 97,96 1,02% , e a mediana foi 98% . l , e a mediana foi 3,65 l. a mdia do vef6 foi de 4,45 0,87 l , e a mediana foi 4,38 l. a mdia da relao vef1/vef6 foi de 0,83 0,08 , e a mediana foi 0,82 . em todos os participantes , o vef6 medido situou - se acima do limite inferior da cvf prevista ( conforme se determinou por meio das equaes desenvolvidas para a populao brasileira ) , e todos apresentaram vef1/vef6 0,8 . esses resultados permitem - nos supor que nenhum dos participantes tinha doena pulmonar restritiva ou obstrutiva ( tabela 1 ) . tabela 1 comparao dos dois grupos ( com e sem dessaturao de oxignio).a a iswd mdia foi de 958,30 146,32 m , e a mediana foi 1.020 m. a mdia da fc ps - iswt foi de 162,41 18,24 bpm , e a mediana foi 166 bpm . a mdia da spo2 ps - iswt foi de 96,27 2,21% . em 11 participantes , os valores de saturao de oxignio ps - iswt foram maiores do que os valores basais . em 17 participantes , a saturao de oxignio diminura 4% ao final do teste . tabela 2 porcentagem de indivduos que atingiram mais de 85% ou 85% ou menos da fc mxima prevista nos dois grupos.a a populao estudada foi dividida em dois grupos com base na presena de dessaturao de oxignio 4% aps o iswt . no foram encontradas diferenas entre os dois grupos quanto idade , gnero , vef1 , vef6 , vef1/vef6 , saturao de oxignio inicial ( spo2 em repouso ) , iswd , fc em repouso , fc aps o iswt e porcentagem da fc mxima ( tabela 1 ) . o imc foi significativamente maior naqueles que apresentaram dessaturao de oxignio ( p = 0,01 ) , e houve diferena significativa entre os dois grupos no tocante spo2 aps o iswt ( p < 0,0001 ) . em 66 participantes , os valores de saturao de oxignio aps o iswt foram bastante semelhantes aos basais , um achado que era esperado por causa da intensidade do exerccio realizado . em 11 indivduos , os valores da spo2 aps o iswt foram maiores que os valores basais . esse resultado no incomum , uma vez que a atividade fsica melhora a ventilao e o recrutamento alveolar . no entanto , 17 ( 20,7% ) dos 83 indivduos da amostra estudada apresentaram queda significativa da saturao de oxignio durante o iswt ( "sat 4% ) . esse resultado foi inesperado , e h poucas informaes na literatura sobre o que acontece com a saturao de oxignio aps o iswt em indivduos saudveis . a hipoxemia induzida pelo exerccio em atletas arbitrariamente definida como uma diminuio da pao2 de aproximadamente 7,5 mmhg , ( ) sao2 abaixo de 95% ou ambas ; casos extremos apresentam sao2 < 88% . ( ) o consumo de oxignio aumenta durante o exerccio para satisfazer s necessidades impostas pelo aumento da taxa metablica e correlaciona - se com a intensidade do trabalho at que todos os indivduos alcancem o consumo mximo de oxignio . ( ) cada etapa do transporte de oxignio do ar ambiente para as clulas pode limitar o consumo de oxignio do corpo todo , e a circulao foi considerada o mais importante fator que limita o consumo mximo de oxignio durante o exerccio que envolve grande massa muscular . a diminuio da pao2 e sao2 bastante comum durante o teste de esforo mximo em remoergmetro . ( ) nesses indivduos , o dbito cardaco pode exceder 30 l / min ; nessas circunstncias , a capacidade de renovar o ar alveolar e manter elevadas presses parciais de oxignio , a resistncia de difuso ao oxignio na membrana alveolocapilar , a reduo do tempo de trnsito das hemcias no capilar pulmonar e o aumento da probabilidade de desequilbrio entre a ventilao e a perfuso so extremamente importantes para o consumo de oxignio . ( ) a prevalncia de hipoxemia induzida pelo exerccio parece ser elevada , chegando a 50% . ( ) a dessaturao de oxignio tambm mais pronunciada durante o exerccio que envolve o corpo todo , como o remo ou a corrida , do que durante o exerccio de membros inferiores , e o exerccio de membros inferiores mais capaz de induzir hipoxemia do que o exerccio de membros superiores . ( ) isso sugere que a quantidade de massa muscular envolvida no exerccio influencia o surgimento de dessaturao de oxignio . ( ) estudaram a incidncia de hipoxemia arterial induzida pelo exerccio em 16 corredores saudveis altamente treinados que eram capazes de atingir e manter taxas metablicas muito altas , inclusive um consumo mximo de oxignio de 72 2 ml / kg . os gases arteriais e o estado cido - bsico foram determinados a cada carga de um teste de esforo progressivo de curto prazo e repetidamente determinados durante a corrida em esteira rolante com carga constante . foram encontrados trs tipos de respostas , as quais foram bastante reprodutveis dentre os indivduos testados : quatro corredores mantiveram a pao2 a 10 mmhg dos valores em repouso ; outros quatro apresentaram reduo de 10 - 15 mmhg na pao2 e os demais oito corredores apresentaram pao2 notavelmente diminuda . a reduo da pao2 variou de 21 mmhg a 35 mmhg ; a pao2 caiu para menos de 75 mmhg em todos os casos e para menos de 60 mmhg em dois casos . durante o exerccio com carga constante , a pao2 frequentemente se manteve durante os 30 s iniciais , quando a hiperventilao foi maior ; posteriormente , houve hipoxemia , cuja gravidade , na maioria dos casos , permaneceu igual ou piorou ao longo dos 3 - 4 min que se seguiram . a hipoxemia mais grave durante o exerccio pesado associou - se a uma diferena entre a presso parcial alveolar de oxignio ( estimada ) e a pao2 ( medida ) superior a 40 mmhg . a anlise de todas as variveis medidas durante a investigao levou os autores hiptese de que a hipoxemia observada atribua - se a uma limitao de difuso secundria a tempos muito curtos de trnsito de hemcias em pelo menos uma parte da circulao pulmonar ; esses tempos de trnsito curtos podem ocorrer em altas taxas metablicas . a respirao corrente durante o exerccio pesado pode frequentemente exceder a curva fluxo - volume mxima , e a hiperventilao pode ser limitada pela carga mecnica sobre a parede torcica secundria a um aumento da impedncia pulmonar , uma situao que tambm pode prejudicar a renovao do ar alveolar . embora tenha sido descrito como um teste incremental de campo de caminhada que produz desempenho mximo limitado por sintomas , o iswt no pode ser comparado com os testes de esforo mximo citados acima . no obstante , 17 voluntrios saudveis apresentaram dessaturao de oxignio aps o iswt no presente estudo . a nica diferena significativa entre os indivduos que apresentaram dessaturao de oxignio e os que no o fizeram foi o imc , que foi maior naqueles . durante atividades fsicas que fazem com que a fc chegue o mais prximo possvel da fc mxima que se espera de determinado indivduo , so incapazes de arterializar o sangue que flui rapidamente , especialmente em indivduos cujas necessidades so ampliadas em virtude da massa corporal maior . embora durand et al . ( ) no tenham encontrado diferenas em altura , peso e volume pulmonar entre atletas que apresentaram dessaturao de oxignio e aqueles que no o fizeram , pode - se argumentar que as porcentagens dessas variveis so mais importantes do que seus valores absolutos . nossos resultados e os dados da literatura levam a uma possibilidade preocupante : a dessaturao de oxignio durante a atividade aerbica provavelmente mais comum do que se pensava e pode representar uma ameaa sistematicamente negligenciada aos atletas de alto desempenho . embora a ocorrncia de dessaturao de oxignio seja reconhecida em estudos que datam da segunda metade do sculo passado , nenhum desses estudos esmiuou os potenciais efeitos nocivos de tais perodos de hipoxemia intermitente . no temos conhecimento de nenhum protocolo de avaliao para treinamento aerbio de alto desempenho que inclua a determinao da dessaturao de oxignio induzida pelo exerccio . como a morte sbita relativamente comum em atletas , parece - nos justificvel a investigao da dessaturao de oxignio . a morte sbita cardaca ( msc ) considerada a principal causa de morte em atletas jovens . os estudos nos quais se relatou a maior incidncia estimaram a ocorrncia de at 110 mortes por ano em atletas jovens , o que equivale a 1 morte a cada 3 dias nos estados unidos . ( ) as evidncias disponveis apontam para uma anormalidade estrutural cardaca como causa bsica de msc . a cardiomiopatia hipertrfica e anomalias das artrias coronrias so responsveis por aproximadamente 25% e 14% , respectivamente , de todas as msc nos estados unidos . ( ) a displasia / cardiomiopatia arritmognica ventricular direita uma doena cardaca caracterizada por necrose miocrdica seguida de substituio fibrogordurosa . essas reas miocrdicas alteradas constituem o substrato anatmico para circuitos de reentrada que propiciam o aparecimento de arritmias ventriculares . ( ) as arritmias ventriculares podem ser particularmente importantes no contexto do presente estudo . a dessaturao de oxignio durante a atividade fsica intensa pode causar episdios repetidos de vasoconstrio pulmonar hipxica e hipertenso pulmonar . as paredes do ventrculo direito podem sofrer durante esses episdios , a ponto de necrose miocrdica , e a substituio fibrogordurosa a consequncia esperada desse tipo de estresse . outra causa comum de msc a sndrome de brugada , ( ) que se caracteriza por uma elevao do segmento st nas derivaes precordiais direitas no eletrocardiograma , seguida de uma onda t negativa . estima - se que a prevalncia mundial da sndrome de brugada seja de 1 - 5 por 10.000 habitantes , embora seja maior no sudeste asitico . ( ) a sndrome de brugada tradicionalmente considerada uma doena cardaca eltrica primria do miocrdio estruturalmente normal . no entanto , a ressonncia magntica , a tomografia por emisso de psitrons , e a avaliao anatomopatolgica de fragmentos de bipsia identificaram anormalidades estruturais em muitos pacientes com diagnstico de sndrome de brugada , incluindo a substituio fibrogordurosa da parede livre do ventrculo direito e bloqueio fibrtico do ramo direito do feixe . est estabelecido na literatura que a sndrome de brugada resultado de uma mutao autossmica dominante do gene scn5a no cromossomo 3 , a qual resulta em anormalidade de perda de funo do canal de sdio . ( ) torna - se cada vez mais claro que a expresso gnica de canais inicos altamente dinmica e pode responder a muitos estmulos ambientais . portanto , uma predisposio gentica para arritmia cardaca no impede a sobreposio de hipoxemia causando a morte sbita de jovens ( atletas ou no ) . os dados do presente estudo e o conhecimento acumulado sobre a dessaturao de oxignio durante a atividade fsica levantam a hiptese de que a hipoxemia durante o exerccio pode ser perigosa e sugerem que aconselhvel incluir um teste de deteco de dessaturao de oxignio nos protocolos de avaliao para atletas de resistncia . em suma , em virtude da possibilidade de dessaturao de oxignio em indivduos saudveis submetidos ao iswt , o uso do iswt para prever a presena de alteraes respiratrias sutis no detectadas por testes submximos como o tc6 pode ser enganador . a constatao de que a dessaturao de oxignio comum em indivduos saudveis submetidos ao iswt soma - se ao conhecimento de que a dessaturao de oxignio durante a atividade fsica intensa muito comum e pode ter efeitos deletrios .

objective : to determine the probability of oxygen desaturation in healthy individuals undergoing the incremental shuttle walk test ( iswt ) . methods : we enrolled 83 healthy subjects : 55 males ( including 1 smoker ) and 28 females . we determined pre - iswt fev1 , fev6 , hr and spo2 , as well as post - iswt hr and spo2 . results : mean values overall were as follows : age , 35.05 12.53 years ; body mass index , 24.30 3.47 kg / m2 ; resting hr , 75.12 12.48 bpm ; resting spo2 , 97.96 1.02% ; fev1 , 3.75 0.81 l ; fev6 , 4.45 0.87 l ; fev1/fev6 ratio , 0.83 0.08 ( no restriction or obstruction ) ; incremental shuttle walk distance , 958.30 146.32 m ; post - iswt hr , 162.41 18.24 bpm ; and post - iswt spo2 , 96.27 2.21% . in 11 subjects , post - iswt spo2 was higher than was pre - iswt spo2 . in 17 subjects , there was a 4% decrease in spo2 after the iswt . there were no statistically significant differences between the groups with and without post - iswt oxygen desaturation in terms of age , gender , fev1 , fev6 , fev1/fev6 , pre - iswt spo2 , incremental shuttle walk distance , hr , or percentage of maximal hr . in the individuals with post - iswt oxygen desaturation , the body mass index was higher ( p = 0.01 ) and post - iswt spo2 was lower ( p = 0.0001 ) . conclusions : healthy individuals can present oxygen desaturation after the iswt . using the iswt to predict subtle respiratory abnormalities can be misleading . in healthy subjects , oxygen desaturation is common after the iswt , as it is during any intense physical activity .

Introduction Methods Discussion OBJETIVO: MTODOS: RESULTADOS: CONCLUSES: Introduo Mtodos Resultados Discusso

PMC4180860

virtual reality technique can be combined with targeted exergames development with the aim to promote motor rehabilitation . recently , a number of researchers have pointed to the potential of virtual reality applications in health care [ 6 , 20 , 28 , 36 , 48 , 50 ] and hence sparked the introduction of this technology within rehabilitation medicine . exergamesvirtual reality games that involve physical exercise has been proposed as a valuable instrument to encourage participation in rehabilitation and improve the adherence to therapy programs because of engaging the user [ 6 , 12 , 48 ] . for example , a study by rizzo and kim demonstrated that virtual reality - based games reduce patients ' dreariness and simultaneously increase their motivation for rehabilitation practice . accordingly , virtual reality provides the capacity to simulate scenarios that are effective in attracting the performers ' attention . simulated circumstances can be used to elicit a thrilling ambience , whereas the patient can still perform movement and behaviors in a safe and controlled environment . for best results in rehabilitation , video games specifically designed for therapy should be used . because conventional video games were primarily developed for entertainment purposes , most of these are not practical for rehabilitation . one hurdle facing the successful use of exergames in rehabilitation is that many off - the - shelf video games are too complex for use by functionally impaired persons or elderly people . video games must therefore be developed to take into consideration the cognitive and physical limitations , as well as the interest sets , of the trainees . to date , however , the vast majority of research has focused on games developed for the entertainment market which fails to adapt the gameplay according to patients ' rehabilitation requirements . fp7 is the short name for the seventh framework programme for research and technological development , the european union 's main instrument for funding research in europe running from 2007 to 2013 . fp7 is also designed to respond to europe 's employment needs , competitiveness , and quality of life [ c.f . rehabilitative wayout in responsive home environments ( rewire ) , which is funded by fp7 , aims to develop , integrate , and field test an innovative virtual reality - based rehabilitation platform for people with stroke . the platform should allow patients , discharged from the hospital , to continue intensive rehabilitation at home under remote monitoring by the hospital . the main idea is to combine off - the - shelf components ( e.g. , the tracking device microsoft kinect , the force plate tymo ) in a robust and reliable way and render a system that can be used in the stroke patients ' homes . in the context of the rewire project when creating a virtual reality - based stroke treatment approach , the development of suitable exergames is undoubtedly an important requirement . there have been , to date , some studies that deal with the development of video games adequate for therapy purposes . based on specific game design principles , they intend to provide engaging and challenging treatment conditions to achieve a high level of motivation for rehabilitation practicing [ 1 , 6 , 7 , 24 , 33 , 43 , 59 ] . moreover , it is important that the developed exergames are functionally integrated in a well - elaborated therapy program . on the one hand that is , it should be aimed at those goals that are considered to be essential for ( and by ) stroke victims . on the other hand , the rehabilitation program should be consistent with established training principles to successfully reach these goals . in this article , we describe how tailored exergames for stroke patients can be developed based on a theoretical framework . we will discuss key exergame design and training program content considerations for patients with stroke , which may be theoretically linked to impaired walking performance due to the stroke event . worldwide , there are about 4.8 million survivors of stroke of whom about 1.1 million suffer lasting functional disabilities . the specific disabilities caused by stroke vary greatly depending on the brain area that is damaged . hemiparesis a paralysis that characteristically affects an arm and leg on one side of the body is one of the most common stroke - induced impairments and often leads to a number of negative walking - related consequences . the typical hemiparetic gait post - stroke is associated with a reduced walking velocity , cadence and stride length , with gait asymmetry , and with a prolonged double - support and stance - phase duration of both lower extremities [ 15 , 26 , 27 , 45 , 61 ] . it is for this reason that many stroke victims show marked deficits in walking abilities . there is general agreement that an association exists between the degree of independent mobility and quality of life [ 47 , 54 , 56 ] . consequently , a central goal of stroke rehabilitation should be to improve gait characteristics and to retrain the patient to the highest possible level of walking ability [ 16 , 34 ] . the findings demonstrated by vincent et al . confirmed the importance of enhancing stroke patients ' functional mobility through rehabilitation . vincent and colleagues set out to reveal rehabilitation needs from the perspective of four different parties involved in the stroke rehabilitation process ( stroke patients , caregivers , health professionals , and healthcare managers ) so as to better plan the post - stroke treatment service . according to patients ' statements which , in our view , should have high priority when designing rehabilitation programs continued rehabilitation should focus primarily on motor activities , such as walking . good balance skills are an important determinant of walking performance and impaired balance ability is assumed to be related to a decreased locomotor function [ 18 , 62 ] . considering that hemiparesis not only affects gait characteristics , but also often leads to diminished balance skills , a post - stroke rehabilitation program targeting deficits in static and dynamic balance may be an effective way to restore independent functional walking . in recent years , another factor contributing to walking recovery has been suggested : several authors noted that task - related activities lead to greater improvements in post - stroke walking competency than non - task - related practices [ 13 , 42 , 49 ] . specifically , they suggested that intervention protocols that include actual walking tasks improve walking skills to a greater extent than rehabilitation programs that do not . additionally , there is robust evidence in the motor learning literature that the best outcomes in terms of long - term retention and transfer of skills are achieved when principles of motor learning are integrated into treatment protocols . in rehabilitation , it is widely accepted that more practice is better and that an intense structured therapy program with numerous repetitions of various , challenging tasks supports motor skill acquisition . specifically , two important elements in our consideration are the motor learning principles variable practice and progression . these principles are known to positively affect gait rehabilitation and , hence , should be considered in developing a rehabilitation program . the literature to date has found that variable practice leads to better transfer and retention of motor skills than if a constant practice structure is used [ 22 , 23 , 31 , 38 , 52 ] . instead of performing one task repeatedly and always in the same manner ( constant practice ) , a specific task should be practiced differently throughout a treatment session by varying the conditions of practice ( variable practice ) . krakauer in his contribution published in the current opinion in neurology stated that variable practice is a fundamental principle in terms of retaining learning over time and that a consistent agreement in the literature exists indicating that varied practice is superior to repetitive identical tasks when it comes to motor learning . the principle of progression holds that motor learning and rehabilitation programs benefit from a continuous adaptation of task difficulty to increasing skill level . for successful learning , an optimal challenging training situation should be given providing an appropriate task difficulty level according to individuals ' capacities . furthermore , it can be hypothesized that the training effects will be enhanced when the locomotor practice gets combined with direct feedback ; e.g. , visual feedback . visual feedback can be used to provide information about a patient 's movement or the result of a movement and is known to promote postural control and stability [ 2 , 19 , 25 , 53 , 62 ] . from the above , it follows that rehabilitation programs for stroke survivors are well - advised to train balance and walking skills by means of a variety of balance- and walking - specific exercises , the difficulty of which is progressively adapted to patients ' skill level , in combination with direct visual feedback on performance . although previous studies have emphasized the importance of theory - based practice and rehabilitation [ 3 , 11 , 37 , 57 ] , there is still a lack of established concepts underlying the practical implementation of motor skill learning principles and theories in rehabilitation . a desirable template would be one that is simple and feasible and that facilitates the task - specific , progressive , and variable training of balance and walking skills . the motor skill taxonomy proposed by gentile seems to constitute just such a template for rehabilitation programs because it provides a two - dimensional basis for classifying a variety of motor skills . based on the above , the aim of this article is to develop and describe a tailored exergames - based stroke rehabilitation program that is based on a theoretical framework . three of these categorize motor skills according to one common characteristic of the skill and lead to one - dimension classification systems . in contrast , gentile 's taxonomy considers two general skill characteristics and offers , therefore , a broader concept leading to a two - dimensional classification system of motor skills . to highlight the high potential of gentile 's two - dimensional approach , it is worthwhile to focus at first on the one - dimensional classification systems and discus some of their limitations : a description of these classification systems is given by magill ( pp . 516 ) . one one - dimension classification system differentiates skills depending on the sizes of the primary muscle groups required to produce an action ( gross motor skills vs. fine motor skills ) . a second one - dimensional system considers the specificity of a movement 's beginning and end points to categorize motor skills ( continuous motor skills vs. discrete motor skills ) . the third one - dimension classification system makes a distinction according to the stability of the environmental context in which an action is being performed ( open motor skills vs. closed motor skills ) [ 35 , 39 ] . these one - dimensional classification systems raise the problem that they fail to capture the complexity of many motor skills by only focusing on a single aspect of motor skills . gentile presented a systematic classification system to categorize motor skills and movement according to two general dimensions of physical actions . the first dimension , environmental context , refers to the environmental conditions to which the performer has to react in order to successfully perform a task . this dimension is characterized by two indicators : ( a ) regulatory conditions and ( b ) intertrial variability . the regulatory conditions indicate relevant environmental features that constrain movement execution and may either be stationary ( stationary regulatory conditions ) or moving ( in - motion regulatory conditions ) . with the indicator intertrial variability , gentile 's taxonomy differentiates between regulatory conditions that change between trials ( intertrial variability ) and those that do not ( no intertrial variability ) . the second dimension , action function , is also characterized by two indicators : ( a ) body orientation and ( b ) object manipulation . body orientation indicates whether an action requires the performer to move from one location to another ( body transport ) or not ( body stability ) . object manipulation indicates whether an object has to be controlled during the action performance ( object manipulation ) or not ( no object manipulation ) . through the interaction of the resulting four environmental context characteristics and four action function characteristics , gentile defines 16 different motor skill categories that provide a comprehensive template to classify motor skills ( table 1 ) . the taxonomy is a good means of becoming aware of the skill characteristics that make skills distinct from , as well as related to , other skills , and is an excellent guide for establishing practice or training routines .table 1gentile 's taxonomy of motor skills gentile 's taxonomy of motor skills according to gentile , the easiest skill category can be found at the top left position ( 1a ) . moving either rightward or downward in the table renders the skill category more difficult , so that the most difficult skill category can be found at the bottom right of the table ( 4d ) . for the action function dimension , this implies that body transport is more difficult than body stability and object manipulation more difficult than no object manipulation . importantly , it also assumes a hierarchy in the action function characteristics : skills involving body transport but no object manipulation are more difficult than those involving object manipulations but not body transport . for the environmental context dimension , the same pattern of progression is assumed . thus , gentile 's taxonomy allows a systematic progression in difficulty of motor tasks and meets the demand of the motor learning principle progression . moreover , each of the 16 categories is associated with unique features based on the two - dimensional approach and , consequently , the taxonomy involves task variations . obviously , gentile 's classification system is consistent with the motor learning principle variable practice . there are several reasons for assuming that this taxonomy provides a valuable tool for developing a theory - based rehabilitation program using virtual reality . considering the explanations above , it is evident that gentile 's taxonomy is in accordance with the generally accepted motor learning principles progression and variable practice . the taxonomy provides a well - structured framework and the 4 4 table can serve as a useful guide for preparing a systematically coherent motor learning concept in a simple manner . for implementing gentile 's taxonomy - based approach , exergames might demonstrate an optimal opportunity for providing beneficial treatment conditions . on the one hand , as we referred above , there is high potential for creating a motivating rehabilitation environment using virtual reality . on the other hand , the taxonomy seems suited for virtual reality applications where the environment , task complexity , and other contextual factors related to exercise performance can be manipulated . exergame parameters can be easily modified and , thereby , adjusted to the demands of the taxonomy - included skill categories . to provide a post - stroke rehabilitation program focused on improved balance skills in standing and walking in accordance with gentile 's framework , we designed six basic exergames . based on these six basic exergames , we created 16 virtual reality scenarios , either by making game parameter modifications or by substituting another basic game . each scenario corresponds to one of the 16 skill categories included in the taxonomy ( table 2).table 2exergame scenarios corresponding to gentile 's skill categories exergame scenarios corresponding to gentile 's skill categories in accordance with a phased iterative approach suggested by campbell et al . , this paper demonstrates the theoretical phase the first step when developing and evaluating complex research - based interventions to improve health . thus , a set of six basic exergames appears to be appropriate in the research process of designing an exergame - based stroke rehabilitation program . it keeps the number of games that have to be developed manageable and with the same game being implemented across adjacent skill categories it renders systematic progression more straightforward . below , we will briefly describe the content of each of the 16 skill categories.the first phase of the rehabilitation program focuses on the basic physical skill of standing quietly . in this exergame , the patient is represented as a scarecrow avatar and is required to maintain a cursor indicating the body 's center of pressure ( cop ) within a predefined marked circle on a computer screen . the exergame focuses on stance steadiness ( sway ) that demonstrates a relevant aspect related to an individual 's balance ability . due to hemiparesis after stroke , patients often show an increased amount of postural sway .whereas the game scarecrow described for skill category 1a demands a centered and stable cop position , this game ( called blueberry collector ) uses controlled cop displacements for successful performance . this exergame targets stroke patients ' dynamic stability , because subjects with hemiparesis often suffer from reduced limits of stability . the virtual environment represents a field of blueberries , which have to be collected by a virtual farmer . the farmer 's moving is controlled by the patient 's weight shifting tasks and , in this way , the blueberries can be gathered . because the position of the berries in the field is fixed and unvarying between the different exergame trials , stationary regulatory conditions are given and no intertrial variability . to meet the requirement of object manipulation , a virtual basked filled with blueberries is placed on the farmer 's head . in order to prevent berries from falling down , the patient must adopt an ideal upright body posture.previous exercise interventions have shown that stepping exercises indicate an effective treatment method to improve postural stability and balance [ 9 , 29 , 41 ] , accordingly , this exergame ( called frog jumping ) requires the control of the cop displacements during stepping movements . the virtual environment represents a pond with several stones placed in it . by doing steps in a specific direction , the patient can control a frog jumping from one stone to another . a virtual arrow indicates which particular stone has to be targeted by the patient . in accordance with the taxonomy conditions , the position of each stone within the pond is fixed ( stationary regulatory conditions ) and the order of the targeted stones unchangeable from one exergame trial to the next ( no intertrial variability).according to gentile 's taxonomy , the skill category 1d can be seen as a progression of 1c . thus , the game frog jumping described for 1c can be extended by integrating an object manipulation task . specifically , in this game , the virtual frog is wearing a crown and it is the patient 's task to perform stepping tasks with an upright body position in order to keep the virtual crown correctly positioned.the game blueberry collector described above is not only adequate for skill category 1b , but also for category 2a by making small adaptations . on the one hand , the virtual basket on the farmer 's head has to be removed to get the condition no object manipulation . on the other hand , because intertrial variability is required , the position of the berries varies within exergame trials.for skill category 2b , the exergame blueberry collector for category 2a can be extended with an object manipulation task . thus , the virtual farmer is balancing a basket on his head such as in the exergame described for skill category 1b.we use the game frog jumping described for category 1c with the only modification of providing a random order of targeted stones marked by the arrow to realize intertrial variability . consequently , each time when the game has been played , a unique step performance pattern is required by the patient.to meet the taxonomy requirements in skill category 2d , the game frog jumping of category 2c can easily be adapted . hence , in this game , the frog is wearing a crown for which the patient has to control his / her position while playing the game.for skill category 3a , we created a game called tractor driver . the exergame presents a moving tractor for which the driving speed is defaulted by the video game . the direction of driving is controlled by the patient 's weight shifting movements . when playing this exergame , the ability to move the cop in a standing posture without loss of balance ( dynamic stability ) is being trained . specifically , the purpose of this game is to fork up hay bales through directing the moving tractor from one hay bale to another . the hay bales are widely spread over the soil . considering that the speed of the moving tractor is default and can not be manipulated by the patient themselves , in - motion regulatory conditions are given . there is a fixed position of each hay bale unchangeable from one exergame trial to the next to ensure no intertrial variability.in this exergame ( called fruit catcher ) , the patient is represented as an avatar balancing a fruit basket on the head . virtual apples are falling down from a tree ( in - motion regulatory conditions ) that should be caught by the basket . the patient has to perform target - oriented cop shifts to get an optimal avatar position and the apples are falling into the basket . this exergame focuses on dynamic stability due to controlled weight shifting movements to selected targets . for presenting no intertrial variability , the game parameters are always the same , even when the game is repeatedly played.according to gentile 's taxonomy , this exergame ( called worm hurdler ) requires stepping tasks . specifically , the patient represented as an avatar has to overstep a crawling worm coming closer alternately from the right and the left side ( in - motion regulatory conditions ) . the parameters of the game are unchangeable and , thus , no intertrial variability is given.the exergame worm hurdler described in 3c can easily be modified for skill category 3d . by integrating a virtual water jar that has to be balanced on the avatar 's head , an object manipulation task is integrated . to avoid water spillage , the patient has to adopt an ideal upright body posture.for skill category 4a , we adapted the game tractor driver described for category 3a by implementing intertrial variability . specifically , to provide varying regulatory conditions , the hay bale positions change from one exergame trial to another.the exergame fruit catcher described for skill category 3b needed a slight modification to meet the category requirements for 4b . in this game , there are not only falling apples which have to be caught , but various kinds of fruits . on the one hand , the fruits have different sizes and , on the other hand , different falling speeds . obviously , intertrial variability is given.when playing the game worm hurdler designed for category 3c , but with changing regulatory conditions between the exergame trials , intertrial variability is ensured . accordingly , in this game , the worms are crawling with varying speeds and are coming either from the right or the left side in random order.for integrating an object manipulation task , the exergame worm hurdler described for skill category 4c can be advanced by balancing a virtual water jar on the avatar 's head . consequently , while overstepping the randomly approaching crawling worms , the patient has to adopt an ideal upright body position ensuring that no water is spilling out ( from the jar ) . the first phase of the rehabilitation program focuses on the basic physical skill of standing quietly . in this exergame , the patient is represented as a scarecrow avatar and is required to maintain a cursor indicating the body 's center of pressure ( cop ) within a predefined marked circle on a computer screen . the exergame focuses on stance steadiness ( sway ) that demonstrates a relevant aspect related to an individual 's balance ability . due to hemiparesis after stroke , patients often show an increased amount of postural sway . whereas the game scarecrow described for skill category 1a demands a centered and stable cop position , this game ( called blueberry collector ) uses controlled cop displacements for successful performance . this exergame targets stroke patients ' dynamic stability , because subjects with hemiparesis often suffer from reduced limits of stability . the virtual environment represents a field of blueberries , which have to be collected by a virtual farmer . the farmer 's moving is controlled by the patient 's weight shifting tasks and , in this way , the blueberries can be gathered . because the position of the berries in the field is fixed and unvarying between the different exergame trials , stationary regulatory conditions are given and no intertrial variability . to meet the requirement of object manipulation , a virtual basked filled with blueberries is placed on the farmer 's head . in order to prevent berries from falling down previous exercise interventions have shown that stepping exercises indicate an effective treatment method to improve postural stability and balance [ 9 , 29 , 41 ] , accordingly , this exergame ( called frog jumping ) requires the control of the cop displacements during stepping movements . by doing steps in a specific direction , the patient can control a frog jumping from one stone to another . a virtual arrow indicates which particular stone has to be targeted by the patient . in accordance with the taxonomy conditions , the position of each stone within the pond is fixed ( stationary regulatory conditions ) and the order of the targeted stones unchangeable from one exergame trial to the next ( no intertrial variability ) . according to gentile 's taxonomy thus , the game frog jumping described for 1c can be extended by integrating an object manipulation task . specifically , in this game , the virtual frog is wearing a crown and it is the patient 's task to perform stepping tasks with an upright body position in order to keep the virtual crown correctly positioned . the game blueberry collector described above is not only adequate for skill category 1b , but also for category 2a by making small adaptations . on the one hand , the virtual basket on the farmer 's head has to be removed to get the condition no object manipulation . on the other hand , because intertrial variability is required , the position of the berries varies within exergame trials . for skill category 2b , the exergame blueberry collector for category 2a can be extended with an object manipulation task . thus , the virtual farmer is balancing a basket on his head such as in the exergame described for skill category 1b . we use the game frog jumping described for category 1c with the only modification of providing a random order of targeted stones marked by the arrow to realize intertrial variability . consequently , each time when the game has been played , a unique step performance pattern is required by the patient . to meet the taxonomy requirements in skill category 2d , the game frog jumping of category 2c hence , in this game , the frog is wearing a crown for which the patient has to control his / her position while playing the game . the exergame presents a moving tractor for which the driving speed is defaulted by the video game . the direction of driving is controlled by the patient 's weight shifting movements . when playing this exergame , the ability to move the cop in a standing posture without loss of balance ( dynamic stability ) is being trained . specifically , the purpose of this game is to fork up hay bales through directing the moving tractor from one hay bale to another . the hay bales are widely spread over the soil . considering that the speed of the moving tractor is default and can not be manipulated by the patient themselves , in - motion regulatory conditions are given . there is a fixed position of each hay bale unchangeable from one exergame trial to the next to ensure no intertrial variability . in this exergame ( called fruit catcher ) , the patient is represented as an avatar balancing a fruit basket on the head . virtual apples are falling down from a tree ( in - motion regulatory conditions ) that should be caught by the basket . the patient has to perform target - oriented cop shifts to get an optimal avatar position and the apples are falling into the basket . this exergame focuses on dynamic stability due to controlled weight shifting movements to selected targets . for presenting no intertrial variability , the game parameters are always the same , even when the game is repeatedly played . according to gentile 's taxonomy specifically , the patient represented as an avatar has to overstep a crawling worm coming closer alternately from the right and the left side ( in - motion regulatory conditions ) . the parameters of the game are unchangeable and , thus , no intertrial variability is given . the exergame worm hurdler described in 3c can easily be modified for skill category 3d . by integrating a virtual water jar that has to be balanced on the avatar 's head , an object manipulation task is integrated . to avoid water spillage , the patient has to adopt an ideal upright body posture . for skill category 4a , we adapted the game tractor driver described for category 3a by implementing intertrial variability . specifically , to provide varying regulatory conditions , the hay bale positions change from one exergame trial to another . the exergame fruit catcher described for skill category 3b needed a slight modification to meet the category requirements for 4b . in this game , there are not only falling apples which have to be caught , but various kinds of fruits . on the one hand , the fruits have different sizes and , on the other hand , different falling speeds . when playing the game worm hurdler designed for category 3c , but with changing regulatory conditions between the exergame trials , intertrial variability is ensured . accordingly , in this game , the worms are crawling with varying speeds and are coming either from the right or the left side in random order . for integrating an object manipulation task , the exergame worm hurdler described for skill category 4c can be advanced by balancing a virtual water jar on the avatar 's head . consequently , while overstepping the randomly approaching crawling worms , the patient has to adopt an ideal upright body position ensuring that no water is spilling out ( from the jar ) . when implementing a therapeutic program to improve stroke victims motor skills , therapists must select exercises that are tailored to the demands of the individual patient . to achieve optimal outcomes in rehabilitation , the activities should be matched to a patient 's functional abilities and limitations . in a well - elaborated training plan , the ( exercise ) tasks which have to be performed should maintain an optimal challenge for the patient [ 7 , 19 ] . obviously , for providing a perfectly tuned exercise difficulty level at any point in time throughout the rehabilitation period , modifying the therapy plan is an essential need and an ongoing process . when a patient is making progress , more challenging tasks should be involved into the rehabilitation program . for selecting functionally appropriate activities during rehabilitation , we highlight the practical value of using gentile 's taxonomy . based on the consideration that the taxonomy - based skill categories present a structure going from simple to complex motor tasks , we provide a formal procedure for guiding patients from the top left of the table to the bottom right . closer inspection of table 1 reveals that the taxonomy can be divided into seven difficulty levels . more specifically , starting from 1 of the 16 skill categories , a category - based progression in task difficulty can be achieved either through a horizontal shift to the right or a vertical shift downward . accordingly , seven levels of difficulty can be distinguished which define a progressive increase of complexity in diagonal direction ( table 3).table 3gentile 's taxonomy of motor skills divided into seven levels of difficulty ( ordered diagonally ) gentile 's taxonomy of motor skills divided into seven levels of difficulty ( ordered diagonally ) considering the allocation of the skill categories to one of the seven difficulty levels , gentile 's taxonomy offers a simple and easy - to - follow way for incorporating an ongoing progression into a rehabilitation process . specifically , for selecting appropriate activities that provide rehabilitation at the optimal level of challenge , we propose the following procedure : a patient may only move up one level of difficulty when all skill categories within the current level are completed . for clarification , beginning with skill category 1a in difficulty level 1 , the patient is required to move to the skill categories in difficulty level 2 ( i.e. , 1b and 2a ) as soon as category 1a is successfully performed . within level 2 , patients and treating therapists are free to choose the order of execution of the belonging skill categories . once a patient is capable to perform both skill categories 1b and 2a successfully , the next difficulty level 3 has to be incorporated into rehabilitation . this procedure can be continued until the patient finally achieves skill category 4d and , consequently , the highest level of difficulty ( i.e. , level 7 ) . in this way , challenging situations are ensured at any point in time during rehabilitation and the patients are encouraged to exercise towards their functional limits . following the procedure suggested to guide someone 's therapy plan , one ensures that the selection of skill categories is constrained . due to this constraint , a variation of both general dimensions defined by gentile 's taxonomy will be considered during the rehabilitation process . accordingly , the procedure prevents a skill category - based progression only in one direction and includes the modification of both the environmental context and the action function characteristics . apart from these constraints , this approach also enables a certain degree of self - determination by therapy - involved actors . within a specific level of difficulty , this autonomy in decision - making increases the potential of tailoring the rehabilitation process to the demands of each individual patient and leaves freedom of choice to the treating therapists for the most adequate exercises that should currently be trained . using gentile 's taxonomy for designing a rehabilitation program that targets balance and walking deficits of stroke patients , an aspect must be considered critically . according to gentile 's framework , a horizontal shift to the right or a vertical shift downward within the taxonomy provides an increase of task complexity . however , when we focus on a motor task such as standing still on one leg , a potential inconsistency in gentile 's approach emerges . standing still on one leg can be categorized by the taxonomy dimension body stability that represents less complex tasks than motor skills that require body transport ( e.g. , walking ) . in our view , maintaining a one - leg stance position needs good balance skills . therefore , this activity might be perceived by some stroke patients as an advanced motor task which challenges them more than a body transport activity . furthermore , where this theoretical approach focused on the general stroke patient population , we are aware of the fact that different accompanying diseases and impairments ; e.g. , dizziness or muscle weakness due to stroke , may cause differences in how patients perceive tasks as being more or less difficult . it can be hypothesized that some patients have more difficulty maintaining a given posture without loss of balance whereas others experience performing a body transport task as more difficult . we think , however , that these potential differences may be resolved by clinicians that treat and monitor patients and decide , together with the patients and according their skill levels , how and when to progress through the taxonomy . it is clear to us that several pilot studies assessing feasibility and usability in several reference and subpopulations are needed as necessary next step in our program development process . feasibility studies are comparative randomized trials designed to provide preliminary evidence on the clinical efficacy of a drug or intervention . usability evaluation is a way to ensure that interactive systems are adapted to the users and should be part of a fundamental step in the user centered design process . in this paper , we elucidate the theoretical basis for a theory - driven rehabilitation program to improve stroke sufferers ' balance skills and walking capability using exergames . according to the framework proposed by campbell et al . , investigating relevant theory prior to an exploratory trial is of great importance . considering the theoretical knowledge gathered , an optimal study intervention design for pilot studies can be worked out that focus on the relevance and effectiveness of the rehabilitation program suggested . depending on the usability and feasibility testing results obtained , refinements and extensions of the exergames can be made and the present set of six basic exergames may be extended to a maximum of 16 different exergames . hence , a comprehensive and practical theory - driven stroke rehabilitation program based on gentile 's taxonomy can be achieved . with stroke , the changes in the central nervous system and lifestyle may impair an individual 's ability to regain and/or maintain certain levels of motor functioning . by using virtual reality , we might be able to offer continued rehabilitation in an attractive and meaningful way . specific targeted exergames have been designed that aim to minimize stroke - induced walking impairments . considering the aim of presenting a well - elaborated training concept with a strong theoretical rationale , in this paper we demonstrate that gentile 's taxonomy presents a feasible template providing assistance for developing and implementing a theory - driven rehabilitation program . the motor skill taxonomy suggested by gentile defines two general dimensions and structures a table that consists of 16 different skill categories ( table 1 ) . on the one hand , based on these skill categories , thus , a therapy program which meets the demand of the motor learning principle variable practice can be created . on the other hand , accordingly , the motor learning principle of progression is explicitly considered . due to the advantages of gentile 's two - dimensional approach , the taxonomy suits our purposes and was used as underlying framework to build up a post - stroke exergame rehabilitation program for enhancing balance and walking skills . based on the literature reviewed here , clinical approaches to maintaining and improving physical functioning over longer time periods in persons with stroke

virtual rehabilitation approaches for promoting motor recovery has attracted considerable attention in recent years . it appears to be a useful tool to provide beneficial and motivational rehabilitation conditions . following a stroke , hemiparesis is one of the most disabling impairments and , therefore , many affected people often show substantial deficits in walking abilities . hence , one of the major goals of stroke rehabilitation is to improve patients ' gait characteristics and hence to regain their highest possible level of walking ability . because previous studies indicate a relationship between walking and balance ability , this article proposes a stroke rehabilitation program that targets balance impairments to improve walking in stroke survivors . most currently , available stroke rehabilitation programs lack a theory - driven , feasible template consistent with widely accepted motor learning principles and theories in rehabilitation . to address this hiatus , we explore the potential of a set of virtual reality games specifically developed for stroke rehabilitation and ordered according to an established two - dimensional motor skill classification taxonomy . we argue that the ensuing exergame-based rehabilitation program warrants individually tailored balance progression in a learning environment that allows variable practice and hence optimizes the recovery of walking ability .

Introduction Stroke-induced motor impairments and their treatment approach Methods Gentile's motor skill taxonomy Gentile's taxonomy-based exergames to improve stroke patients' balance and walking skills Practical application of the taxonomy-based rehabilitation program using exergames Limitations and future directions Conclusion

PMC4942665

survival of individuals infected with hiv1 has been dramatically improved since the introduction of highly active antiretroviral therapy ( haart ) , but this increased survival has been associated with the development of chronic disorders . one example is the early development of emphysema in hiv1 smokers , with a greater prevalence compared to smokers who are hiv1 [ 25 ] . relevant to the pathogenesis of this hiv1 smoker - associated early emphysema , alveolar macrophages ( am ) of hiv1 smokers release increased levels of matrix metalloproteinases ( mmps ) , enzymes thought to play a key role in the pathogenesis of emphysema by virtue of their ability to degrade extracellular matrix and basement membrane components and modulate the recruitment of leukocytes into the lung [ 68 ] . in the context that lung epithelial lining fluid ( elf ) of hiv1 smokers has increased levels of a variety of cytokines [ 5 , 710 ] and that several cytokines mediate the activation state of am [ 11 , 12 ] , we hypothesized that , in the presence of cigarette smoke , upregulation of mmp-9 is associated with increased levels of proinflammatory cytokines and that their interplay may be contributing to the early development of emphysema in hiv1 smokers . based on recent studies demonstrating the importance of il-23 and the th17 immune response in the development of chronic obstructive pulmonary disease [ 1315 ] , their role in the progression of hiv-1 infection [ 1620 ] , and the effect of il-23 on increased lung mmp-9 in mice , we focused on a possible role of il-23 in the upregulation of mmp-9 in am of hiv1 smokers . assessment of inflammatory / immune cells recovered by lavage and lower respiratory tract elf of hiv1 healthy nonsmokers , hiv1 nonsmokers , hiv1 healthy smokers , hiv1 smokers with low dlco and hiv1 smokers with low dlco demonstrates that the upregulation of the mmp-9 by am in hiv smokers is likely derived , at least in part , by am expression of il-23 working in concert with t lymphocytes to induce am to express mmp-9 , a known contributor to the pathogenesis of emphysema . all individuals were evaluated at the weill cornell nih clinical and translational science center and the department of genetic medicine clinical research facility , using institutional review board - approved clinical protocols . individuals underwent an initial screening evaluation including history , complete physical exam , blood studies , urine analysis , chest x - ray , pulmonary function tests , and electrocardiogram . individuals with any significant prior use of addictive drugs ( other than nicotine ) in the previous 6 months were excluded . blood studies included a complete blood count , coagulation parameters , serum electrolytes , liver and kidney function tests , serum evaluation for human immunodeficiency virus antibodies , hiv1 viral load , cd4 count , hepatitis profile ( a , b , and c ) , anti - nuclear antibodies , sedimentation rate , and rheumatoid factor . current smoking status was verified by measurement of urinary levels of nicotine and its derivative cotinine and blood carboxyhemoglobin . pulmonary function tests were carried out according to american thoracic society guidelines [ 21 , 2326 ] . individuals who smoked and had a diffusing capacity < 80% predicted were further evaluated with noncontrast high resolution computed tomography ( ct ) of the chest . for all groups , no subjects had active pulmonary symptoms or a history of lung infections . this group ( n = 11 ) included individuals who were hiv1 and were current smokers . all subjects reported taking haart . each of these individuals had lung function studies with normal forced expiratory volume in 1 sec ( fev1 ) , forced vital capacity ( fvc ) , fev1/fvc , and total lung capacity ( tlc ) , but with diffusing capacity < 80% predicted as a physiological correlate of early emphysema [ 27 , 28 ] . assessment of the chest high - resolution ct ( hrct ) of this group had visual evidence of minimal to mild emphysema in 8/11 subjects confirmed by two experienced pulmonary physicians and a radiologist who was blinded to the subjects ' history . this group ( n = 18 ) were characterized by the same pulmonary function as for the hiv1 smokers . a chest hrct was available for the majority of subjects in the study group and revealed visual changes of minimal to mild emphysema in 9/14 . these individuals ( n = 32 ) included current smokers with a normal screening evaluation , normal pulmonary function tests and chest x - ray , and a positive urine screen for smoking . these individuals ( n = 17 ) were lifelong nonsmokers with a normal screening evaluation , normal pulmonary function tests and chest x - ray , and a negative urine screen for smoking . this group ( n = 5 ) consisted of lifelong hiv1 nonsmokers with an otherwise normal screening evaluation , normal pulmonary function tests and chest x - ray , and a negative urine screen for smoking . for the in vitro analyses , each assay was performed on a random subset of the overall study group ; the number of subjects used for each analysis is indicated . fiberoptic bronchoscopy was performed to obtain inflammatory / immune cells and elf from the lower respiratory tract using bronchoalveolar lavage , as previously described . the lavage fluid was filtered through 2 layers of gauze and centrifuged at 1250 rpm for 5 min , 4c . cells were suspended in 5 ml ack lysing buffer ( invitrogen , grand island , ny ) and then washed twice in rpmi 1640 containing 10% fetal bovine serum , 50 u / ml penicillin , 50 g / ml streptomycin , and 2 mm glutamine ( invitrogen ) . cell viability was estimated by trypan blue exclusion and expressed as a percentage of the total cells recovered . cells were suspended in media ( 10 cells / ml ) and an aliquot of 250 l with 250 l fetal bovine serum ( invitrogen ) was used for a differential cell count assessed by cytocentrifugation ( cytospin 11 ; shandon instruments , pittsburgh , pa ) and stained with diffquik ( baxter healthcare , miami , fl ) . cells recovered from bal fluid were seeded in 12-well plastic culture dishes ( 10/well ) and alveolar macrophages ( am ) were enriched by adherence for 12 hr at 37c in a 5% co2 humidified incubator . the remaining cells consisted of lymphocytes , neutrophils , nk cells , or eosinophils as assessed by flow cytometric analysis ( not shown ) . because this adherence method for am isolation is known to contain a small number of lymphocytes , we refer to the cultures as am / lymphocyte cocultures . all cells including am , t cells recovered from the lower respiratory tract , and t cells from blood were cultured from 6 h to 48 h at 37c in a 5% co2 humidified incubator in rpmi 1640 medium supplemented with 10% fetal bovine serum , 50 u / ml penicillin , 50 g / ml streptomycin , and 2 mm glutamine ( invitrogen ) . lavage fluid supernatant was concentrated 10-fold on centricon filters ( millipore , billerica , ma ) . the concentrated supernatant was assessed for 36 cytokines with a human cytokine array panel a ( r&d systems , minneapolis , mn ) . the supernatant was mixed with a cocktail of biotinylated detection antibodies and incubated on a nitrocellulose membrane , embedded with cognate capture antibodies . chemiluminescent detection reagents were added ( ge healthcare , piscataway , nj ) and the array was developed . relative gene expressions of il-23 , il-23r , and mmp-9 were evaluated by measuring specific mrna levels via taqman real - time reverse transcriptase ( rt ) pcr analysis . total rna was extracted using a modified version of the trizol method ( invitrogen , grand island , ny ) , in which rna is purified directly from the aqueous phase ( rneasy minelute rna purification kit , qiagen , valencia , ca ) . rna samples were stored in rna secure ( ambion , austin , tx ) at 80c . rna integrity was determined by running an aliquot of each rna sample on an agilent bioanalyzer ( agilent technologies , palo alto , ca ) . the concentration was determined using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies , wilmington , de ) . a first - strand cdna was synthesized from 2 g of total rna in 50 l reaction volume , using the taqman reverse transcriptase reaction kit ( applied biosystems , foster city , ca ) , with random hexamers as primers . the primers specific for each mrna ( mmp-9 : hs00234579_m1 , il-23a : hs00900829_g1 , and il-23r : hs00332759_m1 ) and the endogenous controls human -2-microglobulin ( vic/mgb probe , primer limited ) and human -actin endogenous control ( vic / mgb probe , primer limited ) were purchased from applied biosystems . for each individual sample , two conditions were used : 1 : 10 and 1 : 100 dilution of the cdna reaction , and each dilution was assayed in triplicate wells . the threshold cycles ( cts ) were calculated as an average of the triplicate reactions for each condition , and the ct was calculated for each sample using the rrna as an endogenous reference . ct was calculated by subtracting the calibrator from the ct in each individual sample using the algorithm provided by applied biosystems . after removal of nonadherent cells from the lavage fluid , cultured am containing small numbers of lymphocytes from both hiv1 and hiv1 individuals were incubated with rpmi media in 12-well plates at 37c in a 5% co2 humidified incubator . am were treated with serum - free media alone or with serum - free media containing rhil-23 ( r&d systems ) at various concentrations . treated am / lymphocytes were cultured overnight for quantitative cdna analysis or 48 hours for the analysis of substrate ( mmp-9 ) concentration in conditioned media . for analysis of mmp-9 activity , gelatin zymography was carried out by loading equal amounts of protein per sample onto 8% sodium dodecyl sulfate polyacrylamide gels impregnated with 0.1% gelatin ( invitrogen ) and separated using nondenaturing electrophoresis . after electrophoresis , the gels were soaked in renaturing buffer with 2.7% triton x-100 in distilled water ( invitrogen ) . gels were then developed and stained with colloidal blue ( invitrogen ) which reveals the enzymatic activity with white bands against a dark background . recombinant mmp-9 protein in latent and active forms ( chemicon , temecula , ca ) was used as a positive control . optical densities of gel zymograms were quantified with image j software . am ( 2 - 3 10/well ) were infected with the hiv-1 laboratory strain , jrfl , at 10 median tissue culture infectious doses ( tcid50 ) per well , a dose known to infect cells of monocytic origin . after 12 days of incubation at 37c , the cells were washed and the rna was isolated . productive hiv1 infection by jrfl hiv1 was determined by quantifying p24 antigen in the media supernatant by elisa ( beckman coulter , miami , fl ) at 012 days after hiv1 jrfl infection . t cells used in functional experiments ( t cells added to cultures of am ) were isolated directly from bal using t cell rosetting with neuraminidase - treated sheep red blood cell as per standard protocol [ 31 , 32 ] . t cells used for cdna analysis were isolated directly from bal via positive selection using dynabeads cd2 pan t ( invitrogen dynal , life technologies , grand island , ny ) as per manufacturer 's recommendations . t cells isolated by this method had purity > 80% and were used for cdna quantitative pcr analysis . purification of am was based on positive selection of cells expressing cd68 , a glycoprotein expressed intracellularly as well as on the surface of macrophages but not on lymphocytes . eugene , or ) leaf mouse anti - human anti - cd68 antibody ( biolegend , san diego , ca ) was used to attach to cd68 + am . addition of flowcomp flexi detachable beads ( invitrogen dynal , life technologies , grand island , ny ) was used to positively isolate cd68 + am . estimated purity of positively selected cells prior to the detachment of magnetic beads was estimated to be > 90% . cells directly isolated from bal or cultured am / lymphocytes isolated by adherence were detached from plates using ethylenediaminetetraacetic acid ( edta ; 5 mm ) for 3 min with vigorous washing and were thereafter washed with 0.1% bovine serum albumin ( bsa ) . the cells were then labeled with monoclonal anti - cd-68-pe and anti - human il-23r - pe ( r&d systems ) at 4c for 30 min . data on a minimum of 20,000 cell fluorescence events were acquired and analyzed using a facscalibur instrument ( becton - dickinson , san jose , calif ) and cellquest software . immediately prior to the analysis , analysis was performed using affymetrix ( santa clara , ca ) microarray hg - u133 plus 2.0 and associated protocols . an aliquot of each rna sample was run on an agilent bioanalyzer ( agilent technologies , palo alto , ca ) to visualize and quantify the degree of rna integrity . the concentration was determined using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies , wilmington , de , usa ) . strict quality control criteria were used for an rna sample to be accepted for further processing . double - stranded cdna was synthesized from 3 g of total rna using the genechip one - cycle cdna synthesis kit , followed by cleanup with genechip sample cleanup module , in vitro transcription reaction using the genechip ivt labeling kit , and cleanup and quantification of the biotin - labeled crna yield by spectrophotometric analysis . hybridizations to test chips and to microarrays were performed according to affymetrix protocols , and microarrays were processed by the affymetrix fluidics station and scanned with the affymetrix genechip scanner 3000 7 g . the overall microarray quality was verified by the criteria : ( 1 ) 3/5 ratio for gapdh < 3 ; ( 2 ) scaling factor range no more than 2.5 standard deviations ( sd ) from the mean for all microarrays ; and ( 3 ) expression level for all 100 housekeeping genes ( as defined by affymetrix , http://www.affymetrix.com/estore/ ) with coefficient of variation of < 40% . after scanning , the data on each individual microarray were scaled to an arbitrary target intensity as recommended by affymetrix , using the microarray suite version 5.0 software . analysis of the interleukin-23 receptor ( il-23r ) gene was carried out using a database of massive parallel sequencing ( rna - seq ) of the transcriptome of am of hiv1 nonsmokers . the resultant reads were aligned to homo sapiens high coverage assembly grch37 using bowtie v 0.12 . reads per kilobase of exon model per million mapped reads ( rpkm ) were used to quantify transcript levels of the gene . results of elf cytokine protein arrays were expressed as mean values of protein concentration standard deviation relative to an array internal standard and were displayed on a logarithmic scale . the significance of differences of mean values of demographic data between the 5 groups was analyzed by anova . taqman analysis results were expressed as a relative gene expression in comparison to control and/or the lowest expression in particular experiments . the significance of differences in relative gene expression between different phenotypes was tested via anova or kruskal - wallis test when the number of compared groups was > 2 ; student t - test or wilcoxon signed - rank test was used to compare relative gene expression when 2 groups were compared . a multicytokine array was used to evaluate elf levels in 27 individuals , including hiv1 and hiv1 nonsmokers , smokers , and smokers with low dlco ( see table 1 for demographic data ) . as expected , a relatively small number of cytokines were detectable in the elf of hiv1 healthy smokers and hiv1 healthy nonsmokers . in contrast , smokers with low dlco independent of their hiv1 status had a significantly higher proportion of detectable cytokines ( supplemental figure 1 ; see supplemental table i for definitions of all cytokines in the arrays , at supplementary material available online at http://dx.doi.org/10.1155/2016/3463104 ) . in stark contrast to hiv1 nonsmokers or hiv1 healthy smokers , hiv1 nonsmokers had detectible amounts of more than 2/3 of the cytokines represented on the array and more than twice as many compared to hiv1 healthy nonsmokers . interestingly , the cytokine profiles of hiv1 nonsmokers , hiv1 smokers with low dlco , and hiv1 smokers with low dlco were very similar and were characterized by the presence of ifn- as well as the th17 related cytokines il-17 and il-23 that were not present in hiv1 nonsmokers or hiv1 smokers ( figure 1 ) . these data support the concept that proinflammatory cytokines are increased in the lungs of smokers with low dlco , in both hiv1 and hiv1 individuals . hiv1 infection itself leads to a broad upregulation of cytokines detectable in elf , even in the absence of smoking . the presence of il-23 and il-17 that characterizes hiv1 individuals independent of their smoking status , as well as hiv1 smokers with low dlco , suggests a potential role of th17 cellular immune responses in the pathogenesis of emphysema in hiv1 smokers . in addition to ifn- and th17 cytokines , the cytokine profile of hiv1 smokers with low dlco shared other features with hiv1 nonsmokers and hiv1 smokers with low dlco including the presence of chemokine ( c - c motif ) ligand 1 ( ccl-1 ) and chemokine ( c - x - c motif ) ligand 11 ( cxcl-11 ) , both chemotactic for activated t cells [ 36 , 37 ] . the presence of both of these chemokines is consistent with a role for adaptive immunity and t cells in the early development of emphysema in hiv1 smokers . to evaluate whether the il-23 gene was expressed in human am , relative expression of am il-23 was quantified in a subset of all phenotypes using taqman real - time rt - pcr . il-23 was upregulated in both hiv1 phenotypes compared to hiv1 phenotypes ( hiv1 nonsmokers ( n = 5 ) , hiv1 smokers ( n = 5 ) , hiv1 nonsmokers ( n = 4 ) , hiv1 smokers with low dlco ( n = 4 ) , and hiv1 smokers with low dlco ( n = 4 ; p < 0.05 , kruskal - wallis ; figure 2 ) ) . these data are consistent with the results of hg - u133 plus 2.0 gene microarray analysis where il-23 expression in am from hiv1 smokers with low dlco ( n = 11 ) was significantly higher compared to its expression in hiv1 smokers with low dlco ( n = 23 ; not shown ) . in contrast , there was no significant difference in the extent of upregulation of il-23 in smoking compared to nonsmoking phenotypes . to assess the effect of hiv1 infection on am production of il-23 , we used an established model of in vitro infection of am with an hiv1 laboratory strain , jrfl . il-23 expression in jfrl - infected am was compared to il-23 expression in uninfected control am . at day 12 , il-23 expression was increased 10-fold compared to uninfected am , showing a positive trend , but without statistical significance due to high variability associated with jrfl - infected am ( p = 0.06 , wilcoxon signed - rank test , figure 3 ) . this may provide one possible explanation for the increased presence of il-23 in the elf of hiv1 individuals independently from their smoking history . this observation is also consistent with the results of experiments in which we measured the levels of il-23 in conditioned media of am cultured for 48 hr . additionally , in a set of experiments with lps , we also confirmed that il-23 is inducible in normal am without hiv1 infection ( supplemental figure 2 ) . this can explain how , in the setting of chronic inflammation of the airways as in copd , il-23 may be present at increased levels , consistent with previously published literature [ 13 , 15 , 20 , 21 ] . while our experimental design did not target recruitment of individuals with poorly controlled hiv disease , two available cultures of am from individuals with measurable viral loads had spontaneous il-23 release that was 10-fold higher than am from hiv1 individuals ( not shown ) . the gelatinase mmp-9 is capable of degrading extracellular matrix proteins and also can activate a number of bioactive molecules such as chemokines that are not a part of the matrix [ 38 , 39 ] . based on the data shown in figure 1 and our prior observations that expression of am mmp-9 is significantly increased in both hiv1 smokers with low dlco and hiv1 smokers with low dlco compared to both hiv1 healthy smokers and hiv1 healthy nonsmokers , we analyzed the effect of il-23 on am mmp-9 production . we used am / lymphocyte cocultures obtained from hiv1 healthy smokers , stimulated the cultures with il-23 , and then analyzed the relative expression of mmp-9 mrna with taqman rt - pcr . treatment of am / lymphocyte cocultures with il-23 leads to a mild but consistent upregulation of mmp-9 relative gene expression ( n = 13 , p < 0.01 , anova , figure 4(a ) ) . to assess whether il-23 causes increased secretion of mmp-9 , we analyzed the concentrations of mmp-9 in conditioned media via gelatin zymography ( figure 4(b ) ) . stimulation of am / lymphocyte cocultures with il-23 leads to an increase in mmp-9 concentration in conditioned media in a dose - dependent fashion ( n = 24 , p < 0.001 , anova , figure 4(c ) ) . this increase in mmp-9 concentration showed a similar pattern in all tested phenotypes including hiv1 nonsmokers , hiv1 smokers , and hiv1 smokers with low dlco . il-23 is produced by a variety of cell types including am and its primary target is il-23 receptor ( il-23r ) expressed on lymphocytes . as several reports indicate the presence of il-23r on alveolar macrophages in diseased lung [ 40 , 41 ] , we investigated the presence and expression of this molecule on normal am . using genespring affymetrix analysis with a p - call value of 20% for the threshold for detection of gene expression , we analyzed the am transcriptome of 87 individuals ( 24 hiv1 nonsmokers , 34 hiv1 smokers , 18 hiv1 smokers with low dlco , and 11 hiv1 smokers with low dlco ) . the data demonstrated that the il-23r was not expressed on am ( not shown ) . this was consistent with the absence of il-23r expression in the am transcriptome of 9 individuals ' am using rna - seq ( not shown ) . while il-23r has been reported to be expressed by am in sarcoidosis , we did not find any evidence of il-23r expression by am in normal nonsmokers , hiv1 smokers with early emphysema , or hiv1 individuals . this conclusion was based on an extensive evaluation of il-23r gene expression ( microarray analysis , rna sequencing , and taqman rt - pcr ) and flow cytometric analysis of il-23r expression on the am . by flow cytometric analysis of freshly isolated cells from bal , il-23r was not identified in a gated population of am , but in a small population of lymphocytes gated by size , il-23r staining was positive as shown in a single - specimen representative analysis ( figure 5(a ) ) . the absence of il-23r on the surface of am implies that the mechanism of il-23 induced upregulation of mmp-9 is not a consequence of a direct stimulation of il-23r on the surface of am , since il-23r is present neither on am of hiv1 individuals nor on am of smokers with low dlco independent of their hiv1 status . the response of am to il-23 was further evaluated by enrichment of am in the cultures . antibodies targeting cd68 , a glycoprotein expressed on macrophages but not on lymphocytes , attached to magnetic beads were used to positively select cd68 + am . after detaching the beads , purified am were plated on plastic and stimulated with il-23 in parallel with am / lymphocyte cocultures obtained by the adherence method . am purified by immunomagnetic bead separation showed no mmp-9 upregulation upon stimulation with il-23 , in contrast to the effect clearly observed in the am / lymphocytes obtained by adherence ( figure 5(b ) ) . these data indicate that the downstream effects of il-23 on mmp-9 upregulation are dependent upon nonmacrophage cells residing in the environment . in order to assess the relative proportion of am in the am / lymphocyte cocultures obtained by adherence , we analyzed the cultures via flow cytometry and found ( 1 ) the proportion of am was high , similar to or the same as in published literature ( > 97% ) ; ( 2 ) a small , but clearly identifiable population of lymphocytic cells was present in the cultures ( not shown ) . the largest proportion of the nonadherent cells are t lymphocytes as confirmed by positive cd2 and cd4/cd8 staining ( data not shown ) . memory t cells are known to express il-23r and to be the primary target for the th17 modulating effect of il-23 [ 40 , 4244 ] . using il-23r antibody for labeling the cells from bal , we observed that il-23r is expressed in a population of lymphocytes present in hiv1 and hiv1 smokers with early emphysema . we propose that the effect of il-23 on cocultures of am / lymphocytes involves t cells as the primary targets for il-23 . to further assess whether cells other than am contribute to mmp-9 upregulation , we evaluated the effects of phytohemagglutinin ( pha ) , a lectin that acts as a specific mitogen for t lymphocytes , on am / lymphocyte cocultures mmp-9 expression . am / lymphocyte cocultures treated with pha alone do not upregulate mmp-9 . am / lymphocyte cocultures stimulated with both il-23 and pha had increased mmp-9 expression in comparison to am / lymphocyte cocultures stimulated with either pha or il-23 alone ( p < 0.05 , kruskall - wallis , figure 6(a ) ) . based on these data and the fact that t cell - related chemokine levels were elevated in the elf of hiv1 smokers with low dlco ( figure 1 ) , we investigated the effect of adding additional t cells on the release of mmp-9 by am / lymphocyte cocultures stimulated with il-23 . t cells were isolated from bal fluid via rosetting with neuraminidase - treated sheep rbcs . am / lymphocyte cocultures obtained by adherence were treated with il-23 in the presence or absence of purified t cells from the bal and the resulting mmp-9 expression was analyzed via taqman rt - pcr . addition of purified t cells from bal to am / lymphocyte cocultures leads to a significantly increased expression of mmp-9 ( n = 6 , p < 0.01 , anova , figure 6(b ) ) . since mmp-9 is produced by blood t cells , we compared the gene expression of mmp-9 in two cell types used in this experiment , am and bal t cells purified via cd2-labeled magnetic beads . while bal t cells did produce some mmp-9 , there was greater than 1 log higher mmp-9 expression in am in comparison to alveolar t cells ( p < 0.05 , wilcoxon signed - rank test , figure 6(c ) ) . in addition , the t cell effect on mmp-9 upregulation is unlikely to be based on t cell mmp-9 expression , because as the percentage of t cells in am cultures was increased from 5% to 10% and to 40% of total cells mmp-9 expression decreased ( figure 6(d ) ) . thus , higher percentages of t cells in cultures of am at the time of harvesting rna may have simply diluted the overall mmp-9 expression in these cultures as t cells express lower levels of mmp-9 mrna in comparison to am as discussed above . alternatively , t cells may exert a biphasic effect on am mmp-9 expression . while we can not exclude a possible contributing role from other cell types such as nk cells or neutrophils , these results show that t cells , while not a major direct contributor to the expression of mmp-9 , may represent an important target for il-23 and may significantly affect overall upregulation of mmp-9 in am - t - lymphocyte cocultures . taken together , these observations suggest that , upon stimulation with il-23 , t cells interact with am leading to the upregulation of am mmp-9 via a t lymphocyte - am interaction ( figure 7 ) . increased am and elf mmp-1 and mmp-9 expression have been linked to emphysema in humans and in animal models [ 7 , 22 , 38 , 46 , 47 ] . were the first to observe increased lung tissue expression of mmp-9 in autopsies of hiv1 individuals with emphysema who died of aids . smokers with emphysema have both upregulated am expression of mmps and upregulated and activated mmps in elf compared to hiv1 smokers with emphysema . hiv1 individuals are also known to have increased lung cytokine expression [ 5 , 7 , 912 ] . since several cytokines can upregulate monocyte - derived macrophage mmp expression , it is not unreasonable to hypothesize that these observations may all be directly related [ 22 , 4547 ] . our cytokine array data indicating substantial upregulation of lung cytokines in hiv1 individuals are consistent with the broad upregulation of elf cytokines observed by others [ 912 ] . interestingly , in the elf cytokine profiles , hiv1 smokers with low dlco displayed detectable th17 related cytokines as a common feature with hiv1 nonsmokers and hiv1 smokers with low dlco . this increase is not a feature of the cytokine profile of hiv1 healthy nonsmokers or hiv1 healthy smokers . the presence of a group of proinflammatory cytokines in the elf of hiv1 nonsmokers which are similar to the cytokine profile in the elf of hiv1 smokers with low dlco likely plays an important role in the premature development of emphysema in hiv1 individuals who smoke . the observation of increased th17 cytokines in the elf of hiv1 smokers with low dlco is not surprising , as there is emerging evidence of the relevance of th17 cellular responses in lung pathology [ 13 , 4951 ] . the th17 cellular immune response has been described with recent modification of the th1/th2 paradigm based on the identification of il-23 and il-17 involvement in autoimmune diseases [ 44 , 5254 ] . th17 cells , characterized by the release of effector - cytokines il-17 , il-22 and the presence of regulatory - cytokines such as il-23 , are functionally distinct from th-1 cells and th-1-related cytokines such as il-12 and ifn- . depending on the timing , the tissue , and the local microenvironment , il-17-secreting cells appear to play both beneficial and detrimental roles in lung immunity and disease . th17 cells may have a protective role in host defenseagainst certain extracellular bacteria and fungi [ 44 , 51 , 55 , 56 ] . th17 cells are localized at the mucosal level , including the gut and lung mucosa , and their cytokine production adds to the first line of defense against infectious agents . th17 cells and related effector cytokines may also play a prominent role in the pathogenesis of inflammatory lung diseases , where they are hypothesized to promote tissue destruction [ 50 , 51 ] . given the significant increase in th17 related cytokines in stable copd , it has been inferred that th17 cellular responses may be driving the chronic inflammation seen in copd [ 13 , 58 ] . mice exposed to cigarette smoke exhibit enhanced il-17 production , while il-17 receptor knockout mice are protected from emphysema induced by cigarette smoke exposure . patients with stable copd exhibited elevated numbers of il-22 and il-23 positively staining cells in the bronchial epithelium and il-17 positive cells in the submucosa . in ex - smokers with mild to moderate copd , the number of il-17 cells in the bronchial submucosa is increased compared with nonsmoking control subjects . in the same study , a significant , albeit small , increase in sputum il-17 has been correlated with airflow obstruction and lung function impairment , but not airway inflammation . th17 cells are also relevant in hiv1 infection as deterioration of the th17 lineage may correlate to the rapid progression of the disease in infected individuals . in hiv1 infection , th17 cells are depleted from the gut but remain present in blood and lung . increased susceptibility of hiv1-infected individuals to opportunistic pathogens targeting the respiratory tract such as pneumocystis carinii and m. tuberculosis for which th17 cells exhibit antigenic specificity suggests that a dysfunction of th17 cellular responses may correlate with progression of the disease [ 17 , 62 , 63 ] . il-23 is one of the key cytokines characterizing the th17 immune response and it represents an important link between the innate and adaptive immune responses [ 44 , 64 ] . it is a heterodimeric molecule consisting of the p40 subunit shared in common with il-12 , combined with a unique p19 subunit . while both cytokines can induce ifn- expression in cd4 + cells , il-23 , although not crucial for th17 differentiation , plays an important role in maintaining th17 effector function . il-23 is expressed mostly by activated macrophages and dendritic cells while its receptor , il-23r ( the receptor for the p19 subunit ) , is expressed in memory t cells of the th17 subtype . il-23 deregulation may lead to inability to adequately protect the organism from certain pathogens while its overexpression may be linked to various autoimmune phenomena [ 58 , 67 ] . il-23 gene expression is increased in patients with copd as compared to nonsmokers and , in an animal model , cigarette smoking increases the lung expression of il-23 . increased levels of il-23 correlate with increased levels of mmps as detected in mouse models such as tlr4 knock - out mice and in human peripheral blood of patients with relapsing - remitting multiple sclerosis . in support of this concept , intranasal administration of il-23 protein to mice resulted in increased lung mmp-9 , a protease linked to the influx of inflammatory cells into the lung in copd as well as the destruction of lung tissue in emphysema [ 21 , 63 ] . based on this background , we hypothesized that , in the lower respiratory tract in hiv1 smokers , increased il-23 is acting on t - lymphocytes to induce increased am mmp-9 gene expression and consequently elf mmp-9 protein levels . proinflammatory cytokines such as il-1 , tnf- , and il-6 are well known to be inducers of mmp-9 expression in monocyte - derived macrophages [ 47 , 70 , 71 ] . data addressing the effect of il-23 on production of mmps are very limited . il-23 has previously been shown to upregulate mmp-2 in vivo in the ileum of mice infected with toxoplasma gondii . more specifically for the lung , intranasal application of il-23 in a mouse model increased mmp-9 levels in elf and induced il-17 mrna expression . our data support the hypothesis that increased elf levels of il-23 may be responsible , at least in part , for the increased elf mmp-9 in vivo . a limitation of our study is the fact that the observed increase in mmp-9 in the lungs of hiv1 smokers with low dlco may be a consequence of increased il-23 in the elf , but causality has been proven only in vitro . memory t cells are known to express il-23r and to be the primary target for the th17 modulating effect of il-23 [ 40 , 42 , 44 ] . we propose that the effect of il-23 on cocultures of am / lymphocytes involves t cells as the primary targets for il-23 . our observations suggest that , upon stimulation with il-23 , t cells interact with am leading to the upregulation of am mmp-9 . there are several possible mechanisms that might explain this relationship between am and t cells . given its identification in elf in the same phenotypes in which il-23 is present , ifn- was evaluated as a potential target cytokine . while ifn- is indeed upregulated in t cells upon il-23 stimulation consistent with previously reported data and confirmed by our in vitro experiments , ifn- does not induce upregulation of mmp-9 in human am ( not shown ) . in addition , we were unable to passively transfer the effect on am mmp-9 upregulation by adding conditioned media from t cell cultures stimulated with il-23 . the relevance of the t - lymphocyte / monocyte interaction for the induction of mmp-9 expression has been described [ 6 , 74 , 75 ] . various stimuli induce t - lymphocytes to activate monocytes indirectly via released cytokines and directly via direct cellular contact that may include antigen recognition on antigen specific t cell clones , cross - linking of surface receptors , or membrane - associated cytokines . in addition to membrane - associated cytokines , other surface molecules have been assessed for their ability to activate monocytes upon contact with stimulated t - cells , for example , leukocyte function antigen- ( lfa- ) 1/intercellular adhesion molecule-1 , cd2/lfa3 , cd40/cd40l , and lymphocyte activation - antigen-3 . cd40 is a costimulatory protein found on antigen presenting cells and is required for their activation . the binding of cd154 ( cd40l ) on th cells to cd40 activates antigen presenting cells and induces a variety of downstream effects . cd40-ligand ( cd-40l ) is a member of the tumor necrosis factor ( tnf ) superfamily and activated cd4 + t cells are the predominant cd40l expressing population . il-23 administration to peripheral blood t cells in vitro upregulated cd40l expression ( not shown ) . one possibility is that this upregulation of cd40l may lead to enhancement of the direct t cell - am activation which may further induce the upregulation of mmp-9 in am . this effect is indirect and requires both am and t lymphocytes . taken together , these data suggest that the th17 pathway may play a role in the early development of emphysema in hiv1 individuals .

rationale . matrix metalloproteinase-9 ( mmp-9 ) expression is upregulated in alveolar macrophages ( am ) of hiv1 + smokers who develop emphysema . knowing that lung epithelial lining fluid ( elf ) of hiv1 + smokers contains increased levels of inflammatory cytokines compared to hiv1 smokers , we hypothesized that upregulation of lung cytokines in hiv1 + smokers may be functionally related to increased mmp-9 expression . methods . cytokine arrays evaluated cytokine protein levels in elf obtained from 5 groups of individuals : hiv1 healthy nonsmokers , hiv1 healthy smokers , hiv1 smokers with low diffusing capacity ( dlco ) , hiv1 + nonsmokers , and hiv1 + smokers with low dlco . results . increased levels of the th17 related cytokine il-23 were found in hiv1 smokers with low dlco and hiv1 + smokers and nonsmokers . relative il-23 gene expression was increased in am of hiv1 + individuals , with greater expression in am of hiv1 + smokers with low dlco . infection with hiv1 in vitro induced il-23 expression in normal am . il-23 stimulation of am / lymphocyte cocultures in vitro induced upregulation of mmp-9 . lung t lymphocytes express receptor il-23r and interact with am in order to upregulate mmp-9 . conclusion . this mechanism may contribute to the increased tissue destruction in the lungs of hiv1 + smokers and suggests that th17 related inflammation may play a role .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions

PMC1747179

consequently , signal transduction pathways are vital to an organism 's survival and function . in microorganisms , they control the majority of cellular functions including chemotaxis , respiration , development , osmoregulation , transport , metabolism , virulence , host - recognition and antibiotic resistance . such signal transduction pathways behave as information processing circuits that link input ( stimulus nutrients , temperature , redox , etc . ) and output ( regulatory response gene expression , enzyme activity , flagellar motor switch , etc . ) the detection of a signal ( input ) and coupling this with an adaptive cellular response ( output ) is common to all signal transduction systems ; however , microorganisms employ diverse mechanisms for linking these events . these range from single - domain transducers to several interacting proteins and multi - protein complexes . the most widely recognized signaling systems are the so - called two - component systems that utilize protein phosphorylation as the fundamental signaling mechanism ( 2,3 ) . the prototypical two - component system consists of two proteins : a membrane - bound , sensor histidine kinase and a cytoplasmic , response regulator . the sensor kinase detects environmental signals via one or more , amino - terminal sensory domains . subsequently , the sensor kinase undergoes a conformational shift that results in an atp - dependent autophosphorylation of a conserved histidine residue within its carboxy - terminal , transmitter domain . the cognate response regulator then catalyzes the transfer of this phosphoryl group to a conserved aspartate residue within its amino - terminal , receiver domain . phosphorylation of the receiver domain activates the response regulator 's output domain(s ) and effects a particular adaptive response typically regulation of gene expression at the transcriptional level ( 1 ) . despite the importance of two - component systems , most signal transduction events in prokaryotes are carried out by one - component systems that consist of a single protein molecule containing both input and output domains but lacking the phosphotransfer domains typical of two - component systems ( 4 ) . many one - component systems have been extensively studied including the laci lactose operon repressor ( 5 ) and the catabolite activator , cap , of escherichia coli ( 6 ) ; the arginine catabolism regulator , rocr , of bacillus subtilis ( 7 ) ; and the quorum - sensing regulator , trar , of agrobacterium tumefaciens ( 8) . these proteins bind ligands via their input domain and regulate gene expression with their output domain . most one - component systems typically consist of at least one input and one output domain , yet in some cases a single domain is capable of both detecting a ligand and producing a regulatory response . for example , both the metalloregulators , czra of staphylococus aureus ( 9 ) and cmtr of mycobacterium tuberculosis ( 10 ) , consist of a single domain , which directly binds dna in response to metal ligands . one - component systems are more widely distributed among bacteria and archaea , and display a greater diversity of domains than two - component systems ( 4 ) . the domains comprising these proteins may be categorized into four major types according to their function : input , transmitter , receiver and output ( 1 ) . relatively few input domains specific to signal transduction have been characterized and include the following : pas ( 11,12 ) , gaf ( 13 ) , cache ( 14 ) , chase ( 15,16 ) , chase2 through chase6 ( 17 ) , nit ( 18 ) and 4hb_mcp ( 19 ) . these domains exhibit extreme sequence variability due to the broad range of signals they detect and are the least conserved of all the signaling modules . in contrast , transmitter and receiver domains display remarkable sequence conservation , which reflects the conserved phosphorylation reaction between these domain types that links sensor kinases and response regulators ( 20 ) . helix ( hth ) domains because the predominant adaptive response of two- and one - component systems is regulation of gene expression ( 4 ) . several novel output domains have been recently described in response regulators , which implicate these systems in other types of control , such as the regulation of enzyme activity . these include adenylate and diguanylate cyclases , c - di - gmp - phosphodiesterase , phosphohydrolase and other related domains ( 2123 ) . output domains are more conserved than input domains yet still considerably divergent due to their moderate number of regulatory roles ( e.g. gene - regulation , protein protein interactions , etc . ) . one of the first efforts to catalog signal transduction proteins was the sentra database ( 24 ) . it contains information on classical two - component systems and a few other signaling systems that interact via phosphorylation or methylation reactions . currently , sentra includes information on two - component signal transduction proteins for 43 genomes . several other databases attempt to document the signal transduction machinery within various genomes ; however , these projects are usually a part of a larger initiative and therefore limited in scope and/or accuracy . for example , the kegg project ( 25 ) focuses on deriving higher - order information ( e.g. pathways ) from genomic data and has successfully mapped metabolic pathways across multiple genomes , yet the curators have recently begun mapping some regulatory pathways including signal transduction . kegg transfers pathway information among genomes by first defining reference pathways based on scientific literature and then computationally extending these networks across genomic data via orthologous relationships established from sequence similarity searches and the positional correlation of genes . this approach is constrained to experimentally defined pathways and does not provide a complete record of the signal transduction repertoire . in addition , kegg defines orthologous relationships using bi - directional best hits with respect to the entire protein sequence this poorly ascertains related signal transduction proteins , given their highly modular nature and extreme sequence variability . model organism databases such as the e.coli ecocyc ( 26 ) and b.subtilis dbtbs ( 27 ) often contain information about signal transduction for their associated organisms , since these are primarily restricted to literature - based curation efforts and therefore limited in coverage ( albeit with high accuracy ) . finally , as one - component systems have only recently been recognized as a major part of signal transduction ( 4 ) , most database resources when describing signal transduction contain only information on two - component systems . we have developed a novel resource , the mist database that contains a comprehensive compilation of the signal transduction proteins within bacterial genomes . as these proteins are modular in nature , our approach focuses on determining signal transduction proteins from a protein 's domain composition , which we derive using the hmmer software ( 28 ) . from the pfam ( 29 ) and smart ( 30 ) domain libraries , we have designated a set of profile hidden markov models ( hmm ) that represent signaling domains ( e.g. hatpase_c transmitter domain or dna - binding output domains ) and implicate a role in signal transduction . we classify a protein as belonging to signal transduction if it contains one or more of these specific signaling domains . in this manner , we systematically produce the repertoire of signal transduction proteins for microbial genomes ( or any other collection of protein sequences ) . to our knowledge , signal transduction is the only current resource on signal transduction in prokaryotes that provides a thorough catalog of both two- and one - component systems within bacterial genomes . the biological function of a signal transduction protein is determined by specific signaling domains that detect an environmental cue ( input ) , mediate protein protein communication ( transmitter , receiver ) , or initiate a cellular response ( output ) . thus , domains that perform these roles serve as markers and facilitate the identification of signal transduction proteins given two conditions : ( i ) an adequate mechanism for detecting domains in an amino acid sequence and ( ii ) a comprehensive set of domains known or predicted to participate in signal transduction . protein domains may be robustly represented by profile hmms ( 28 ) , which statistically model the primary structure of homologous domain sequences and enable their rapid identification from a protein sequence with tools such as the hmmer software package . furthermore , domain profiles are more sensitive than pairwise sequence comparisons and typically contain manually - curated score thresholds from which the significance and membership of domain matches may be automatically evaluated . to meet the second requirement , we selected 133 signaling domains ( supplementary table s1 ) from the pfam ( 29 ) and smart databases ( 30 ) based on the following : ( i ) known domain function ( information from pfam , smart , interpro ( 31 ) , cog ( 32 ) resources and analysis of literature on signal transduction ) , and ( ii ) predicted domain function based on the association with other signaling domains . mist incorporates a straightforward and systematic methodology for identifying signal transduction proteins within completely sequenced bacterial genomes ( figure 1 ) . perl scripts download all available genomes from ncbi in the xml ( extensible markup language ) format and load this information into the database . each xml file contains the full refseq annotation ( 33 ) for a genome , which includes its associated nucleotide data , genes and translated proteins . secondly , we predict the domain architecture for each protein including signal peptides , transmembrane regions , coiled - coils and low - complexity segments . finally , we identify the set of putative signal transduction proteins by scanning each protein for the presence of transmitter , receiver or output signaling domains ( see above ) . input domains are often found in pathways other than signal transduction ( e.g. metabolic pathways ) and therefore proteins identified solely from an input domain are not classified as belonging to signal transduction . during this process , we also filter out various domain combinations that indicate a role other than signal transduction . for example , due to structural similarities , dna topoisomerase iv often contains a predicted hatpase_c transmitter domain ( pfam accession no . sm00387 ) in addition to other domains suggestive of topoisomerase activity ( dna_gyraseb and dna_gyraseb_c ) , yet this protein is not involved in signal transduction . this pipeline may be executed on a regular schedule such that new genomes are seamlessly integrated into the database and signal transduction blueprints for these organisms are automatically generated . mist is updated on a monthly basis to maintain a current record of the signal transduction repertoire within newly sequenced genomes . first , all complete , bacterial genomes are downloaded from ncbi and loaded into the mist database . finally , a protein is classified as belonging to signal transduction if it contains at least one transmitter , receiver , or output signaling domain . the mist database is implemented using the postgresql ( ) version 8.1.3 relational database management system on the gentoo distribution ( ) of the linux / gnu operating system . all protein domain architectures are derived on our 34-node linux cluster using hmmer version 2.3.2 ( 28 ) and the pfam version 19.0 ( 29 ) and smart version 5.0 ( 30 ) domain libraries . every year , the domain architectures and signal transduction predictions will be updated based on the latest releases of the pfam and smart databases . phobius version 1.01 ( 34 ) is used to predict signal peptides and transmembrane regions . coiled - coils and regions of low - complexity are predicted with the coils version 2.2 ( 35 ) and seg ( 36 ) programs , respectively . php and cgi scripts running on the apache web server version 2.0.58 ( ) deliver web content to users via the internet . as of august 2006 , mist version 1.0 contains 69 720 predicted signal transduction proteins22 868 proteins that belong to two - component regulatory systems and 46 402 one - component regulators from 365 bacterial and archaeal genomes . excluding chemotaxis proteins , there are almost equal numbers of sensor kinases and response regulators : 9317 ( 51.8% ) and 9025 ( 49.2% ) , respectively . these display a strong , positive , linear relationship ( figure 2 ) . using several chemotaxis - related domains from the pfam database , we found 4526 chemotaxis proteins , out of which 328 are putative class ii histidine kinases ( chea - like ) ( 37 ) . mist contains more than twice as many one - component proteins as two - component proteins . when considering the number of systems based on their output activity , the number of one - component systems exceeds two - component systems by a factor of five . this is slightly higher than our initial analysis of the signaling transduction proteins derived from 145 prokaryotic genomes ( 4 ) . the taxonomic distribution of two- and one - component systems within the mist database is given in supplmentary table s2 . scatterplot showing the linear , positive relationship ( r = 0.92 ) between the number of predicted histidine kinases and response regulators within representative bacterial genomes ( excluding chemotaxis proteins ) . in addition to specific information about the signal transduction repertoire for each microbial genome , mist stores the large amount of pre - computed data used to identify this class of proteins . this includes any predicted pfam and smart domains , protein secondary features ( e.g. transmembrane regions , signal peptides , coiled - coils , low - complexity regions ) , chromosomal position of the corresponding gene , statistical information and the complete refseq annotation . we have also indexed the ncbi non - redundant database to provide cross - referencing support . therefore , while mist focuses primarily on the classification and analysis of signal transduction proteins , this information along with its web interface ( see below ) enables users to effectively use mist as a general resource for exploring microbial genomes . to provide an effective means for exploring and utilizing the mist database , we designed and implemented a user - friendly web interface oriented toward experimental biologists ( ) . a separate page for each microbe displays both general and specific information about its signal transduction repertoire including : descriptive data about the genome and proteome ( e.g. size , replicons , etc . ) , a graphical representation of its signal transduction profile , querying options and a summary table of the signal transduction protein counts for each replicon ( figure 3 ) . the profile represents an overall view of the number of signaling domains separated into functional categories . for example , e.coli o157:h7 contains 272 dna - binding domains , clearly indicating a substantial degree of gene - regulation . each of the headings and signal transduction counts within the summary table are linked to the particular slice of data it represents . clicking on a replicon displays the signal transduction proteins associated with that replicon . selecting the heading two - component proteins displays all the predicted two - component proteins for this organism . clicking on a signal transduction count the resulting list for each protein contains genbank identifier ( gi ) number , refseq description , set of input and/or output domains and visualized domain architecture . following an individual protein link provides an expanded overview of that particular protein and its associated gene . in addition to basic sequence data , this page contains the full protein / gene refseq annotation , domain architectures and graphical view of the gene neighborhood . the domain architecture section presents a linear representation of the protein 's domain architecture and other secondary features ( e.g. transmembrane regions , low - complexity regions ) . each arrow is hyperlinked to its respective gene / protein page for evaluating neighboring genes . queries can be made with various search types and terms ( upper right corner ) . relevant data may be accessed in several ways : ( i ) choosing an organism from the complete list of bacteria in mist , ( ii ) searching for a particular organism , or ( iii ) searching by a genbank gi number . in addition , multiple species may be selected and simultaneously searched using a sophisticated , taxonomy - driven , selection tool . users may query for particular pfam or smart domains , keyword , locus , gi number or internal mist identifier . using sensitive profile hmm searches and utilizing the available knowledge on microbial signal transduction from the literature and on - line resources , we were able to identify more than 69 000 proteins that are predicted to comprise signal transduction networks . we have developed a comprehensive database , which captures , stores and makes this information available to the scientific community . we expect mist to become a widely - used resource significantly enhancing our knowledge within this important area of research as well as improving current genome annotation and contributing to important medical applications , such as antimicrobial drug development . future mist development will focus on incorporating user- and literature - based curation , go ontology driven annotations ( 38 ) , addition of new domain models , mapping functional residues , pathway support and improvements to the web interface .

signal transduction pathways control most cellular activities in living cells ranging from regulation of gene expression to fine - tuning enzymatic activity and controlling motile behavior in response to extracellular and intracellular signals . because of their extreme sequence variability and extensive domain shuffling , signal transduction proteins are difficult to identify , and their current annotation in most leading databases is often incomplete or erroneous . to overcome this problem , we have developed the microbial signal transduction ( mist ) database ( ) , a comprehensive library of the signal transduction proteins from completely sequenced bacterial and archaeal genomes . by searching for domain profiles that implicate a particular protein as participating in signal transduction , we have systematically identified 69 270 two- and one - component proteins in 365 bacterial and archaeal genomes . we have designed a user - friendly website to access and browse the predicted signal transduction proteins within various organisms . further capabilities include gene / protein sequence retrieval , visualized domain architectures , interactive chromosomal views for exploring gene neighborhood , advanced querying options and cross - species comparison . newly available , complete genomes are loaded into the database each month . mist is the only comprehensive and up - to - date electronic catalog of the signaling machinery in microbial genomes .

INTRODUCTION HIGH-THROUGHPUT IDENTIFICATION OF SIGNAL TRANSDUCTION PROTEINS DATABASE CONTENTS WEB INTERFACE CONCLUSIONS AND FUTURE WORK SUPPLEMENTARY DATA

PMC4027930

osteoarthritis ( oa ) is among the diseases with the highest rates of comorbidity.1,2 comorbidity can be defined as any distinct additional clinical entity that has existed or that may occur during the clinical course of a patient who has the index disease under study.3 common comorbidities in oa include cardiovascular diseases , diabetes , obesity chronic obstructive pulmonary disease ( copd ) , chronic pain , depression , and visual and hearing impairments.4 comorbidity in older adults with oa is associated with more pain , greater limitations in daily activities , and a worse prognosis with respect to these limitations.5,6 performing exercises is one of the key recommendations in current guidelines for the management of knee oa;7,8 this has been found to relieve pain and to reduce activity limitations.9 comorbidity may interfere with the application of exercise therapy in oa , however;10 for example , in persons with heart failure , only moderate - intensity resistance training is recommended , and the last repetitions should not be straining.11 furthermore , the warming - up and cooling - down sessions should be prolonged ; perceived exertion and/or dyspnea scales should take precedence over heart rate and work rate targets ; and isometric exercises should be avoided.12 because comorbidities have a significant influence on prognosis6 and may influence treatment , they should be routinely taken into account.13 unfortunately , there is no evidence - based protocol available for the treatment of patients with knee oa and comorbidity.14 current oa guidelines do not offer specific recommendations concerning comorbidity - associated exercise adaptations.7,8,15 it is often not feasible to combine different disease - specific treatment guidelines , since one treatment might interact negatively with another treatment or affect the natural course of a coexisting disease.16 furthermore , in clinical practice , older adults with knee oa and ( severe ) comorbidity are seldom referred for exercise therapy ; often drop out at an early stage of the treatment ; or may be treated inadequately ( eg , therapists may reduce the intensity of treatment to an ineffective level ) . when dealing with comorbidity , a patient - centered rather than a disease - oriented approach , in which the process of decision making should be based on clinical reasoning , is preferred.16 the hypothesis - oriented algorithm for clinicians ( hoac ) ii17 describes a framework for clinical decision making in physical therapy ; it addresses examination , evaluation , diagnosis , prognosis , and intervention in a specific patient . although the hoac ii gives general direction in clinical reasoning , specific advice concerning comorbidity - adapted oa exercise therapy and comorbidity is not available in the literature . therefore , there is a need for comorbidity - adapted protocols for exercise therapy in older adults with knee oa and comorbidity . these protocols are expected to improve the application of oa - specific exercise therapy , may help to avoid adverse events , and may improve the outcome of exercise therapy . the evaluation of complex interventions requires a phased approach , because of specific difficulties in developing , identifying , documenting , and reproducing the intervention . to design a complex intervention , we used the medical research council ( mrc ) framework , which was developed to help researchers to define clearly where they are in the research process.18 the framework describes four phases in the design and evaluation of complex interventions : the preclinical or theoretical phase ; phase i , or the modeling phase ; phase ii , or the exploratory trial ; and phase iii , or definitive randomized controlled trial . in the preclinical or theoretical phase , the evidence that the intervention might have the desired effect is identified . the theoretical basis for the intervention is reviewed and potentially active ingredients are identified . in phase i , or the modeling phase , the components of the intervention are defined and tested , using qualitative techniques ( eg , case studies ) . in phase ii , or the exploratory trial , the optimum intervention is developed , based on the information gathered in phase i. phase iii consists of the definitive randomized controlled trial , and phase iv the long - term implementation of the intervention.18 in a previous study , restrictions and contraindications for exercise therapy for patients with knee oa and comorbidity ( theoretical phase ) were identified in the literature.10 the purpose of the present study was to develop comorbidity - adapted exercise protocols for older adults with knee oa and comorbidity ( phase i , modeling phase ) . first , based on previous work,4 we selected comorbidities in oa that 1 ) are common ( present > 5% ) , and 2 ) have impact on pain and/or affect daily functioning . the following comorbidities were selected : cardiac diseases ; hypertension ; type 2 diabetes ; obesity ; copd ; low back pain ; chronic pain ; depression ; and visual or hearing impairments.4 second , a literature search in the pubmed ( publication date range 19662009 ) database was conducted to make an inventory of restrictions and contraindications for exercise therapy in patients with oa of the knee and highly prevalent comorbidities . the method and the results of this search based on the results of the first two steps , comorbidity - related adaptations to the diagnosis and treatment of oa were described . guidelines on exercise therapy in each comorbidity ( eg , cardiac disease , diabetes , copd , and nonspecific low back pain ) were consulted.1923 if there was no exercise therapy guideline available for a specific comorbidity , an available medical guideline was used ( eg , guidelines for depression or hypertension).24,25 the principles described in these guidelines were incorporated into the adapted protocols for exercise therapy in oa of the knee . fourth , the preliminary versions of the protocols were discussed with clinical experts in the fields of each comorbid disease and , subsequently , based on their feedback , further improved . the experts had extensive experience in the fields of cardiac rehabilitation , diabetes , copd rehabilitation , chronic nonspecific pain , and visual and hearing impairments . advice was sought on the treatment of each comorbidity and on how the principles of exercise therapy and training of the comorbid diseases should be incorporated into the exercise regimen for oa of the knee . after optimizing the protocols , the clinical experts were consulted again for the collection of feedback and to gain final consensus on the protocols . fifth , the draft protocols were field - tested in a pilot study in patients with knee oa and the target comorbidities . thereafter , the protocols were further improved , based on the feedback from therapists and patients , leading to a final version of the protocols . the method for field - testing of the protocols in this pilot study participants were referred to our rehabilitation center by their general practitioner because of persistent knee problems . physical measurements were carried out by a research assistant and questionnaires were filled out by the participants . the study was approved by the medical ethical review board of the slotervaart hospital and reade , amsterdam , the netherlands . all participants gave written informed consent and the study was conducted in accordance with the handbook for good clinical research practice of the world health organization and declaration of helsinki principles.26 fourteen participants were recruited . inclusion criteria were : 1 ) diagnosis of knee oa according to the clinical american college of rheumatology criteria;27 2 ) presence of at least one of the target comorbidities , ie , coronary disease , heart failure , hypertension , type 2 diabetes , obesity , copd , chronic pain , nonspecific low back pain , depression , and vision and/or hearing impairment ( diagnosed by a medical specialist ) ; 3 ) severity score 2 of the comorbidity on the cumulative illness rating scale,28 indicating that the comorbidity has an impact on daily activities ; and 4 ) the primary treatment goal should be oa related ( instead of comorbidity related ) . exclusion criteria were : 1 ) indication for total knee replacement ; 2 ) inability to participate in treatment , eg , due to transport problems ; 3 ) insufficient capacity in the dutch language . the protocols were applied and evaluated by three qualified physical therapists with extensive experience ( 3 , 8 , and 12 years ) in knee / hip rehabilitation oa treatment . in addition , two of the three therapists were members of the committee for hip and knee oa guideline development for the royal dutch society of physical therapy . to evaluate the treatment process , the therapists completed a weekly registration form , providing information about the duration of the treatment period , content of the treatment , adaptations in the treatment due to the comorbidity , and any problems encountered in applying the protocols . adverse events , defined as any undesirable experience occurring in a subject during the study ( regardless of whether or not this was related to the treatment ) , were registered . to evaluate the feasibility of the protocols , semi - structured interviews were held by the first author ( mdr ) along with therapists and participants . topic lists were used to structure the interview ( see table 1 ) . to evaluate patient outcome after treatment , performance - based tests were performed and self - reported questionnaires were filled in by participants at baseline and directly after treatment . the western ontario and mcmaster universities osteoarthritis index ( womac ) is a disease - specific , self - administered questionnaire , developed to study patients with hip and knee oa.29,30 the womac consists of 24 questions grouped into three subscales ( pain : five questions ; stiffness : two questions ; and physical function : 17 questions ) and scaled in a 5-point likert ( lk ) scale . the maximum score in the lk scale is 20 points for pain , 8 points for stiffness , and 68 points for physical function . the womac is widely used in clinical research , and has been shown to be reliable , valid , and responsive for use in patients with oa.2931 the patient - specific functioning scale was used to evaluate limitations in activities of the individual patient.32 patients were asked , which activities do you perceive as important and were hampered by knee pain during the last week ? a list of activity suggestions was offered to support recall , and patients were allowed to provide other limited activities that were not on the list . of these activities , the difficulty of performance of the main activities were scored by self - assessment on a numeric rating scale ( [ nrs from 010 ] 0= no problems to perform the activity ; 10= impossible to perform the activity ) . patient - specific functional scale is an efficient and valid measure for assessing limitations in activities and change in limitations in persons with knee dysfunction.32 the get up and go test33,34 was performed with subjects seated on a high standard chair ( seat height 49 cm ) . the subjects were instructed to stand up without the help of the arms on the command go and walk 15 m along an unobstructed corridor as fast as possible without running . patients who normally used walking devices were allowed to use them during the test . a longer time taken to perform the test the total distance covered in meters during 6 minutes of walking was scored.35,36 quality of life was assessed with the 36-item short - form health survey ( sf-36).37 the sf-36 is a widely applied generic instrument for measuring health status and consists of eight dimensions : physical functioning , social functioning , physical role , emotional role , mental health , energy , pain and general health perception . the sf-36 gives scores on a 0100 scale , with higher scores indicating better health . the reliability ( median reliability coefficient 0.85 for all subscales ) for the sf-36 has been established,3840 and its validity has been shown in an elderly population , in which the instrument distinguished between those with and without poor health.41 psychological functioning was assessed with the hospital anxiety and depression scale ( hads).42 the hads is a self - report rating scale of 14 items on a 4-point lk scale ( range : 03 ) . it is designed to measure anxiety and depression ( seven items for each subscale ) . the total score is the sum of the 14 items , and , for each subscale , the score is the sum of the respective seven items ( ranging from 021 ) . the hads is widely used in clinical research and has been shown to be reliable , valid , and responsive for use as a screening tool in patients with oa.43 pain was assessed with a subscale of the womac . muscle strength was assessed for flexion and extension of the knee using an isokinetic dynamometer ( enknee ; enraf - nonius b.v . , patients performed a maximum of three test repetitions to measure the strength of the quadriceps and hamstrings for each knee . mean muscle strength per leg was calculated to obtain a measure of overall leg muscle strength ( in nm ) . subsequently , mean muscle strength was divided by the patient s weight to control for the correlation between muscle strength and weight . excellent intra - rater reliability ( intraclass correlation coefficient 0.93 ) has been reported for this measure in knee oa patients.45 at the end of the treatment , patients were asked to rate global perceived effect ( gpe ) of the treatment46 on a scale of 19 , with a score of 1 meaning much better , 5 meaning no change , and 9 meaning much worse . patient satisfaction with the kind of treatment was measured by the nrs ( 010 ) , with higher scores indicating greater satisfaction . additional data recorded were age , sex , and duration of complaints . the weight ( kg ) and height ( m ) were measured in standing position . comorbidity was scored with the cumulative illness rating scale.28 radiographs of the knee were scored using the grading scales proposed by kellgren and lawrence ( k&l).47 in order to evaluate the treatment process , a descriptive analysis of the treatment registration forms was performed . the feasibility of the protocols was evaluated by analyzing the notes that were taken during the interviews with the therapists and participants . a faithful depiction of the experiences of the participants and therapists was achieved by verifying with the participant or therapist whether the remarks were interpreted in a correct way by giving a summary at the end of the interview . to analyze the patient outcomes after treatment , because the data were not normally distributed pre- and posttreatment scores were analyzed with a nonparametric wilcoxon signed - rank test ( p - value < 0.05 ) . first , based on previous work,4 we selected comorbidities in oa that 1 ) are common ( present > 5% ) , and 2 ) have impact on pain and/or affect daily functioning . the following comorbidities were selected : cardiac diseases ; hypertension ; type 2 diabetes ; obesity ; copd ; low back pain ; chronic pain ; depression ; and visual or hearing impairments.4 second , a literature search in the pubmed ( publication date range 19662009 ) database was conducted to make an inventory of restrictions and contraindications for exercise therapy in patients with oa of the knee and highly prevalent comorbidities . the method and the results of this search based on the results of the first two steps , comorbidity - related adaptations to the diagnosis and treatment of oa were described . guidelines on exercise therapy in each comorbidity ( eg , cardiac disease , diabetes , copd , and nonspecific low back pain ) were consulted.1923 if there was no exercise therapy guideline available for a specific comorbidity , an available medical guideline was used ( eg , guidelines for depression or hypertension).24,25 the principles described in these guidelines were incorporated into the adapted protocols for exercise therapy in oa of the knee . fourth , the preliminary versions of the protocols were discussed with clinical experts in the fields of each comorbid disease and , subsequently , based on their feedback , further improved . the experts had extensive experience in the fields of cardiac rehabilitation , diabetes , copd rehabilitation , chronic nonspecific pain , and visual and hearing impairments . advice was sought on the treatment of each comorbidity and on how the principles of exercise therapy and training of the comorbid diseases should be incorporated into the exercise regimen for oa of the knee . after optimizing the protocols , the clinical experts were consulted again for the collection of feedback and to gain final consensus on the protocols . fifth , the draft protocols were field - tested in a pilot study in patients with knee oa and the target comorbidities . thereafter , the protocols were further improved , based on the feedback from therapists and patients , leading to a final version of the protocols . the method for field - testing of the protocols in this pilot study participants were referred to our rehabilitation center by their general practitioner because of persistent knee problems . physical measurements were carried out by a research assistant and questionnaires were filled out by the participants . the study was approved by the medical ethical review board of the slotervaart hospital and reade , amsterdam , the netherlands . all participants gave written informed consent and the study was conducted in accordance with the handbook for good clinical research practice of the world health organization and declaration of helsinki principles.26 fourteen participants were recruited . inclusion criteria were : 1 ) diagnosis of knee oa according to the clinical american college of rheumatology criteria;27 2 ) presence of at least one of the target comorbidities , ie , coronary disease , heart failure , hypertension , type 2 diabetes , obesity , copd , chronic pain , nonspecific low back pain , depression , and vision and/or hearing impairment ( diagnosed by a medical specialist ) ; 3 ) severity score 2 of the comorbidity on the cumulative illness rating scale,28 indicating that the comorbidity has an impact on daily activities ; and 4 ) the primary treatment goal should be oa related ( instead of comorbidity related ) . exclusion criteria were : 1 ) indication for total knee replacement ; 2 ) inability to participate in treatment , eg , due to transport problems ; 3 ) insufficient capacity in the dutch language . the protocols were applied and evaluated by three qualified physical therapists with extensive experience ( 3 , 8 , and 12 years ) in knee / hip rehabilitation oa treatment . in addition , two of the three therapists were members of the committee for hip and knee oa guideline development for the royal dutch society of physical therapy . to evaluate the treatment process , the therapists completed a weekly registration form , providing information about the duration of the treatment period , content of the treatment , adaptations in the treatment due to the comorbidity , and any problems encountered in applying the protocols . adverse events , defined as any undesirable experience occurring in a subject during the study ( regardless of whether or not this was related to the treatment ) , were registered . to evaluate the feasibility of the protocols , semi - structured interviews were held by the first author ( mdr ) along with therapists and participants . topic lists were used to structure the interview ( see table 1 ) . to evaluate patient outcome after treatment , performance - based tests were performed and self - reported questionnaires were filled in by participants at baseline and directly after treatment . the western ontario and mcmaster universities osteoarthritis index ( womac ) is a disease - specific , self - administered questionnaire , developed to study patients with hip and knee oa.29,30 the womac consists of 24 questions grouped into three subscales ( pain : five questions ; stiffness : two questions ; and physical function : 17 questions ) and scaled in a 5-point likert ( lk ) scale . the maximum score in the lk scale is 20 points for pain , 8 points for stiffness , and 68 points for physical function . the womac is widely used in clinical research , and has been shown to be reliable , valid , and responsive for use in patients with oa.2931 the patient - specific functioning scale was used to evaluate limitations in activities of the individual patient.32 patients were asked , which activities do you perceive as important and were hampered by knee pain during the last week ? a list of activity suggestions was offered to support recall , and patients were allowed to provide other limited activities that were not on the list . of these activities , the patient selected three main activities and ranked them in order of importance . the difficulty of performance of the main activities were scored by self - assessment on a numeric rating scale ( [ nrs from 010 ] 0= no problems to perform the activity ; 10= impossible to perform the activity ) . patient - specific functional scale is an efficient and valid measure for assessing limitations in activities and change in limitations in persons with knee dysfunction.32 the get up and go test33,34 was performed with subjects seated on a high standard chair ( seat height 49 cm ) . the subjects were instructed to stand up without the help of the arms on the command go and walk 15 m along an unobstructed corridor as fast as possible without running . patients who normally used walking devices were allowed to use them during the test . a longer time taken to perform the test the total distance covered in meters during 6 minutes of walking was scored.35,36 quality of life was assessed with the 36-item short - form health survey ( sf-36).37 the sf-36 is a widely applied generic instrument for measuring health status and consists of eight dimensions : physical functioning , social functioning , physical role , emotional role , mental health , energy , pain and general health perception . the sf-36 gives scores on a 0100 scale , with higher scores indicating better health . the reliability ( median reliability coefficient 0.85 for all subscales ) for the sf-36 has been established,3840 and its validity has been shown in an elderly population , in which the instrument distinguished between those with and without poor health.41 psychological functioning was assessed with the hospital anxiety and depression scale ( hads).42 the hads is a self - report rating scale of 14 items on a 4-point lk scale ( range : 03 ) . it is designed to measure anxiety and depression ( seven items for each subscale ) . the total score is the sum of the 14 items , and , for each subscale , the score is the sum of the respective seven items ( ranging from 021 ) . the hads is widely used in clinical research and has been shown to be reliable , valid , and responsive for use as a screening tool in patients with oa.43 pain was assessed with a subscale of the womac . muscle strength was assessed for flexion and extension of the knee using an isokinetic dynamometer ( enknee ; enraf - nonius b.v . , patients performed a maximum of three test repetitions to measure the strength of the quadriceps and hamstrings for each knee . mean muscle strength per leg was calculated to obtain a measure of overall leg muscle strength ( in nm ) . subsequently , mean muscle strength was divided by the patient s weight to control for the correlation between muscle strength and weight . excellent intra - rater reliability ( intraclass correlation coefficient 0.93 ) has been reported for this measure in knee oa patients.45 at the end of the treatment , patients were asked to rate global perceived effect ( gpe ) of the treatment46 on a scale of 19 , with a score of 1 meaning much better , 5 meaning no change , and 9 meaning much worse . patient satisfaction with the kind of treatment was measured by the nrs ( 010 ) , with higher scores indicating greater satisfaction . additional data recorded were age , sex , and duration of complaints . the weight ( kg ) and height ( m ) were measured in standing position . comorbidity was scored with the cumulative illness rating scale.28 radiographs of the knee were scored using the grading scales proposed by kellgren and lawrence ( k&l).47 in order to evaluate the treatment process , a descriptive analysis of the treatment registration forms was performed . the feasibility of the protocols was evaluated by analyzing the notes that were taken during the interviews with the therapists and participants . a faithful depiction of the experiences of the participants and therapists was achieved by verifying with the participant or therapist whether the remarks were interpreted in a correct way by giving a summary at the end of the interview . to analyze the patient outcomes after treatment , change scores were determined by subtracting the baseline scores from the posttreatment scores . because the data were not normally distributed pre- and posttreatment scores were analyzed with a nonparametric wilcoxon signed - rank test ( p - value < 0.05 ) . participants were referred to our rehabilitation center by their general practitioner because of persistent knee problems . physical measurements were carried out by a research assistant and questionnaires were filled out by the participants . the study was approved by the medical ethical review board of the slotervaart hospital and reade , amsterdam , the netherlands . all participants gave written informed consent and the study was conducted in accordance with the handbook for good clinical research practice of the world health organization and declaration of helsinki principles.26 inclusion criteria were : 1 ) diagnosis of knee oa according to the clinical american college of rheumatology criteria;27 2 ) presence of at least one of the target comorbidities , ie , coronary disease , heart failure , hypertension , type 2 diabetes , obesity , copd , chronic pain , nonspecific low back pain , depression , and vision and/or hearing impairment ( diagnosed by a medical specialist ) ; 3 ) severity score 2 of the comorbidity on the cumulative illness rating scale,28 indicating that the comorbidity has an impact on daily activities ; and 4 ) the primary treatment goal should be oa related ( instead of comorbidity related ) . exclusion criteria were : 1 ) indication for total knee replacement ; 2 ) inability to participate in treatment , eg , due to transport problems ; 3 ) insufficient capacity in the dutch language . the protocols were applied and evaluated by three qualified physical therapists with extensive experience ( 3 , 8 , and 12 years ) in knee / hip rehabilitation oa treatment . in addition , two of the three therapists were members of the committee for hip and knee oa guideline development for the royal dutch society of physical therapy . to evaluate the treatment process , the therapists completed a weekly registration form , providing information about the duration of the treatment period , content of the treatment , adaptations in the treatment due to the comorbidity , and any problems encountered in applying the protocols . adverse events , defined as any undesirable experience occurring in a subject during the study ( regardless of whether or not this was related to the treatment ) , were registered . to evaluate the feasibility of the protocols , semi - structured interviews were held by the first author ( mdr ) along with therapists and participants . topic lists were used to structure the interview ( see table 1 ) . to evaluate patient outcome after treatment , performance - based tests were performed and self - reported questionnaires were filled in by participants at baseline and directly after treatment . the western ontario and mcmaster universities osteoarthritis index ( womac ) is a disease - specific , self - administered questionnaire , developed to study patients with hip and knee oa.29,30 the womac consists of 24 questions grouped into three subscales ( pain : five questions ; stiffness : two questions ; and physical function : 17 questions ) and scaled in a 5-point likert ( lk ) scale . the maximum score in the lk scale is 20 points for pain , 8 points for stiffness , and 68 points for physical function . the womac is widely used in clinical research , and has been shown to be reliable , valid , and responsive for use in patients with oa.2931 the patient - specific functioning scale was used to evaluate limitations in activities of the individual patient.32 patients were asked , which activities do you perceive as important and were hampered by knee pain during the last week ? a list of activity suggestions was offered to support recall , and patients were allowed to provide other limited activities that were not on the list . of these activities , the patient selected three main activities and ranked them in order of importance . the difficulty of performance of the main activities were scored by self - assessment on a numeric rating scale ( [ nrs from 010 ] 0= no problems to perform the activity ; 10= impossible to perform the activity ) . patient - specific functional scale is an efficient and valid measure for assessing limitations in activities and change in limitations in persons with knee dysfunction.32 the get up and go test33,34 was performed with subjects seated on a high standard chair ( seat height 49 cm ) . the subjects were instructed to stand up without the help of the arms on the command go and walk 15 m along an unobstructed corridor as fast as possible without running . patients who normally used walking devices were allowed to use them during the test . a longer time taken to perform the test the total distance covered in meters during 6 minutes of walking was scored.35,36 quality of life was assessed with the 36-item short - form health survey ( sf-36).37 the sf-36 is a widely applied generic instrument for measuring health status and consists of eight dimensions : physical functioning , social functioning , physical role , emotional role , mental health , energy , pain and general health perception . the sf-36 gives scores on a 0100 scale , with higher scores indicating better health . the reliability ( median reliability coefficient 0.85 for all subscales ) for the sf-36 has been established,3840 and its validity has been shown in an elderly population , in which the instrument distinguished between those with and without poor health.41 psychological functioning was assessed with the hospital anxiety and depression scale ( hads).42 the hads is a self - report rating scale of 14 items on a 4-point lk scale ( range : 03 ) . it is designed to measure anxiety and depression ( seven items for each subscale ) . the total score is the sum of the 14 items , and , for each subscale , the score is the sum of the respective seven items ( ranging from 021 ) . the hads is widely used in clinical research and has been shown to be reliable , valid , and responsive for use as a screening tool in patients with oa.43 pain was assessed with a subscale of the womac . muscle strength was assessed for flexion and extension of the knee using an isokinetic dynamometer ( enknee ; enraf - nonius b.v . , patients performed a maximum of three test repetitions to measure the strength of the quadriceps and hamstrings for each knee . mean muscle strength per leg was calculated to obtain a measure of overall leg muscle strength ( in nm ) . subsequently , mean muscle strength was divided by the patient s weight to control for the correlation between muscle strength and weight . excellent intra - rater reliability ( intraclass correlation coefficient 0.93 ) has been reported for this measure in knee oa patients.45 at the end of the treatment , patients were asked to rate global perceived effect ( gpe ) of the treatment46 on a scale of 19 , with a score of 1 meaning much better , 5 meaning no change , and 9 meaning much worse . patient satisfaction with the kind of treatment was measured by the nrs ( 010 ) , with higher scores indicating greater satisfaction . the weight ( kg ) and height ( m ) were measured in standing position . comorbidity was scored with the cumulative illness rating scale.28 radiographs of the knee were scored using the grading scales proposed by kellgren and lawrence ( k&l).47 in order to evaluate the treatment process , a descriptive analysis of the treatment registration forms was performed . the feasibility of the protocols was evaluated by analyzing the notes that were taken during the interviews with the therapists and participants . a faithful depiction of the experiences of the participants and therapists was achieved by verifying with the participant or therapist whether the remarks were interpreted in a correct way by giving a summary at the end of the interview . to analyze the patient outcomes after treatment , change scores were determined by subtracting the baseline scores from the posttreatment scores . because the data were not normally distributed pre- and posttreatment scores were analyzed with a nonparametric wilcoxon signed - rank test ( p - value < 0.05 ) . eleven draft protocols for exercise therapy in persons with knee oa and comorbidity were developed based on our literature search and consultation with experts . regular oa exercise therapy as recommended in oa guidelines7,8,15 was the basis of the protocols . in the protocols , it was made explicit 1 ) how comorbidity compromises the regular application of exercise therapy in oa of the knee , and 2 ) how the therapist should consider the whole system , consisting of integrated body structures / functions and activities instead of separate organs , for all phases of treatment ( examination , evaluation , diagnosis , prognosis , and intervention ) . the hoac ii framework was used to incorporate principles of clinical reasoning into the protocols.17 the protocols on exercise therapy in persons with knee oa and comorbidities consist of a diagnostic phase and an intervention phase . each step in the protocols encourages clinical reasoning in order to tailor the diagnostic and intervention phase to the individual person . to facilitate this process , we designed a flowchart for the diagnostic and intervention phase ( figure 1 ) . the diagnostic phase includes an anamnesis , physical examination , establishment of treatment goals , and determination of the treatment strategy . during the anamnesis , oa - related problems , comorbidity - related restrictions and contraindications for exercise therapy thereafter , a clinical decision is made as to whether physical examination is possible , or whether the referring physician needs to be consulted because of contraindications for physical examination or the need for further medical information . with respect to the latter , test results of a maximum symptom - limited exercise test may be required ( for example , for persons with heart failure ) to establish an appropriate training intensity . if there are no contraindications for physical examination , comorbidity - related examination is performed according to the protocols ( eg , foot examination in patients with type 2 diabetes ) . subsequently , a decision is made as to whether there are contraindications or restrictions for exercise therapy . in case of a contraindication , if there are comorbidity - related restrictions for exercise therapy , a comorbidity - adapted program is indicated . in this phase , the therapist also considers whether referral to professionals in other disciplines ( eg , a dietician , psychologist , or occupational therapist ) is indicated . with regard to the intervention phase , the basic intervention in persons with knee oa consists of regular exercise therapy , according to the royal dutch society for physical therapy s guideline for physical therapy in patients with knee oa,15 which is similar to international guidelines.7,8 regular exercises for patients with oa comprise exercises aiming at improvement of muscle strength , aerobic capacity , flexibility , and ability to perform daily weight - bearing activities such as walking , stair climbing , and transfers ( eg , sitting down or standing up from a chair ) . individual therapy is given two times per week for between 30 to 60 minutes per session . the training intensity is increased from 40%85% of the maximum oxygen uptake ( vo2max ) or the heart rate reserve . the increase of training intensity is monitored by using the borg rpe scale ( 620 ) or heart rate frequency . the treatment ends when treatment goals are achieved or when no further improvement is feasible . the regular oa exercises are adapted to the comorbidity by changes in the duration , frequency , intensity , and type ( content ) of exercise therapy . the exact adaptations depend on restrictions for exercise therapy identified by the therapist in the diagnostic phase ( anamnesis and physical examination ) . the specific options for adaptations to oa exercises are listed in the protocols and summarized in table 1 . three participants dropped out , one because of ocular problems due to diabetes and two others because of comorbidities not included in this study ( hemochromatosis and cancer ) . nine of the eleven remaining participants had two or more comorbidities ( table 2 ) . the normal duration of the treatment in persons with knee oa without comorbidity in our center is 12 weeks . there were no adverse events reported during the study . in participants with comorbidities resulting in physiological impairments ( coronary disease , heart failure , type 2 diabetes , copd , and obesity ; n=6 ) , four were referred back to the general practitioner or specialist because of a high or fluctuating blood pressure . in these cases , while medication was adjusted , aerobic and strength exercises were postponed during the first 4 to 6 weeks of treatment . whole - body training or arm training was applied when loadability of the lower extremities was extremely low . this occurred mostly in participants with more than two comorbidities . in participants with comorbidities resulting in behavioral impairments ( chronic pain and nonspecific low back pain ; n=4 ) , adaptations were made by using a combined behavioral approach with regular oa exercises . in a time - contingent manner , the amount of physical activity was gradually increased combined with a gradual increase in the level of regular oa exercise , such as strengthening exercises of the lower limbs . adaptations were made by giving extra attention to providing positive feedback , stimulating a positive attitude toward physical activities , and gradually increasing the level of physical activity . in one participant with low vision , environmental restrictions led to adaptations in training equipment , training conditions ( eg , lighting ) , and treatment location . no specific adaptations of the oa exercise program were needed in participants with hearing impairments ( two of eleven participants ) . with regard to the feasibility of the protocols , the physical therapists who tested the protocols found that they offered guidance in setting up a treatment plan / strategy , making clinical decisions , and adapting the treatment to the comorbid disease ( table 3 ) . the following quote is from one of the physical therapists : by using the protocol i had more knowledge about the physical capabilities of the person with oa and this specific comorbidity . because of this i was able to design a more adequate training program and to better estimate the training intensity . this enabled me to treat the patient more intensively than i would have done without the use of the protocol . all physical therapists indicated that the list of restrictions for exercise therapy was a conveniently arranged checklist for the diagnostic and treatment phases . the list was also helpful in the process of clinical decision making , especially when more than one comorbidity was present . if more than one comorbidity was present , more emphasis was placed on the protocol(s ) for the comorbidity with the highest impact on physical functioning . importantly , the therapists agreed with the suggestion to increase feasibility by reducing the protocols to three main protocols . protocol a concerned physiological adaptations ( for persons with coronary artery disease , heart failure , hypertension , type 2 diabetes , obesity , and/or copd ) . protocol b concerned behavioral adaptations ( for persons with chronic pain , nonspecific low back pain , and/or depression ) . three of eleven participants would have been excluded from treatment in the absence of the protocols . they tailored the programs according to the individual s capacity , hereby preventing adverse events . the protocols were feasible in persons with mild ( k&l grade 1 ) to severe ( k&l grade 4 ) oa . all participants were satisfied with the applicability of the protocols , as indicated by a mean score of 8 points ( range : 710 ) on the nrs of satisfaction . nine of eleven participants mentioned that the therapists appeared to have a good level of knowledge about their health condition(s ) , which gave them more confidence in performing exercises . the following quote is from one participant : i felt more confident in performing exercises and was less afraid to get hypoglycemia during or after the training , because the therapist had more knowledge about my diseases and training possibilities . when i was treated in primary care for my knee complaints , i dropped out in an early phase of the treatment because my knee pain was getting worse due to the high training intensity at the beginning of the program . in addition , i was afraid when feeling an increase in my heart rate during the exercises and of becoming hypoglycemic . patient outcomes after treatment are presented in table 4 . on the womac physical functioning scale , a statistically significant improvement ( p<0.05 ) was found with an average increase of 18% above the baseline score . for the 6-minute walking test , the average increase was 13% above the baseline score ( p<0.05 ) . there was also a statistically significant decrease in pain , as measured with the womac pain subscale , where the average was 16% above the baseline score ( p<0.05 ) . the main activity limitation ( as ranked by each participant as being most important ) on the patient - specific functioning scale questionnaire also showed a statistically significant improvement ( p<0.05 ) . with regard to the extent to which symptoms changed over the period of treatment ( global perceived effect scale ) , four patients indicated that they were much or moderately improved after treatment ; four patients reported little improvement after the treatment ; and two patients reported no change in symptoms after treatment . eleven draft protocols for exercise therapy in persons with knee oa and comorbidity were developed based on our literature search and consultation with experts . regular oa exercise therapy as recommended in oa guidelines7,8,15 was the basis of the protocols . in the protocols , it was made explicit 1 ) how comorbidity compromises the regular application of exercise therapy in oa of the knee , and 2 ) how the therapist should consider the whole system , consisting of integrated body structures / functions and activities instead of separate organs , for all phases of treatment ( examination , evaluation , diagnosis , prognosis , and intervention ) . the hoac ii framework was used to incorporate principles of clinical reasoning into the protocols.17 the protocols on exercise therapy in persons with knee oa and comorbidities consist of a diagnostic phase and an intervention phase . each step in the protocols encourages clinical reasoning in order to tailor the diagnostic and intervention phase to the individual person . to facilitate this process , we designed a flowchart for the diagnostic and intervention phase ( figure 1 ) . the diagnostic phase includes an anamnesis , physical examination , establishment of treatment goals , and determination of the treatment strategy . during the anamnesis , oa - related problems , comorbidity - related restrictions and contraindications for exercise therapy thereafter , a clinical decision is made as to whether physical examination is possible , or whether the referring physician needs to be consulted because of contraindications for physical examination or the need for further medical information . with respect to the latter , test results of a maximum symptom - limited exercise test may be required ( for example , for persons with heart failure ) to establish an appropriate training intensity . if there are no contraindications for physical examination , comorbidity - related examination is performed according to the protocols ( eg , foot examination in patients with type 2 diabetes ) . subsequently , a decision is made as to whether there are contraindications or restrictions for exercise therapy . in case of a contraindication , if there are comorbidity - related restrictions for exercise therapy , a comorbidity - adapted program is indicated . in this phase , the therapist also considers whether referral to professionals in other disciplines ( eg , a dietician , psychologist , or occupational therapist ) is indicated . with regard to the intervention phase , the basic intervention in persons with knee oa consists of regular exercise therapy , according to the royal dutch society for physical therapy s guideline for physical therapy in patients with knee oa,15 which is similar to international guidelines.7,8 regular exercises for patients with oa comprise exercises aiming at improvement of muscle strength , aerobic capacity , flexibility , and ability to perform daily weight - bearing activities such as walking , stair climbing , and transfers ( eg , sitting down or standing up from a chair ) . individual therapy is given two times per week for between 30 to 60 minutes per session . the training intensity is increased from 40%85% of the maximum oxygen uptake ( vo2max ) or the heart rate reserve . the increase of training intensity is monitored by using the borg rpe scale ( 620 ) or heart rate frequency . the treatment ends when treatment goals are achieved or when no further improvement is feasible . the regular oa exercises are adapted to the comorbidity by changes in the duration , frequency , intensity , and type ( content ) of exercise therapy . the exact adaptations depend on restrictions for exercise therapy identified by the therapist in the diagnostic phase ( anamnesis and physical examination ) . the specific options for adaptations to oa exercises are listed in the protocols and summarized in table 1 . three participants dropped out , one because of ocular problems due to diabetes and two others because of comorbidities not included in this study ( hemochromatosis and cancer ) . nine of the eleven remaining participants had two or more comorbidities ( table 2 ) . the normal duration of the treatment in persons with knee oa without comorbidity in our center is 12 weeks . there were no adverse events reported during the study . in participants with comorbidities resulting in physiological impairments ( coronary disease , heart failure , type 2 diabetes , copd , and obesity ; n=6 ) , four were referred back to the general practitioner or specialist because of a high or fluctuating blood pressure . in these cases , while medication was adjusted , aerobic and strength exercises were postponed during the first 4 to 6 weeks of treatment . whole - body training or arm training was applied when loadability of the lower extremities was extremely low . this occurred mostly in participants with more than two comorbidities . in participants with comorbidities resulting in behavioral impairments ( chronic pain and nonspecific low back pain ; n=4 ) , adaptations were made by using a combined behavioral approach with regular oa exercises . in a time - contingent manner , the amount of physical activity was gradually increased combined with a gradual increase in the level of regular oa exercise , such as strengthening exercises of the lower limbs . adaptations were made by giving extra attention to providing positive feedback , stimulating a positive attitude toward physical activities , and gradually increasing the level of physical activity . in one participant with low vision , environmental restrictions led to adaptations in training equipment , training conditions ( eg , lighting ) , and treatment location . no specific adaptations of the oa exercise program were needed in participants with hearing impairments ( two of eleven participants ) . with regard to the feasibility of the protocols , the physical therapists who tested the protocols found that they offered guidance in setting up a treatment plan / strategy , making clinical decisions , and adapting the treatment to the comorbid disease ( table 3 ) . the following quote is from one of the physical therapists : by using the protocol i had more knowledge about the physical capabilities of the person with oa and this specific comorbidity . because of this i was able to design a more adequate training program and to better estimate the training intensity . this enabled me to treat the patient more intensively than i would have done without the use of the protocol . all physical therapists indicated that the list of restrictions for exercise therapy was a conveniently arranged checklist for the diagnostic and treatment phases . the list was also helpful in the process of clinical decision making , especially when more than one comorbidity was present . if more than one comorbidity was present , more emphasis was placed on the protocol(s ) for the comorbidity with the highest impact on physical functioning . importantly , the therapists agreed with the suggestion to increase feasibility by reducing the protocols to three main protocols . protocol a concerned physiological adaptations ( for persons with coronary artery disease , heart failure , hypertension , type 2 diabetes , obesity , and/or copd ) . protocol b concerned behavioral adaptations ( for persons with chronic pain , nonspecific low back pain , and/or depression ) . three of eleven participants would have been excluded from treatment in the absence of the protocols . they tailored the programs according to the individual s capacity , hereby preventing adverse events . the protocols were feasible in persons with mild ( k&l grade 1 ) to severe ( k&l grade 4 ) oa . all participants were satisfied with the applicability of the protocols , as indicated by a mean score of 8 points ( range : 710 ) on the nrs of satisfaction . nine of eleven participants mentioned that the therapists appeared to have a good level of knowledge about their health condition(s ) , which gave them more confidence in performing exercises . the following quote is from one participant : i felt more confident in performing exercises and was less afraid to get hypoglycemia during or after the training , because the therapist had more knowledge about my diseases and training possibilities . when i was treated in primary care for my knee complaints , i dropped out in an early phase of the treatment because my knee pain was getting worse due to the high training intensity at the beginning of the program . in addition , i was afraid when feeling an increase in my heart rate during the exercises and of becoming hypoglycemic . patient outcomes after treatment are presented in table 4 . on the womac physical functioning scale , a statistically significant improvement ( p<0.05 ) was found with an average increase of 18% above the baseline score . for the 6-minute walking test , there was also a statistically significant decrease in pain , as measured with the womac pain subscale , where the average was 16% above the baseline score ( p<0.05 ) . the main activity limitation ( as ranked by each participant as being most important ) on the patient - specific functioning scale questionnaire also showed a statistically significant improvement ( p<0.05 ) . no significant changes were found for the other measurements . with regard to the extent to which symptoms changed over the period of treatment ( global perceived effect scale ) , four patients indicated that they were much or moderately improved after treatment ; four patients reported little improvement after the treatment ; and two patients reported no change in symptoms after treatment . oa . nevertheless , no evidence - based recommendations are available concerning comorbidity - adapted exercises in patients with knee oa . the present study concerns the development of comorbidity - adapted exercise protocols in patients with knee oa . the protocols were found to be feasible and helpful in clinical reasoning and adapting oa exercises . to our knowledge , this is the first time that comorbidity - adapted protocols have been developed for exercise therapy in patients with oa of the knee and comorbidity . evidence - based diagnostics and treatment strategies generally overlook comorbidity.13,14 the interacting effects of diseases and their management require more complex and individualized care than simply the sum of separate guideline components . eleven comorbidity - adapted exercise protocols were developed for patients with knee oa and comorbidity . the protocols were found to provide guidance in clinical reasoning to direct both the diagnostic and treatment phases in persons with oa and complex , comorbidity - related health problems . the results of our field - testing revealed that the eleven protocols could be reduced to three main protocols due to overlap in diagnostics and/or treatment - related adaptations of the comorbidities and to improve user - friendliness . protocol a concerned physiological adaptations ( for persons with coronary artery disease , heart failure , hypertension , type 2 diabetes , obesity , and/or copd ) . protocol b concerned behavioral adaptations ( for persons with chronic pain , nonspecific low back pain , and/or depression ) . protocol c concerned environmental adaptations ( for persons with visual and/or hearing impairments ) . the protocols encourage physical therapists to think in advance about 1 ) how comorbidity compromises the regular application of exercise therapy by using the list of restrictions for exercise therapy of the comorbid disease and 2 ) how to adapt the exercise . as expected , in participants with physiological impairments ( eg , coronary disease ) , the training intensity and frequency and type of exercises were adapted to the comorbidity . in participants with behavioral impairments ( eg , chronic pain ) , a combination of regular oa exercises with a behavioral approach was preferred , in which the level of physical activity was gradually increased in a time - contingent manner . in one participant with visual impairments , environmental adaptations were applied ( eg , adapting the lighting in the exercise hall ) . an average increase of 18% on the physical functioning subscale and a decrease of 16% on the pain subscale were found with womac , which can be regarded as clinically important , relevant change.48 the treatment was safe and , by using the protocols , more patients with oa and comorbidity could participate in the exercise therapy . when taking comorbidity into account , adequate clinical reasoning is essential in order to deal with persons with a complex health status . physical therapists need to be alert to changes in health conditions that may necessitate further adaptations of the exercises . physical therapists with experience in dealing with chronic conditions may have an advantage in clinical reasoning and in the adaption of exercise programs in accordance with the comorbidity . physical therapists need to have an advanced understanding of complex system interrelationships regarding multiple morbidities . therefore , therapists should receive specific training to increase their knowledge about various comorbidities and their effects on oa exercise therapy . a number of remarks can be made about the usage and further development of the protocols . first , part of the results are based on personal opinions of three physical therapists working in a rehabilitation setting . to make the protocols broadly applicable , testing among various physical therapists practicing in different settings second , the protocols were tested on eleven patients with knee oa and various comorbidities . to compare the effectiveness of the protocols to usual care , a randomized clinical trial should be performed . comorbidity - adapted exercise protocols for patients with knee oa were developed that can provide guidance in clinical reasoning with regard to diagnostics and treatment . to evaluate the effectiveness of treatment in line with our protocols , a randomized clinical trial should be performed .

backgroundexercise therapy is generally recommended for patients with osteoarthritis ( oa ) of the knee . comorbidity , which is highly prevalent in oa , may interfere with exercise therapy . to date , there is no evidence - based protocol for the treatment of patients with knee oa and comorbidity . special protocols adapted to the comorbidity may facilitate the application of exercise therapy in patients with knee oa and one or more comorbidities.purposethe purpose of this study was to develop comorbidity - adapted exercise protocols for patients with knee oa and comorbidity.methodseveral steps were undertaken to develop comorbidity - adapted protocols : selection of highly prevalent comorbidities in oa , a literature search to identify restrictions and contraindications for exercise therapy for the various comorbid diseases , consultation of experts on each comorbid disease , and field testing of the protocol in eleven patients with knee oa and comorbidity.resultsbased on literature and expert opinion , comorbidity - adapted protocols were developed for highly prevalent comorbidities in oa . field testing showed that the protocols provided guidance in clinical decision making in both the diagnostic and the treatment phase . because of overlap , the number of exercise protocols could be reduced to three : one for physiological adaptations ( coronary disease , heart failure , hypertension , diabetes type 2 , chronic obstructive pulmonary diseases , obesity ) , one for behavioral adaptations ( chronic a - specific pain , nonspecific low back pain , depression ) , and one for environmental adaptations ( visual or hearing impairments ) . evaluation of patient outcome after treatment showed significant ( p<0.05 ) and clinically relevant improvements in activity limitations and pain.conclusioncomorbidity-adapted exercise protocols for patients with knee oa were developed , providing guidance in clinical reasoning with regard to diagnostics and treatment . to evaluate the effectiveness of treatment in line with our protocols , a randomized clinical trial should be performed .

Introduction Methods Development of comorbidity-adapted protocols Field-testing Procedure Participants Therapists Measurements Analysis Results Results of the development of the protocols Results from the field-testing Discussion Conclusion

PMC2921000

tusc was co - expressed with gst - spc110p as described13,21 , except that complexes were eluted by cleavage of the gst tag with tev protease as the final purification step . tusc rings were formed by 30 minute incubation on ice after dilution to 0.2 m in brb80 ( 80 mm pipes ph 6.9 , 1 mm egta , 1 mm mgcl2 ) . the cryo - em reconstruction was performed essentially as described by egelman17 and sasche , et al.25 .

microtubules are nucleated in vivo by -tubulin complexes . the 300 kda -tubulin small complex ( tusc ) , consisting of two molecules of -tubulin and one copy each of the accessory proteins spc97p and spc98p , is the conserved , essential core of the microtubule nucleating machinery1,2 . in metazoa multiple tuscs assemble with other proteins into -tubulin ring complexes ( turcs ) . the structure of turc suggested that it functions as a microtubule template25 . because each tusc contains two molecules of -tubulin , it was assumed that the turc - specific proteins are required to organize tuscs to match thirteen - fold microtubule symmetry . here , we show that tusc forms rings even in the absence of other turc components . the yeast adaptor protein spc110p stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions . the 8 cryo - em reconstruction of the filament reveals thirteen -tubulins per turn , matching microtubule symmetry , with plus ends exposed for interaction with microtubules , implying that one turn of the filament constitutes a microtubule template . the domain structures of spc97p and spc98p suggest functions for conserved sequence motifs , with implications for the turc - specific proteins . the tusc filaments nucleate microtubules at a low level , and the structure provides a strong hypothesis for how nucleation is regulated , converting this less active form to a potent nucleator .

Methods Summary Supplementary Material

PMC3868094

the international pilot study of schizophrenia estimated that 70% of schizophrenia patients suffered from hallucinations . 25 - 30% of the auditory hallucinations in schizophrenia are refractory to traditional antipsychotic drug treatment . even with the advent of newer antipsychotics , a significant minority of patients continue to experience persistent hallucinations . apart from pharmacological treatments , other therapies such as transcranial magnetic stimulation , cognitive behavior therapy , and hallucination - focused integrative treatment tiapride , is a substituted benzamide antipsychotic drug and has a selective d2 and d3 dopamine receptor antagonist activity in limbic brain areas and the locus ceruleus . the usual indications of tiapride use are the presence of extrapyramidal symptoms or other dyskinesias , hyperkinesias , patients with huntington 's chorea and also geriatric agitation and restlessness . the common side - effects with tiapride are drowsiness , extrapyramidal symptoms , dizziness , and orthostatic hypotension . here , we report two cases of schizophrenia with persistent auditory hallucinations that responded favorably to tiapride as an add on drug to their already existing drug treatments . a 37-year - old right handed female christian graduate was brought to the out - patient department by family members with chief complains of hearing voices inaudible to others , suspiciousness toward others as though they are talking about her and plotting against her since the past 5 years . she would hear voices of known and unknown people saying sometimes good things and majority of times saying bad things to her . she would get commanding hallucinations telling her to tear her books , bible , certificates , etc . , she also complained of occasional anger outbursts , decreased sleep , and stopped doing household activities . she was diagnosed as having paranoid schizophrenia as per diagnostic and statistical manual for the classification of psychiatric disorders 4 edition text revised ( dsm - iv tr ) ( dsm - iv tr ) criteria and was started on olanzapine 10 mg / day , haloperidol 10 mg / day and trihexyphenidyl 4 mg / day all in oral formulations . gradually , the doses were increased weekly to olanzapine 20 mg / day , haloperidol 20 mg / day , and trihexyphenidyl 6 mg / day . clozapine 25 mg at night was added , which was gradually increased to 100 mg at night . her hallucinations persisted , but there was some improvement in that the number of people whose voices she used to get reduced . the patient and relatives were not willing for electroconvulsive therapy ( ect ) as a treatment option . hence , tiapride 50 mg / day was added after 6 months of addition of clozapine 100 mg . after 2 weeks of tiapride therapy , the dose was increased to 100 mg / day in divided doses . olanzapine was reduced as she complained of excessive sedation with it . currently , the patient is on haloperidol 20 mg / day , olanzapine 10 mg / day , trihexyphenidyl 6 mg / day , clozapine 100 mg at night and tiapride 100 mg / day . she was assessed for hallucinations using the hallucinations subscale of the psychopathology rating scale ( psyrats ) . her score on the hallucination subscale improved from 28 to 11 after the addition of tiapride . a 32-year - old married 10 standard educated female was brought to the out - patient department by her sister with complaints of suspiciousness toward the neighbors that they are talking about her and that they would kill her and her family members since the past 6 months . she would feel that they are keeping a watch on her by cameras fitted in her house by them . she would also hear voices inaudible to others commenting on her actions and sometimes giving running commentary also . she was diagnosed as having paranoid schizophrenia as per dsm - iv tr criteria and was started on risperidone 4 mg / day and trihexyphenidyl 4 mg / day . gradually risperidone was increased to 8 mg / day in weekly increments and trihexyphenidyl to 6 mg / day . her symptoms showed no improvements after 1 month and olanzapine 10 mg / day was added , which was increased gradually to 20 mg / day . she and her family members were not consenting for ect and tiapride 50 mg / day in divided doses was added to her medication . she started feeling better , her voices began to reduce and the intensity and frequency of hallucinations decreased . the patient claimed 80% improvement in auditory hallucinations after 2 months of addition of tiapride to her medication . her total score on the psyrats - hallucination subscale improved from 41 to 25 after addition of tiapride . a 37-year - old right handed female christian graduate was brought to the out - patient department by family members with chief complains of hearing voices inaudible to others , suspiciousness toward others as though they are talking about her and plotting against her since the past 5 years . she would hear voices of known and unknown people saying sometimes good things and majority of times saying bad things to her . she would get commanding hallucinations telling her to tear her books , bible , certificates , etc . , she also complained of occasional anger outbursts , decreased sleep , and stopped doing household activities . she was diagnosed as having paranoid schizophrenia as per diagnostic and statistical manual for the classification of psychiatric disorders 4 edition text revised ( dsm - iv tr ) ( dsm - iv tr ) criteria and was started on olanzapine 10 mg / day , haloperidol 10 mg / day and trihexyphenidyl 4 mg / day all in oral formulations . gradually , the doses were increased weekly to olanzapine 20 mg / day , haloperidol 20 mg / day , and trihexyphenidyl 6 mg / day . clozapine 25 mg at night was added , which was gradually increased to 100 mg at night . her hallucinations persisted , but there was some improvement in that the number of people whose voices she used to get reduced . the patient and relatives were not willing for electroconvulsive therapy ( ect ) as a treatment option . hence , tiapride 50 mg / day was added after 6 months of addition of clozapine 100 mg . after 2 weeks of tiapride therapy , the dose was increased to 100 mg / day in divided doses . olanzapine was reduced as she complained of excessive sedation with it . currently , the patient is on haloperidol 20 mg / day , olanzapine 10 mg / day , trihexyphenidyl 6 mg / day , clozapine 100 mg at night and tiapride 100 mg / day . she was assessed for hallucinations using the hallucinations subscale of the psychopathology rating scale ( psyrats ) . her score on the hallucination subscale improved from 28 to 11 after the addition of tiapride . a 32-year - old married 10 standard educated female was brought to the out - patient department by her sister with complaints of suspiciousness toward the neighbors that they are talking about her and that they would kill her and her family members since the past 6 months . she would feel that they are keeping a watch on her by cameras fitted in her house by them . she would also hear voices inaudible to others commenting on her actions and sometimes giving running commentary also . she was diagnosed as having paranoid schizophrenia as per dsm - iv tr criteria and was started on risperidone 4 mg / day and trihexyphenidyl 4 mg / day . gradually risperidone was increased to 8 mg / day in weekly increments and trihexyphenidyl to 6 mg / day . her symptoms showed no improvements after 1 month and olanzapine 10 mg / day was added , which was increased gradually to 20 mg / day . she and her family members were not consenting for ect and tiapride 50 mg / day in divided doses was added to her medication . she started feeling better , her voices began to reduce and the intensity and frequency of hallucinations decreased . the patient claimed 80% improvement in auditory hallucinations after 2 months of addition of tiapride to her medication . her total score on the psyrats - hallucination subscale improved from 41 to 25 after addition of tiapride . tiapride offers good results in patients whose main complains are auditory hallucinations , which have not responded to other antipsychotics . tiapride does not cause sedation ; hence , it can be added easily with other antispychotics and no worries of cumulative sedation effects . to best of our knowledge , there are no literature on use of tiapride for auditory hallucinations , but there is one report on the treatment of visual release hallucinations with tiapride .

hallucinations are considered as core symptoms of psychosis by both international classification of diseases10 ( icd-10 ) and diagnostic and statistical manual for the classification of psychiatric disorders 4th edition text revised ( dsm - iv tr ) . the most common types of hallucinations in patients with schizophrenia are auditory in nature followed by visual hallucinations . few patients with schizophrenia have persisting auditory hallucinations despite all other features of schizophrenia having being improved . here , we report two cases where tiapride was useful as an add - on drug for treating persistent auditory hallucinations .

INTRODUCTION CASE REPORTS Case 1 Case 2 DISCUSSION

PMC3363065

ischemic colitis is an inflammatory disease caused by the lack of colonic blood supply.1,2 being one of the most common form of intestinal ischemias , ischemic colitis is manifested with unspecific clinical symptoms such as acute abdominal pain , diarrhea , or hematochezia . the major causes include hypotension due to dehydration or bleeding and decreased blood flow.3 ischemic colitis occurs mostly in the elderly because of age - related tortuosity of the colonic arteries and so on.4 factors other than age include diseases of decreased blood flow ; small vessel etiology due to underlying diseases such as thrombosis , hypertension , and diabetes mellitus , iatrogenic causes such as surgery , or drugs.2,3 drug , especially estrogen , has been reported as the cause of ischemic colitis in western countries.3,5,6 but in korea , estrogen is a very rare cause of ischemic colitis . we report a case of ischemic colitis with a review of literature , developing after taking combined oral contraceptives without particular comorbidity in a 26-year - old american female living in south korea . a 26-year - old foreign female visited the hospital for sudden occurrence of lower abdominal pain during sleep with more than 10 times of diarrhea , and 3 times of hematochezia . she is unmarried and has been working as an elementary school english teacher in south korea for 2 years with generally good health . blood pressure 130/90 mm hg , pulse rate 60/min , respiratory rate 20/min , body temperature 37.3 , height 173 cm , weight 99 kg , and body mass index ( bmi ) 33 were measured at admission . physical examination revealed acute ill looking appearance , with soft abdomen but tenderness on the lower abdomen . complete blood count test showed leukocyte 8,630/mm , hemoglobin 12.8/dl , and platelet count 210,000/mm . blood chemistry was analyzed as total protein 7.2 g / dl , albumin 4.6 g / dl , total bilirubin 0.3 mg / dl , ast 17 u / l , alt 17 u / l , alkaline phosphatase 47 u / l , amylase 140 u / l , lipase 43 u / l , blood urea nitrogen 10.9 mg / dl , creatinine 0.9 mg / dl , esr 10 mm / hr , and crp 0.77 mg / dl . stool examination showed wbc 0 - 2/hpf and rbc 0 - 2/hpf , with negative culture result . abdominal computerized tomography taken on the day of admission showed small amount of ascites in the pelvic cavity and blood vessels were normal without unusual finding ( fig . 1 ) . sigmoidoscopy on day 2 of admission revealed normal rectum and sigmoid colon , but erythemas and petechias were found along the longitudinal axis from the descending colon to the splenic flexure . biopsy was performed at the descending colon , which revealed loss of the surface epithelium , mucosal necrosis , and hyalinization of lamina propria compatible with ischemic colitis ( fig . the patient was managed conservatively with bowel rest , intravenous fluids , and empirical antibiotics . symptoms of abdominal pain , diarrhea , and hematochezia were relieved and the patient started soft diet on day 4 of admission , and she was discharged on day 5 of admission . after 2 months of asymptomatic period , she visited the outpatient department again with the recurrence of abdominal pain and hematochezia . on repeated carefyl history taking , she gave drug history about taking combined oral contraceptives ( mercilon ; combined ethinyl estradiol and desogestrel ) since coming to korea . the new onset abdominal pain recurred 2 days ago with hematochezia after restarting the use of oral contraceptives 1 month ago . we advised her to stop the use of oral contraceptives after explaining that it might be the possible cause of ischemic colitis . we confirmed by a phone call 6 months later that she was faring well without any recurrence of abdominal pain or hematochezia . ischemic colitis is caused by various predisposing factors including old age , hypotension and decreased blood flow due to diseases , cardiogenic thromboembolism , myocardial infarction , major cardiovascular surgery , hypercoagulable state , drugs , long - distance running , constipation , vasospasm , and snake venom.2,3 the patient of this study was hard to find a reasonable cause of ischemic colitis because she did not have any risk factor and history taking was not sufficient enough to find the combined oral contraceptives taking in unmarried person . it could be said , therefore , that finding predisposing factors of ischemic colitis is the most important to prevent further recurrence . it is impossible and sometimes not necessary to inspect all the above mentioned causes for every ischemic colitis patients . ischemic colitis in the elderly is rarely caused by hypercoagulable state , so the test of it is generally not recommended.3 younger patients , however , require tests on hypercoagulable state and detailed history taking for drugs . venous thrombosis caused by estrogen in oral contraceptives was first reported in the early 1960s.7 in 1990s , it was reported that women with genetic defects such as factor v leiden mutation , prothrombin 20210a , and deficiency of anti - thrombin , protein c and protein s was more susceptible to venous thrombosis while using oral contraceptives.8 - 10 oral contraceptives change the blood concentration of lots of proteins involved in the coagulation.11 it is not clear how these changes of coagulation - related factors clinically affect the venous thrombosis.12 based on the available studies , the mechanism of venous thrombosis caused by oral contraceptives could be explained by the activated protein c ( apc ) resistance.12 the apc resistance is related to the blood concentration of protein s , a tissue factor pathway inhibitor , and has a positive relation with sex hormone binding globulin concentration.12 - 14 each oral contraceptives causes venous thrombosis at different frequencies . it was reported that the risk of venous thrombosis depends on the dose of estrogen before 1995.12 it was reported later that progesterone involves in the development of venous thrombosis , leading to a new concept of ' total estrogenicity ' combining the antiestrogenic effects of estrogen and progesterone.15 if the estrogen is fixed on a certain dose , the frequency of venous thrombosis depends on the combined progesterone , so that the 3rd ( gestodene or desogestrel ) and 4th ( cyproterone acetate or drospirenone ) generation of progesterone increase the risk of venous thrombosis 1.5 - 4 times higher than the 2nd generation progesterone ( levonorgetrel).12 moreover , obese females have 2.4-times higher risk of thrombosis than females with the normal weight . obese females taking oral contraceptives have the risk of developing thrombosis 24 times higher than normal weight females not taking oral contraceptives.16 our patient was taking combined oral contraceptives including desogestrel , which causes venous thrombosis more often , and was obese with bmi value of 33 , contributing the increased risk of thrombosis than normal weight female who were taking the same contraceptives . patients manifest rapid onset of abdominal pain , tenderness around the affected bowel area , and mild to moderate hematochezia , as in this case.3 any part of the colon could be involved , but around 75% shows left colon lesion , 25% of them including splenic flexure.17 our case also showed typical descending colon lesion including splenic flexure . the diagnosis of ischemic colitis is initially suspected by typical clinical presentation , but the clinical manifestations are similar with other various diseases , making it hard to diagnose only by clinical suspicion . computerized tomography is useful to exclude other diseases presenting similar symptoms but difficult to make a definite diagnosis . colonoscopy , on the other hand , is more sensitive by inspecting the location , and mucosal changes and getting biopsy , which are important in the diagnosis of ischemic colitis.3 our patient was also unable to be diagnosed as ischemic colitis by the abdominal computerized tomography but sigmoidoscopy enabled the definite diagnosis . single linear ulcer along the longitudinal axis is a distinctive colonoscopic finding suggestive of ischemic colitis , along with other findings of pale , edematous , petechial mucosa to hemorrhagic nodule and gangrene appearing in various degrees . the histologic patterns of ischemic colitis include mucosal coagulation necrosis and hemorrhage , neutrophil infiltration , and granulation tissue formation ; these patterns are not specific for diagnosis but helpful for predicting the disease progression.18,19 this patient showed mucosal necrosis and hyalinization of lamina propria . accordingly , ischemic colitis should be diagnosed in combination of clinical manifestations , radiologic images , and colonoscopic findings.3 treatment of ischemic colitis depends on the clinical manifestations and colonoscopic findings . most of patients with ischemic colitis improve with conservative managements such as bowel rest , intravenous fluids , and empirical antibiotics . some cases may require surgery for gangrenous colitis or stricture.20 prognosis depends on the extent of injury and comorbidities , but around 85% of patients are improved within 1 - 2 days of conservative management and fully recovered within 2 weeks . our patient recovered with conservative management and did not have the recurrence of the disease after quitting the possible cause . this case represents the repetitive ischemic colitis due to the use of oral contraceptives in young healthy female without comorbidity , suggesting the importance of precise history taking including oral contraceptives and other risk factors for thrombosis .

ischemic colitis is generally considered a disease of the elderly . the causes of ischemic colitis include low - flow states due to cardiac dysfunction or hypovolemia and certain medications including estrogen . here we report a case of ischemic colitis in a 26-year - old woman . she had no specific medical history except taking oral - contraceptives for a long time . the mechanism of estrogen - induced ischemic colitis is not clearly understood . but we recommend that oral - contraceptives should be considered as a cause of ischemic colitis in young women .

INTRODUCTION CASE REPORT DISCUSSION

PMC3649702

brain tumors are the second most common form of childhood cancer , after acute lymphoblastic leukemia ( all ) . given 5-year survival rates that approach 90% for children treated for all and 70% for those treated for brain tumors , there are currently a great many survivors of these cancers that suffer from the consequences of rt , including adverse physiological , psychological , and cognitive side effects that manifest both acutely and years later . these so called late effects result in lowered quality of life ( qol ) in survivors , rt for pediatric cancer has long been acknowledged as a primary cause of neurological complications and neurocognitive decline [ 48 ] . childhood rt is associated with a significant decrease in iq scores [ 814 ] , thought to result from deficits in core processing functions impaired by rt , including processing speed , attention [ 1518 ] , working memory , and other executive functions [ 7 , 19 ] . in addition to cognitive impairments , adult survivors of childhood rt also experience elevated rates of emotional distress , such as anxiety and/or depression [ 20 , 21 ] and posttraumatic stress disorder . these cognitive and emotional consequences of rt result in decreased qol that manifests in a variety of ways . for example , adult survivors of childhood rt are less likely to obtain a college education [ 23 , 24 ] or marry [ 5 , 20 ] and more likely to be unemployed [ 24 , 25 ] . improving qol for survivors necessarily involves attenuating the long - term neural consequences of rt . ionizing radiation damages the brain directly , but in addition , it chronically suppresses cell proliferation , thereby depriving the brain of the raw materials needed for repair . when the brain is irradiated in childhood , there is a further consequence of rt , as suppressed cell proliferation and hostile environmental conditions disrupt ongoing developmental processes . jump - start cell proliferation and foster a neural environment that is conducive to plasticity . exercise may represent one such treatment , and its restorative potential for the post - rt brain is discussed . rt damages the brain regardless of age . however , the brains of children are still developing , and rt profoundly affects ongoing developmental processes . the potential mechanisms underlying this disruption are many , such as perturbations of vasculature and suppression of cell proliferation [ 2729 ] . damage to the endocrine system [ 30 , 31 ] has been shown to play a role , in particular , decreased expression of growth hormone ( gh ) . gh deficiency results from the effects of a brain tumor or of therapy such as surgery , rt , or chemotherapy . report that the peak gh response within 12 months after the initiation of cranial rt depends on hypothalamic dose - volume effects and may be predicted on the basis of a linear model that sums the effects of the entire dose distribution . the rate of decline in the peak gh response may also be influenced by clinical factors indicating the severity of the disease and the type and location of tumor . disruption of brain development could also be due in part to cancer treatment effects on food intake . treatment - induced nausea and vomiting , as well as gastrointestinal toxicity can lead to nutritional deficiency and changes in body composition [ 34 , 35 ] , which may be long - lasting . in contrast , survivors of childhood all are more likely to be obese , compared with age - matched controls . thus , treatment effects on hormone levels and nutritional intake , alone or in combination , are likely important contributors to altered neural development and , ultimately , cognitive impairments . animal models of pediatric rt enable controlled study of mechanisms that contribute to disrupted development and , ultimately , cognitive late effects . to model the effects of rt on the developing brain , we have treated postnatal day 28 ( pnd28 ) rats with whole brain irradiation ( wbi ) , using one of 2 regimens : single dose ( 20 gy ) or fractionated , in which animals received 20 gy over the course of 5 days ( 4 gy / d ) . either regimen results in a profound stunting of brain growth visible to the naked eye ( see figure 1 ) , although the effect is clearly bigger with single dose treatment . using this model , we can probe the cellular , chemical , and structural effects of rt that contribute to decreased brain size and cognitive impairments in adulthood . to enhance translational value , we are using imaging techniques to discover rt - induced changes in vivo that predict future cognitive impairments before they manifest . for example , we are using magnetic resonance imaging ( mri ) and diffusion tensor imaging ( dti ) to assess rt - induced structural changes and h magnetic resonance spectroscopy ( mrs ) to assess chemical changes following rt ( see figure 2 ) . dti has the added advantage of providing information on fractional anisotropy ( fa ) , a measure of the functional integrity of white matter tracts . our preliminary h mrs findings showed changes in glutamate , alanine , and lactate in rt brains , compared to sham controls . in addition , fa analysis showed a significant decline in fimbria volume and mean fimbria fa value in rt brains compared to controls . these changes were observed three months prior to the detected cognitive changes shown in figure 3 , suggesting that imaging changes can be used as early markers of cognitive decline . because ionizing radiation kills dividing cells , it is effective at treating cancer , yet devastating to noncancerous tissue in the brain . although mature neurons are postmitotic and therefore not directly affected by radiation , the brain 's actively dividing neural stem cells ( nsc ) are largely wiped out , even by very low doses . this is problematic , as it decreases the availability of new neurons in neurogenic regions of the brain and of new glia ( oligodendrocytes and astrocytes ) in nonneurogenic areas . the dentate gyrus ( dg ) of the hippocampus , along with the lining of the lateral ventricles ( the subventricular zone , or svz ) , is one of the few neurogenic regions of the adult mammalian brain ( see for review ) . for example , newly generated neurons are kept alive by effortful learning ( for review see ) and are needed for the formation of long - term spatial memory . analysis of postmortem human tissue following cancer treatment shows an almost complete lack of hippocampal neurogenesis , the functional importance of which is attested to by the cognitive impairments observed in survivors . animal models have yielded direct links between rt - induced decrements in hippocampal neurogenesis and cognitive impairments . many studies have focused on deficits in spatial performance ( place learning or spatial memory ) and trace fear conditioning , since these are hippocampus - dependent functions . spatial impairments have been observed in conjunction with decrements in dg cytogenesis following both fractionated [ 43 , 44 ] and single - dose wbi [ 45 , 46 ] . our group also has noted performance deficits in a spatial task after fractionated irradiation ( see figure 3(c ) ) . decreased fear conditioning has also been associated with radiation - induced suppression of dg cytogenesis [ 4749 ] . in sum , an increasing body of evidence implicates suppression of hippocampal neurogenesis as a causative factor in cognitive impairments following rt . however , suppression of hippocampal neurogenesis is likely only part of the story . a plentiful supply of glial cells is essential for neuronal health and function [ 51 , 52 ] , so reduced proliferation of nscs due to rt could contribute to cognitive impairments by reducing the availability of glia . for example , problems with executive functions are widely reported in adult survivors of childhood rt . executive functions develop linearly during adolescence , in apparent conjunction with myelination of the frontal lobes . frontal lobe white matter appears particularly vulnerable to rt , and rt - induced damage to white matter tracts may , in large part , underlie the neurocognitive deficits experienced by adult survivors of childhood cancer [ 19 , 54 ] . myelination is dependent on a ready supply of healthy oligodendrocytes , which is in turn dependent on adequate proliferative activity of nscs . rt - induced ablation of nscs in nonneurogenic regions could therefore contribute to cognitive impairments . to provide direct evidence that rt - induced suppression of gliogenesis contributes to frontal lobe dysfunction , the 5-choice serial reaction time task ( 5-csrtt ) is a reliable means by which to assess prefrontal cognitive processes in the rodent . this automated task measures several aspects of visual attention , specifically divided , sustained , and selective attention , as well as processing speed and impulsivity . the task requires the animal to detect brief flashes of light that appear in one of five apertures ( see figure 3(a ) ) and then nose - poke into the aperture that the light appeared in . the animal is given 5 seconds in which to make the nose - poke response . correct responses are rewarded by a food pellet being dispensed into a magazine at the rear of the testing chamber ( see figure 3(a ) ) . to provide motivation , , there is an intertrial interval ( iti ) , and the animal must inhibit responding during this interval , because premature responses result in a short time - out period during which there are no trials , and thus food reward can not be obtained . in performing this task , the animal has to sustain attention to all 5 of the apertures in order to constantly monitor where the light stimulus will be presented . incorrect responses ( nose - pokes into an aperture other than that in which the light was presented ) indicate impaired attention . measures of impulsivity are collected through responses that are characterized as perseverative and/or premature . processing speed measures are based on various latency times that are collected throughout the task . we have used the 5-csrtt to probe for impairment of prefrontal cognitive processes following fractionated irradiation ( 4 gy / d for 5 days ) . irradiated animals and shams our preliminary findings indicate that the irradiated animals are significantly less accurate at nose - poking into the correct aperture , suggesting that they have attentional impairments ( see figure 3(b ) ) . future experiments will focus on replicating these impairments in irradiated animals and determine whether they are linked to reduced gliogenesis in frontal regions . the microenvironment of the brain is regulated and protected by specific barriers , which include the vascular endothelial barrier ( also called the blood - brain barrier , or bbb ) at the capillary - parenchyma interface and the epithelial barrier ( blood - cerebrospinal fluid barrier ) at the choroid plexus . the bbb is more than a physical barrier : it plays a fundamental role in regulating the movement of substances between the blood and the cns ( see figure 4(a ) ) . the microvascular network is also the site of the bbb , and the endothelial cells ( ecs ) that make up the microvascular network barrier contain few pinocytotic vesicles and adhere to each other via tight junctions . tight junctions limit paracellular transport of hydrophilic compounds into the cns as compared to non - cns vessels [ 58 , 59 ] . also , astrocytes in close proximity to the ecs add another impediment to paracellular transport by biochemically conditioning the ecs and strengthening the tight junctions between them . because of this intertwined fate with the circulatory system , ecs play a unique role in maintaining physiological homeostasis , controlling the movement of substances across from the blood compartment into the different tissues and organs with varying demands and function . the ecs also play an important immune function through leukocyte surveillance and extravasation by regulating adhesion integrins and cytokine production . in particular , they have been shown to directly secrete tumor necrosis factor ( tnf ) . when the barrier between the vascular supply of the brain and the cns parenchyma is disrupted , excess extravasation of proteins , biologic - response molecules ( e.g. , growth factors , cytokines , and clotting factors ) , inflammatory cells , and therapeutic drugs can damage the brain [ 56 , 6466 ] . the disruption of the bbb ( see figure 4(b ) ) has been identified as a consequence of various diseases / injuries such as multiple sclerosis , ischemia , hiv , hypertension , brain tumors , cns injury , and radiation exposure [ 6670 ] , wherein inflammatory cells are able to penetrate the bbb and destroy the myelin surrounding the axons . demyelination and myelin thinning have been reported in the cns following rt [ 7174 ] . have also shown that rt - induced bbb permeability prolonged the induced demyelination of neurons [ 75 , 76 ] . we and others [ 7780 ] have demonstrated that there is an increase in bbb permeability following rt , which is caused in part by ec damage , as expressed by changes in tight junction integrity and by vesicle formation postirradiation . rt - induced ec damage has been investigated [ 8183 ] with the aim of elucidating the effect on initiating and/or sustaining radiation side effects . eissner et al . , as well as others [ 82 , 83 ] , have shown that when irradiated , ecs in vitro and in vivo undergo apoptosis at a higher percentage than other cells . our studies using electron microscopy show that rt causes damage to the tight junctions , which is also connected to the observed increase in bbb permeability . in addition , several studies , including our own , have shown an increase in bbb permeability and an increase in the number of vesicles following fractionated cranial irradiation [ 7880 ] . such damage to the microvasculature and breach of the bbb can disturb the delicate brain microenvironment and expose the brain parenchyma and neural cells to noxious substances [ 70 , 78 , 85 ] . this microenvironment imbalance can set into motion a chain of events ( such as cytokines release ) , magnifying the original signal and finally causing measurable late - term tissue damage in the irradiated brain that may play a role in cognitive impairment . we and others have shown that rt induces an inflammatory response as indicated by an increase in tnf- and intercellular adhesion molecule-1 ( icam-1 ) signaling in the brain [ 8790 ] . we have reported activated astrocytes after treatment with single and fractionated rt [ 78 , 91 ] . prolonged gliosis can create glial scar sites , which have been theorized to inhibit axonal regeneration or remyelination [ 92 , 93 ] . we have demonstrated that this inflammation response is related to an increase in bbb permeability following rt and that it is abrogated when treated with antibodies to tnf- or icam-1 [ 77 , 90 ] . in a histological study on mouse brains we observed significant changes 120 days following fractionated rt : fewer neurons , a significant decrease in myelin suggesting complete destruction of the parts of the white matter at 120 days following rt , and at 90 days following rt , we observed swelling of nerve fibers and increased thickening of the myelin sheaths ( see figure 5(b ) ) indicative of dying axons . given its myriad beneficial effects on the brain , exercise has been suggested as a treatment for a wide variety of brain maladies , from aging to alcoholism . in the case of aging , exercise has been shown to have a remarkable restorative effect , encouraging the resurgence of atrophied regions such as white matter tracts and the hippocampus , and improving cognition . such effects are particularly encouraging for the post - rt brain , since it shares many things in common with the aged brain , such as decreased cell proliferation , decreased growth hormone , and increased inflammation . moreover , in both cases these conditions worsen over time , to ultimately create a neural milieu in which plasticity is suppressed . in aged rodents , exercise can increase proliferation of nscs , suggesting that it has neurogenic potential even in a system in which cell proliferation is drastically reduced . encouragingly , exercise has been reported to increase hippocampal neurogenesis in the irradiated brain in rodent models [ 100 , 101 ] . neurogenesis is tightly linked to the microenvironment and is known to be suppressed under conditions in which there is unchecked inflammation or a lack of trophic or hormonal support . exercise has been shown to increase growth hormone and reduce inflammation , two potential ways in which it could counteract the suppressive environment created by rt . recent research has begun to elucidate the important role that microglia have in maintaining the neurogenic niche [ 106108 ] . unfortunately , radiation severely disrupts microglial distribution , alters their morphology ( see figure 6 ) , and decreases their numbers , effects that likely contribute to rt - induced neurogenesis impairment . it has been shown that , after radiation , microglia in the svz rebound more quickly than those in the dg . this may explain why neurogenesis recovers better in the svz , compared to the dentate . voluntary exercise has been shown to increase microglia , suggesting a further means by which exercise could help to restore a microenvironment conducive to cell proliferation . exercise might also have an enhancing effect on gliogenesis in the post - rt brain . as described above exercise has been shown to enhance cortical gliogenesis in the intact brain , and our future efforts will include determining whether post - rt exercise enhances cortical gliogenesis and ameliorates impairments in the 5-csrtt . in addition to these many direct benefits , it is important for survivors of childhood rt to exercise , in order to counteract chronic conditions that arise from cancer treatment , such as impaired pulmonary and cardiac function . unfortunately , treatment - induced fatigue , cardiorespiratory problems , and muscular deconditioning tend to promote sedentary habits during treatment that linger into adulthood , with the result that childhood cancer survivors are much less physically active than their healthy peers ( see for review ) . cranial rt in particular is associated with sedentary habits in adulthood [ 114 , 115 ] . however , recent studies suggest that physical fitness is an achievable goal for childhood cancer survivors [ 36 , 113 ] , so the beneficial effects of exercise observed in animal models can be followed up in human patients . fortunately , rodents show no reluctance to exercise after rt , and initial studies suggest that exercise is capable of ameliorating rt - induced deficits in both neurogenesis and cognition . voluntary running in adulthood has been shown to restore neurogenesis in mice irradiated early in life , suggesting that exercise may be a feasible means by which to promote cell proliferation in adult survivors of childhood rt . a recent study showed that voluntary running ameliorated radiation - induced spatial memory decline 4 months after radiation as well as partially restored neurogenesis in the dg . while these results are encouraging , continued study of the effects of exercise on the rt brain in animal models is essential . for one thing , it is important to better understand the effects of exercise in the context of the rt brain . in particular , it is necessary to determine whether exercise has an adequate neural substrate on which to work . for example , one well - established effect of exercise is its ability to induce angiogenesis [ 116 , 117 ] , an effect that depends upon the brain 's capacity to produce new ecs . given the suppressive effect of rt on cell proliferation , it is possible that the angiogenic effect of exercise would be limited in the post - rt brain . in short , it is possible that the effects of exercise would be dampened in the post - rt brain , reducing its potential as a stand - alone treatment . for example , stem cell replacement shows promise in the irradiated rodent brain and may eventually be possible in humans if the hostile environment created by rt can be made more permissive for growth and repair . exercise represents a viable means by which to achieve this , and future studies should address the potential of exercise to neutralize the hostile environment created by rt , as a preliminary step in restorative treatments . both human and animal studies indicate that suppressed cell proliferation and the hostile neural environment induced by cranial rt contribute to subsequent cognitive impairments . these effects of rt may be alleviated , at least to some extent , by exercise . it has been well established that exercise can both promote cell proliferation as well as foster a neural environment permissive for plastic change . early evidence from animal models indicates that exercise has the capacity to do both in the post - rt brain . however , further studies are needed , in order to determine whether rt - induced perturbations of the microenvironment could limit the plasticity - enhancing effects exercise has to offer .

brain cancer is a common type of childhood malignancy , and radiotherapy ( rt ) is a mainstay of treatment . rt is effective for tumor eradication , and survival rates are high . however , rt damages the brain and disrupts ongoing developmental processes , resulting in debilitating cognitive late effects that may take years to fully manifest . these late effects likely derive from a long - term decrement in cell proliferation , combined with a neural environment that is hostile to plasticity , both of which are induced by rt . long - term suppression of cell proliferation deprives the brain of the raw materials needed for optimum cognitive performance ( such as new neurons in the hippocampus and new glia in frontal cortex ) , while chronic inflammation and dearth of trophic substances ( such as growth hormone ) limit neuroplastic potential in existing circuitry . potential treatments for cognitive late effects should address both of these conditions . exercise represents one such potential treatment , since it has the capacity to enhance cell proliferation , as well as to promote a neural milieu permissive for plasticity . here , we review the evidence that cognitive late effects can be traced to rt - induced suppression of cell proliferation and hostile environmental conditions , as well as emerging evidence that exercise may be effective as an independent or adjuvant therapy .

1. Introduction 2. RT Disrupts Brain Development 3. RT-Induced Suppression of Cell Proliferation Contributes to Cognitive Impairments 4. RT Creates a Brain Milieu Hostile to Plasticity 5. The Restorative Potential of Exercise for the Post-RT Brain 6. Conclusions

PMC3046376

the velocity of scanning a whole glass slide reaches now in less than one minute with a sufficient number of focus points , and will probably further decrease with the new scanner generation . in addition to its main applications which are research ( such as tissue micro arrays ( tma ) , interdisciplinary case discussion as well as education in anatomy and pathology , the aims listed below are already under consideration : digital storage of routine cases , replacement of the conventional microscope by virtual microscopy , adjuvant automated measurements of stains and in situ hybridization ( immunohistochemistry ; ihc , fluorescent in situ hybridization ( fish ) , necessary for predictive pathological diagnosis ) , as well as the development of so - called diagnosis assistants to further support the pathologist in his / her daily work.[36 ] in addition to precise , robust , and standardized hardware ( scanners and their components such as optics , inbuilt cameras , image standards , and display stations ) , the software must communicate with databank systems and fulfill image - specific properties.[710 ] unfortunately , generally accepted standards do not exist neither for the hardware , nor for display of colored images or monitors . databank handling mainly addresses problems and standardization of storage , velocity , and retrieval of patients ' data , whereas , image - specific properties address features such as brightness , illumination , focus , and image size.[1113 ] the personal adjustment or general standardization of these parameters is mandatory for human - machine interaction , that is , a pathologist who wants to derive a diagnosis from an electronically presented histological image . this standardization is , however , not sufficient to investigate in analysis and potential - automated quantification and extraction of any image information . what is the information of a histological image ? how can it be described and measured ? finally , what are the basic algorithms to proceed to an automated information extraction ? or does this approach belong to the set of questions that can not be answered in principle ? image information in surgical pathology ( or tissue - based diagnosis ) is the morphology of a disease that can be " read by a trained person ( pathologist ) " . the contribution of a microscopic image to the pathologist 's diagnosis ( or to limit the pathologist 's freedom to state any diagnosis ) . obviously , not every image possesses information according to this definition . in addition , there are images which display a strong association with a certain disease , or allow an easy detection of information , others might be related with a broad variety of diseases , and it is difficult to derive a diagnosis . , if its presented information can be easily derived.[1416 ] on the other hand , those images are of poor quality if it is difficult ( or even impossible ) to detect a correlation with a diagnosis . thus , image quality is a main feature that influences the detection of image information . it is usually evaluated by comparing a derived image with the original one [ figure 1 ] . survey of basic image quality measurements - machine - oriented methods commonly deal with signal / noise ratio and compare image copies with the original one , whereas , subjective methods try to evaluate the influence of certain image parameters ( brightness , contrast , etc . ) on the interpretation of the viewer . in interactive virtual microscopy , image parameters can be modified according to the individual taste of the viewing pathologist . in automated application , an original image that serves for virtual slides derived from glass slides of different laboratories does not exist and has to be computed by normalization of grey value range and distribution the standardization of image quality is necessary for any reproducible measurement or interpretation , and the first step if we want to develop a quantitative method on image information . having images of good quality , we could then try to look for image properties that are related to information . in any image these properties are color features in relation to their image position , and include objects , structures , and texture . objects are items that can be identified ( interpreted ) and directly associated with a meaning , for example , trees , animals , buildings , or biological meaningful units such as vessels , cells , nuclei , chromosomes , genes , membranes , etc . if they are not known to the pathologist ( i.e. can not be associated with a diagnosis ) , they can only be described ( or measured ) . often these objects are multiple in an image , and their spatial relationship can be analyzed which are called structures . naturally , structures can be condensed to a new object , if they aggregate and display in translational symmetries such as rings , tubes , lines , etc.[2124 ] the described classic ( commonly applied by pathologists ) information approach correlates image features that are mainly detected objects and structures , with external information . the association function is the knowledge of the viewing pathologist . in other words , an object has to be known by the viewer in order to be identified . it is the implementation of the well - known sentence : you only do see what you see , i.e. , what you have seen and identified previously , and , therefore , you do already know ( < man erblickt nur , was man schon wei und versteht > , in a letter of j.w . illustration of an original image ( epithelioid mesothelioma , immunohistochemically stained with calretinin - a positive marker for mesothelioma ) , segmented objects , derived structure , and image texture ( computed with an autoregressive algorithm ) a different approach is the analysis of the basic image elements which are the pixels ( or voxels ) and their individual grey values . . it is crudely defined , and includes terms like coarseness , contrast , directionality , line - likeness , regularity , and roughness . voss calculated image texture by an autoregressive algorithm which results in reproducible and stable extraction of image information . the prerequisite for a reproducible and stable automated application of any of the mentioned algorithms is an image that has been normalized in order to minimize the influence of different laboratories ( fixation , tissue processing , staining intensity , etc . ) . it is not a simple task to describe image quality , although every pathologist intuitively can judge the quality of a histological image . in the literature , two different terms of image quality are distinguished : a ) that of individual ( emotional ) judgment and b ) that of parameter - based measurements . in addition , the influence of the image display ( monitor ) plays an important role and should be discussed separately . individual judgment of histological virtual slides has been performed in relation to image compression , to brightness , focus , and field of view . an image compression of 1:20 ( in jpg compression technique ) has been considered not to affect the pathologist 's ability to state a diagnosis . image presentation on a monitor should take into account nyquist 's theorem ( relationship between color wavelength and the resolution of the human eye ) as well as the natural field of view which is the angle of overlapping fields of view of the two human eyes ) . the computation yields a maximum display of about 25 cm 35 cm ( height length ) on a monitor at a distance of 1 m to the observer . to evaluate a diagnosis from larger fields of view takes more time and is more tiring , that is , one has to move the eyes or head , smaller ones are felt to have lost information . the color display of virtual slides is of additional significance if a diagnosis should be evaluated , although images derived from histological glass slides do not correspond to an original , that is , natural image . investigations of color measurements between the standard glass slide and the acquired virtual slides revealed striking differences in color presentation between scanners of different companies and even between different scanners of the same company . eosin ( h&e ) stained glass slide could allow an adjustment of display in order to correct electronically the variation in color , etc . such a still missing standardized virtual h and e slide could allow corrections in virtual microscopy prior to being viewed by a pathologist . for other than human - machine interaction purposes such as automated feature extraction , etc . , virtual slides have to be spatially standardized for background grey values ( vignetting ) , the range of grey values in each color space , and for the normalization of the grey value histogram ( having approximately the same grey values in the same number of pixels ) . it is also useful to perform the corrections after a gradient transformation on the original image ( differentiated image ) . the work on a differentiated image is an appropriate technique to search for membranes or other objects automatically . an example on how image standardization affects segmentation and grey value distribution is shown in [ figure 3 ] . example of influence of image standardization ( shading correction ) on the derived grey value histogram and accuracy of object segmentation ( artificial image consisting of randomly distributed balls and fibers ) the average grey value distance between the standardized and the original image can serve as a measure of quality of the original image . kayser et al , have shown that the measured corrections are smaller in fluorescent images compared to h&e stained or ihc ( diaminobenzidine ; dab , alkaline phosphatase ; ap ) stained images . the documentation of the measured aberrations allows the construction of so - called running curves . these can be used to evaluate or control the constancy or potential dynamics of image quality [ figure 4 ] . an internet assessable automated ihc , fish , nuclear organizer regions ( agnor ) , and comet fish measurement system ( eamus , www.diagnomx.eu ) has already implemented the described quality evaluation algorithms . example of monitoring formal image quality ( mean grey value distance of the original image from the corrected one ) having assessed the basic image quality , the next steps of image quality control depend upon the aims of the virtual microscopy system . most of the systems are constructed to segment and identify objects such as nuclei or membranes . the segmentation of objects only makes sense if object features can be derived from the detected objects . these include at the basic level area , mean grey value content , grey value distribution within the object , and the form factor . thus , the amount of pixels that present an object is closely related to the statistical accuracy to measure the basic and derived features . the magnification of a virtual slide should be relative to the size of the objects to be measured . an object should cover about 1000 pixels in order to allow a feature extraction with an accuracy of 3% . the detection of maxima and minima in the grey value histogram is , therefore , an indicator of the accuracy to potentially segment objects . in addition to the normalization of the grey value histogram , the number and significance of grey value maxima and minima are characteristics that describe suitable potential thresholds to separate an object area from the background.[21417183840 ] the grey value difference between a maximum and the associated minimum should amount to at least 5% of the histogram peak , if reproducible object segmentation is aimed . an example of evaluated potential thresholds in a standardized histological image ( epithelioid mesothelioma ) together with the summary of object segmentation prerequisites is depicted in [ figure 5 ] . illustration of grey value histogram , derived segmentation thresholds , and segmented objects ( epithelioid mesothelioma , h and e , same case as displayed in figure 2 ) an additional important , however not frequently implemented , image information parameter is the spatial arrangement of objects , which is called image structure . the majority of automated ( or semi - automated ) image analysis systems do not analyze structures , although the spatial arrangement of objects such as cancer cells is of great biological significance . it reflects , for example , to the approved grading of cancer cell types.[4345 ] a reproducible measurement of an image structure requires a ) reproducible identification of objects , b ) a sufficient number of objects , and c ) a reproducible ( and spatial independent ) definition of the applied neighborhood condition . independent from the applied neighborhood condition , at least 100 objects should serve to construct an image structure . if substructures ( clusters ) should be detected , the minimum number of image objects should amount to 1000 or even greater . virtual slides can meet these conditions as they allow investigations of different magnifications of the same selected tissue area . voronoi 's neighborhood condition ( and the derived dirichlet 's tessellation ) is the most frequently used one . the application of graph theory and the construction of the minimum spanning tree ( with or without distance limitations ) , or o'callaghan 's approach are also useful , especially in detection of clusters within the obtained structure . the viewing pathologist associates a certain meaning with the image information , which can be called the reaction of the pathologist . in mathematical terms , the pathologist 's meaning is a set of discrete items , such as diagnosis and differential diagnosis . the items are overlapping , if the pathologist is only able to derive a differential diagnosis . the maximum of the mapping procedure is directly related to the final or definite diagnosis . in a next step , the different contributors to the set of diagnosis ( texture , object , and structure ) can be separately analyzed . a consideration of the functions extremes reveals : a ) a detectable texture is a prerequisite to segment objects , and b ) segmented objects are a prerequisite to measure the built structure . to furthermore tune the algorithm , a virtual slide can be spatially analyzed for regions which display with no or only a few textures , and consequently do not contain objects and structures . thus , the image can be divided into two non - overlapping areas , namely , the object space and the background . the same procedure can then be applied for the object space only , and sub - areas displaying with the maximum of textures , objects , or structures can be separated . they can be reproducible identified by measures of the texture entropy and structural entropy based upon segmented objects [ figure 6 , as well as by application of spectral analysis , or by mimicking the pathologist 's pathway through a histological image . demonstration of automated selection of roi in a pleural biopsy ( epitheloid mesothelioma , h & e ) . the accuracy of the tested algorithm ( 2227 slides , biopsies obtained from colon , lung , pleura , and stomach ) revealed > 97% ) the traditional histological diagnosis which comprises a reproducible and detailed morphological description of the underlying disease has been extended by the terms " predictive diagnosis " and " risk - associated diagnosis " . risk - associated diagnosis is related to the individual risk of developing a certain disease ( for example , breast cancer associated with the brca1/2 gene expression ) , whereas , predictive diagnosis guides the individual patient 's therapy by analyzing the expression of certain genes in association with that of associated cellular or nuclear receptors . both diagnoses require precise and reproducible measurements or morphology - associated object features , either in the complete virtual slide or in carefully selected rois . algorithms to reproducibly measure the rois and to verify the correct selection have been successfully tested . experienced pathologists have been asked to state a diagnosis viewing the rois only , and , in addition , independently state the diagnosis by viewing the whole image . rois are , in addition , useful to select the biological important areas for tma , which is another technique to invest in predictive diagnosis or so - called individual cancer therapy . its present stage can be described as follows : commercially available scanners to acquire virtual slides and to perform virtual microscopy are in use for image storage and for educational purposes for several years . although they have not been approved by the fda to our knowledge , they have been remarkably matured , and are currently already equipped with software that addresses to reproducible measurements of image objects , especially for application in predictive diagnosis . the implementation of structure analysis is still in its childhood as well as the analysis of textures . image standardization focuses on the implementation of medical standards such as ( digital imaging and communications in medicine , dicom3 ) , and pathology - related changes of picture archiving and communication systems ( pacs ) . the necessary working groups have announced a release of a pathology addressed pacs in the near future . investigations on additional standardization of virtual slides to be applied for automated object segmentation and derived image information are in the stage of individual tests at present . image information in surgical pathology ( or tissue - based diagnosis ) is the morphology of a disease that can be " read by a trained person ( pathologist ) " . the contribution of a microscopic image to the pathologist 's diagnosis ( or to limit the pathologist 's freedom to state any diagnosis ) . obviously , not every image possesses information according to this definition . in addition , there are images which display a strong association with a certain disease , or allow an easy detection of information , others might be related with a broad variety of diseases , and it is difficult to derive a diagnosis . commonly , an image is called to be of good quality , if its presented information can be easily derived.[1416 ] on the other hand , those images are of poor quality if it is difficult ( or even impossible ) to detect a correlation with a diagnosis . thus , image quality is a main feature that influences the detection of image information . it is usually evaluated by comparing a derived image with the original one [ figure 1 ] . survey of basic image quality measurements - machine - oriented methods commonly deal with signal / noise ratio and compare image copies with the original one , whereas , subjective methods try to evaluate the influence of certain image parameters ( brightness , contrast , etc . ) on the interpretation of the viewer . in interactive virtual microscopy , image parameters can be modified according to the individual taste of the viewing pathologist . in automated application , an original image that serves for virtual slides derived from glass slides of different laboratories does not exist and has to be computed by normalization of grey value range and distribution the standardization of image quality is necessary for any reproducible measurement or interpretation , and the first step if we want to develop a quantitative method on image information . having images of good quality , we could then try to look for image properties that are related to information . in any image these properties are color features in relation to their image position , and include objects , structures , and texture . objects are items that can be identified ( interpreted ) and directly associated with a meaning , for example , trees , animals , buildings , or biological meaningful units such as vessels , cells , nuclei , chromosomes , genes , membranes , etc . if they are not known to the pathologist ( i.e. can not be associated with a diagnosis ) , they can only be described ( or measured ) . often these objects are multiple in an image , and their spatial relationship can be analyzed which are called structures . naturally , structures can be condensed to a new object , if they aggregate and display in translational symmetries such as rings , tubes , lines , etc.[2124 ] the described classic ( commonly applied by pathologists ) information approach correlates image features that are mainly detected objects and structures , with external information . the association function is the knowledge of the viewing pathologist . in other words , it is the implementation of the well - known sentence : you only do see what you see , i.e. , what you have seen and identified previously , and , therefore , you do already know ( < man erblickt nur , was man schon wei und versteht > , in a letter of j.w . illustration of an original image ( epithelioid mesothelioma , immunohistochemically stained with calretinin - a positive marker for mesothelioma ) , segmented objects , derived structure , and image texture ( computed with an autoregressive algorithm ) a different approach is the analysis of the basic image elements which are the pixels ( or voxels ) and their individual grey values . . it is crudely defined , and includes terms like coarseness , contrast , directionality , line - likeness , regularity , and roughness . voss calculated image texture by an autoregressive algorithm which results in reproducible and stable extraction of image information . the prerequisite for a reproducible and stable automated application of any of the mentioned algorithms is an image that has been normalized in order to minimize the influence of different laboratories ( fixation , tissue processing , staining intensity , etc . ) . it is not a simple task to describe image quality , although every pathologist intuitively can judge the quality of a histological image . in the literature , two different terms of image quality are distinguished : a ) that of individual ( emotional ) judgment and b ) that of parameter - based measurements . in addition , the influence of the image display ( monitor ) plays an important role and should be discussed separately . individual judgment of histological virtual slides has been performed in relation to image compression , to brightness , focus , and field of view . an image compression of 1:20 ( in jpg compression technique ) has been considered not to affect the pathologist 's ability to state a diagnosis . image presentation on a monitor should take into account nyquist 's theorem ( relationship between color wavelength and the resolution of the human eye ) as well as the natural field of view which is the angle of overlapping fields of view of the two human eyes ) . the computation yields a maximum display of about 25 cm 35 cm ( height length ) on a monitor at a distance of 1 m to the observer . to evaluate a diagnosis from larger fields of view takes more time and is more tiring , that is , one has to move the eyes or head , smaller ones are felt to have lost information . the color display of virtual slides is of additional significance if a diagnosis should be evaluated , although images derived from histological glass slides do not correspond to an original , that is , natural image . investigations of color measurements between the standard glass slide and the acquired virtual slides revealed striking differences in color presentation between scanners of different companies and even between different scanners of the same company . an analysis of a standard image derived from a color standardized hematoxylin / eosin ( h&e ) stained glass slide could allow an adjustment of display in order to correct electronically the variation in color , etc . such a still missing standardized virtual h and e slide could allow corrections in virtual microscopy prior to being viewed by a pathologist . for other than human - machine interaction purposes such as automated feature extraction , etc . , virtual slides have to be spatially standardized for background grey values ( vignetting ) , the range of grey values in each color space , and for the normalization of the grey value histogram ( having approximately the same grey values in the same number of pixels ) . it is also useful to perform the corrections after a gradient transformation on the original image ( differentiated image ) . the work on a differentiated image is an appropriate technique to search for membranes or other objects automatically . an example on how image standardization affects segmentation and grey value distribution is shown in [ figure 3 ] . example of influence of image standardization ( shading correction ) on the derived grey value histogram and accuracy of object segmentation ( artificial image consisting of randomly distributed balls and fibers ) the average grey value distance between the standardized and the original image can serve as a measure of quality of the original image . kayser et al , have shown that the measured corrections are smaller in fluorescent images compared to h&e stained or ihc ( diaminobenzidine ; dab , alkaline phosphatase ; ap ) stained images . the documentation of the measured aberrations allows the construction of so - called running curves . these can be used to evaluate or control the constancy or potential dynamics of image quality [ figure 4 ] . an internet assessable automated ihc , fish , nuclear organizer regions ( agnor ) , and comet fish measurement system ( eamus , www.diagnomx.eu ) has already implemented the described quality evaluation algorithms . example of monitoring formal image quality ( mean grey value distance of the original image from the corrected one ) having assessed the basic image quality , the next steps of image quality control depend upon the aims of the virtual microscopy system . most of the systems are constructed to segment and identify objects such as nuclei or membranes . the segmentation of objects only makes sense if object features can be derived from the detected objects . these include at the basic level area , mean grey value content , grey value distribution within the object , and the form factor . additional features such as moments , entropy , etc . , are derivatives from the basic features . thus , the amount of pixels that present an object is closely related to the statistical accuracy to measure the basic and derived features . the magnification of a virtual slide should be relative to the size of the objects to be measured . an object should cover about 1000 pixels in order to allow a feature extraction with an accuracy of 3% . the detection of maxima and minima in the grey value histogram is , therefore , an indicator of the accuracy to potentially segment objects . in addition to the normalization of the grey value histogram , the number and significance of grey value maxima and minima are characteristics that describe suitable potential thresholds to separate an object area from the background.[21417183840 ] the grey value difference between a maximum and the associated minimum should amount to at least 5% of the histogram peak , if reproducible object segmentation is aimed . an example of evaluated potential thresholds in a standardized histological image ( epithelioid mesothelioma ) together with the summary of object segmentation prerequisites is depicted in [ figure 5 ] . illustration of grey value histogram , derived segmentation thresholds , and segmented objects ( epithelioid mesothelioma , h and e , same case as displayed in figure 2 ) an additional important , however not frequently implemented , image information parameter is the spatial arrangement of objects , which is called image structure . the majority of automated ( or semi - automated ) image analysis systems do not analyze structures , although the spatial arrangement of objects such as cancer cells is of great biological significance . it reflects , for example , to the approved grading of cancer cell types.[4345 ] a reproducible measurement of an image structure requires a ) reproducible identification of objects , b ) a sufficient number of objects , and c ) a reproducible ( and spatial independent ) definition of the applied neighborhood condition . independent from the applied neighborhood condition , at least 100 objects should serve to construct an image structure . if substructures ( clusters ) should be detected , the minimum number of image objects should amount to 1000 or even greater . virtual slides can meet these conditions as they allow investigations of different magnifications of the same selected tissue area . voronoi 's neighborhood condition ( and the derived dirichlet 's tessellation ) is the most frequently used one . the application of graph theory and the construction of the minimum spanning tree ( with or without distance limitations ) , or o'callaghan 's approach are also useful , especially in detection of clusters within the obtained structure . potential histological image features that contain all image information are the described texture , objects , and structure . the viewing pathologist associates a certain meaning with the image information , which can be called the reaction of the pathologist . in mathematical terms , the pathologist 's meaning is a set of discrete items , such as diagnosis and differential diagnosis . the items are overlapping , if the pathologist is only able to derive a differential diagnosis . the maximum of the mapping procedure is directly related to the final or definite diagnosis . in a next step , the different contributors to the set of diagnosis ( texture , object , and structure ) can be separately analyzed . a consideration of the functions extremes reveals : a ) a detectable texture is a prerequisite to segment objects , and b ) segmented objects are a prerequisite to measure the built structure . to furthermore tune the algorithm , a virtual slide can be spatially analyzed for regions which display with no or only a few textures , and consequently do not contain objects and structures . thus , the image can be divided into two non - overlapping areas , namely , the object space and the background . the same procedure can then be applied for the object space only , and sub - areas displaying with the maximum of textures , objects , or structures can be separated . they can be reproducible identified by measures of the texture entropy and structural entropy based upon segmented objects [ figure 6 , as well as by application of spectral analysis , or by mimicking the pathologist 's pathway through a histological image . demonstration of automated selection of roi in a pleural biopsy ( epitheloid mesothelioma , h & e ) . the accuracy of the tested algorithm ( 2227 slides , biopsies obtained from colon , lung , pleura , and stomach ) revealed > 97% ) the traditional histological diagnosis which comprises a reproducible and detailed morphological description of the underlying disease has been extended by the terms " predictive diagnosis " and " risk - associated diagnosis " . risk - associated diagnosis is related to the individual risk of developing a certain disease ( for example , breast cancer associated with the brca1/2 gene expression ) , whereas , predictive diagnosis guides the individual patient 's therapy by analyzing the expression of certain genes in association with that of associated cellular or nuclear receptors . both diagnoses require precise and reproducible measurements or morphology - associated object features , either in the complete virtual slide or in carefully selected rois . algorithms to reproducibly measure the rois and to verify the correct selection have been successfully tested . experienced pathologists have been asked to state a diagnosis viewing the rois only , and , in addition , independently state the diagnosis by viewing the whole image . rois are , in addition , useful to select the biological important areas for tma , which is another technique to invest in predictive diagnosis or so - called individual cancer therapy . its present stage can be described as follows : commercially available scanners to acquire virtual slides and to perform virtual microscopy are in use for image storage and for educational purposes for several years . although they have not been approved by the fda to our knowledge , they have been remarkably matured , and are currently already equipped with software that addresses to reproducible measurements of image objects , especially for application in predictive diagnosis . the implementation of structure analysis is still in its childhood as well as the analysis of textures . image standardization focuses on the implementation of medical standards such as ( digital imaging and communications in medicine , dicom3 ) , and pathology - related changes of picture archiving and communication systems ( pacs ) . the necessary working groups have announced a release of a pathology addressed pacs in the near future . investigations on additional standardization of virtual slides to be applied for automated object segmentation and derived image information are in the stage of individual tests at present . diagnostic surgical pathology or tissue - based diagnosis is changing its face as it combines the latest progress and knowledge of molecular biology and genetics with innovative development of computerized information technology . they serve , however , only as starting point to invest in biological properties and gene expressions of individual patient 's disease , especially cancer . tissue - based diagnosis is now - a - days the trusted guide in the treatment of a broad variety of cancer such as breast , colon , or lung cancer . quantification of content - based image information is the contribution of information technology to furthermore develop and expand the combination of alteration , expression , and disease - associated impact of genes and their interaction . the analysis of this information is based upon three main components only , namely image texture , objects , and structure . it could , therefore , serve for reliable forecasting of disease development . a precise definition of a suitable neighborhood condition that includes functional properties and the evaluation of rare events , which act only as catalytic converters are still missing to our knowledge . therefore , the described , already implemented tools of tissue - based diagnosis and content - based image information analysis can be considered as now - a - days detected entrance into a new world of disease understanding and treatment . as a result , diagnostic surgical pathology is getting more responsibility and closer to the treatment and fate of patients , because it is incorporating recently developed biology and it technology .

virtual microscopy , which is the diagnostic work on completely digitized histological and cytological slides as well as blood smears , is at the stage to be implemented in routine diagnostic surgical pathology ( tissue - based diagnosis ) in the near future , once it has been accepted by the us food and drug administration . the principle of content - based image information , its mandatory prerequisites to obtain reproducible and stable image information as well as the different compartments that contribute to image information are described in detail . automated extraction of content - based image information requires shading correction , constant maximum of grey values , and standardized grey value histograms . the different compartments to evaluate image information include objects , structure , and texture . identification of objects and derived structure depend on segmentation accuracy and applied procedures ; textures contain pixel - based image information only . all together , these image compartments posses the discrimination power to distinguish between object space and background , and , in addition , to reproducibly define regions of interest ( rois ) . rois are image areas which display the information that is of preferable interest to the viewing pathologist . they contribute to the derived diagnosis to a higher level when compared with other image areas . the implementation of content - based image information algorithms to be applied for predictive tissue - based diagnoses is described in detail .

INTRODUCTION Basic Considerations Image Quality and Standardization Extraction of Image Information Applications CONCLUSION

PMC3675674

bacterial toxins of the cholesterol - dependent cytolsysin ( cdc ) superfamily form pores in lipid bilayers through the oligomerization of their membrane attack complex / perforin fold ( macpf ) domain . cdcs are produced by over five different genera of gram - positive bacteria , resulting in a wide range of animal and human diseases ( tweten et al . , 2001 ) . intermedilysin ( ily ) is a cdc secreted by streptococcus intermedius that causes brain and liver abscesses in its host ( nagamune et al . , 1996 ) and is thought to be the major virulence factor for the bacterium ( nagamune et al . , 1996 ) . cholesterol is essential for ily cytolytic activity , and crystal structures of ily and other cdcs show strong structural homology ( bourdeau et al . , 2009 ; polekhina et al . , 2005 ; rossjohn et al . , 1997 ; rossjohn et al . , 2007 ; xu et al . , 2010 ) most cdcs bind to cholesterol - rich membranes via a cholesterol recognition motif ( crm ) and neighboring undecapeptide . this binding triggers the oligomerization and allosteric regulation of the transition to the pore state ( dowd and tweten , 2012 ) . ily , on the other hand , has evolved to recruit the human complement regulator cd59 as a receptor providing the basis for its species specificity ( giddings et al . , 2004 ) . it is cd59-binding that promotes both oligomerization of ily monomers on the cell surface ( lachapelle et al . , 2009 ) and the structural rearrangements required for pore formation ( dowd and tweten , 2012 ) . in contrast , the interaction of cholesterol with the crm and undecapeptide is required for the maintenance of the ily prepore at the membrane as cd59 is released ( lachapelle et al . , mutational analysis of ily has determined residues involved in both lipid and receptor binding ( wickham et al . , 2011 ) as well as those crucial for mediating structural transitions ( dowd and tweten , 2012 ; hotze et al . , 2012 ; lachapelle et al . , 2009 ; our structure of the ily - cd59 complex now provides a framework with which to integrate these biochemical and biophysical data . cd59 is the only membrane - bound inhibitor of the complement terminal pathway ( meri et al . , this small ( 77 amino acid ) gpi - anchored cell - surface receptor binds complement components c8 and c9 ( ninomiya and sims , 1992 ) and directly inhibits the formation of the membrane attack complex ( mac ) , an immune pore that kills microbes by the disruption of lipid bilayers . cd59 is expressed on a variety of cell types , including leukocytes , epithelial , and endothelial cells ( meri et al . , 1991 ) and plays an important role in preventing complement - mediated tissue damage in hosts . cd59 has been implicated in a number of human diseases , including tumor cell evasion during monoclonal antibody treatments ( fishelson et al . , 2003 ; gorter and meri , 1999 ) and paroxysmal nocturnal hemoglobinuria ( hill et al . , 2006 ) . structural studies of soluble cd59 ( fletcher et al . , 1994 ; kieffer et al . , 1994 ; leath et al . , 2007 ) and direct binding assays of cd59 with complement components ( huang et al . , 2006 ) have provided some insight into the role of this receptor in regulating mac assembly . however , a lack of structural information characterizing cd59 with its binding partners has limited our ability to control complement susceptibility . here , we report the crystal structure of cd59 bound to ily , a bacterial toxin that directly competes for mac binding . we define two interfaces on cd59 that coordinate ily monomers and validate this interaction through the development of a liposome - based model system . we also find that an ily - derived peptide based on our structure disrupts the interface and competes for pore formation . in addition , cryo - electron microscopy ( cryo - em ) images of the crystallized ily mutant bound to cd59-decorated liposomes suggest that the linear arrays observed in our crystal could also occur in the context of a membrane . altogether , our structural and functional data provide mechanistic insight into how cd59 engages ily for the formation of a pore in lipid bilayers , providing a basis for the development of new antibiotics as well as potential therapeutics that target cd59 . an early prepore intermediate comprising the initial attachment of ily to cd59 was captured with a disulfide - locked ily mutant ( ily ) and a cd59 construct lacking the gpi anchor . the complex was assembled in vitro , subjected to further purification , and crystallized . the crystals diffracted to a resolution of 3.5 , and the structure was solved by molecular replacement . the final model , consisting of all four domains of ily and the ectodomain of cd59 , was refined to a final rwork / rfree of 21.2%/26.6% and displayed good geometry ( figures 1 and s1a ; table 1 ) . as described previously , ily is comprised of four structural domains , the first three of which are formed from discontinuous stretches of sequence . domain 1 ( d1 ) ( residues 5676 , 117201 , 258299 , and 379399 ) forms a cap at the top of the molecule and is the least well - ordered region of our crystal ( figure s1b ) . domain 2 ( d2 ) ( residues 77116 and 400417 ) consists of three kinked strands that span 50 and bridge domains 1 and 4 . domain 3 ( d3 ) ( residues 202257 and 300378 ) packs against d2 and contains the helical bundles that are predicted to refold into transmembrane -hairpins . this domain , along with d1 , makes up the macpf domain and also contains the site of the engineered disulfide bond ( c346-c361 ) that prevents the structural rearrangements required for full pore formation . finally , domain 4 ( d4 ) ( residues 418528 ) is a discrete sandwich structure extending from the end of d2 . cd59 is a compact , heavily disulfide - bonded structure with a central sheet bookended by a short helix and a two - strand hairpin . the asymmetric unit of the crystal contains two copies of the ily - cd59 complex , which overlay with an rmsd of 1.0 over 530 residues . cd59 predominantly interacts with d4 of ily ( figure 1a ) , which is in agreement with previous studies ( nagamune et al . 2005 ) . strikingly , there are two major ily - cd59 interfaces within the crystal : one within the asymmetric unit and one between neighboring unit cells ( figure 1b ) . the primary ily - cd59 binding site ( 665 buried surface area ) is composed of a side - to - side contact between the hairpin extension of ily d4 ( the 2-3 loop ) and the core sheet of cd59 ( figure 1c ) , effectively creating an extension of the cd59 sheet . the interface is further stabilized by the stacking of hydrophobic side - chains ( ily : y436 , ily : i450 , cd59:f47 , cd59:y61 , and cd59:f42 ) as well as an extensive network of hydrogen bonds ( both sidechain - sidechain and sidechain - backbone ) and salt bridges ( ily : r480 , cd59:e58 ; ily:448 , cd59:k65 ; ily : d445 , and cd59:k66 ) . this interface is supported by two independent mutagenesis studies ( hughes et al . , 2009 ; specifically , ily double ( y434a / y436a ) and triple ( s499n / k500r / n501 t ) mutants located within our primary cd59 interface ( figure 1c ) dramatically inhibited pore formation and decreased the affinity of ily for cd59 . in addition , double- and single - point mutations in cd59 have implicated aromatic residues that map to this interface ( f42 , f47 , and y62 ) as being involved in ily binding ( wickham et al . , 2011 ) . the secondary binding site is formed by the same region of ily ( the 2-3 loop in d4 ) interacting with cd59 from a neighboring unit cell . this interface buries 500 and is stabilized by a series of hydrogen bonds and three salt bridges ( ily : d443 , cd59:r55 ; ily : d445 , cd59:k38 ; ily : e93 , and cd59:k41 ) . the second interface is again validated by previous mutagenesis studies of both ily and cd59 ( wickham et al . , 2011 ) . in particular , a double mutation in ily ( r451a / s452a ) that reduces cd59-binding and hemolysis maps to this region ( figure 1d ) . cd59 mutations d22 and f23 , also located in this interface ( figure 1d ) , play a role in ily binding and function . therefore , cd59 displays two distinct binding sites for ily that can be simultaneously occupied . this suggests that , in addition to localizing ily to the membrane surface , cd59 helps nucleate an early prepore complex by bridging neighboring ily molecules , thereby creating the linear array observed in our crystals . to address the functional significance of the ily - cd59 interfaces observed in our crystal structure , we developed a simple model membrane system to probe ily pore formation in a fluorescence - based assay ( koer et al . , 2007 ) . calcein - containing liposomes were decorated with soluble cd59 and modified to possess a myristoylated lysine - rich cytotopic peptide ( fraser et al . , 2003 ) . lipid attachment of cytotopic cd59 was confirmed by the colocalization of protein in lipid - containing fractions across a ficoll gradient ( figure 2a ) . the fluorescence intensity of calcein released from liposomes upon lysis was used to determine the rate of pore formation . exposure of wild - type ily ( ily ) with cholesterol - containing cd59-decorated liposomes resulted in functional transmembrane pores ( figure 2b ) , whereas undecorated liposomes did not support lysis ( figure 2b ) . furthermore , incubation of liposomes with cytoptic cd59 alone did not leak calcein ( figure 2b ) . to confirm binding of the ily hairpin extension to cd59 , a peptide comprised of ily residues 438452 was synthesized with bridging n- and c - terminal cysteines . the peptide successfully competed for cd59 binding and inhibited ily pore formation with an ic50 of approximately 25 m ( figures 2c and s2 ) . to understand how cd59 coordinates ily monomers to form the pore , we characterized the complex in a lipid environment using electron microscopy . because negatively stained specimens could lead to distortions of liposome curvature and possible liposome collapse , we used cryo - em to visualize membrane - bound complexes . examination of cryo - em fields of ily - cd59-liposome complexes revealed a population of liposomes with multiple instances of embedded circular pores ( figure 3a ) . density is clearly visible for the ily protein rings with obvious disruption to the lipid bilayer . the diameter of the ily pore is approximately 300 , consistent with the dimensions previously reported for other cdc and perforin pores ( law et al . , 2010 ; tilley et al . , 2005 ) . to test the ability of the crystallized ily mutant ( ily ) to form linear arrays in the context of a membrane , we examined two - dimensional cryo - em images of the mutant form bound to cd59-decorated liposomes ( figure 3b ) . as expected , no perforation of the lipid bilayer was observed with this prepore intermediate . protein density on the periphery of liposomes appears to be organized in linear clusters ; however , due to the fragility of the system , we were unable to collect in - plane images to confirm this . reversible linear arrays of a prepore - trapped mutant form of perfringolysin have demonstrated that cdcs undergo transient , yet specific , intermolecular interactions that precede ring closure ( hotze et al . , 2012 ) . these data suggest that the linear organization of ily - cd59 complexes observed in our crystal structure is relevant for understanding how ily engages with cd59 in the context of a membrane . previously reported cryo - em reconstructions of the pneumolysin prepore and pore structures have provided a model for how cdcs oligomerize on membranes ( tilley et al . , 2005 ) . preserving the two ily - cd59 interfaces observed in our crystal structure , we used this as a starting point to turn the linear array of our lattice into a model for the ily early prepore state ( see experimental procedures for details ) . this arrangement places cd59 at the periphery of the ring ( figure 4 ) , and we find that the presence of cd59 would not obstruct ily oligomerization , in concordance with data showing that cd59 remains bound during prepore assembly ( lachapelle et al . , 2009 ) . these data , along with the high concentration of cd59 on the cell surface ( anstee , 1990 ) , have led us to include cd59 in our model of an ily prepore oligomeric ring . the subsequent release of cd59 during the pore transition , observed previously by biophysical studies ( lachapelle et al . , 2009 ) , could be due in part to conformational changes that are incompatible with maintaining cd59 in the complex . in our model , the c - terminal residue of cd59 preceding the gpi anchor is positioned approximately 15 from the plane of the membrane ( figure 4 ) . this observation is consistent with distances measured for other gpi - anchored proteins ( lehto and sharom , 2002 ) and with a model previously proposed for cd59 glycosylation ( rudd et al . , 1997 ) . therefore , our structural and functional data together suggest that the role of cd59 in ily pore formation is to orient the ily crm and undecapeptide proximal to cholesterol - containing lipid bilayers and to propagate ily monomer - monomer interactions through two binding interfaces . polekhina et al . , 2005 ; rossjohn et al . , 1997 ; rossjohn et al . , 2007 ; xu et al . , 2010 ) and components of the complement mac ( aleshin et al . , 2012 ; hadders et al . , 2007 ; 2011 ) reveal that the fundamental pore - forming mechanism of these macpf - containing proteins are similar . however , within this conserved mechanism , one of the most important differences remains in its regulation . for many cdcs , membrane binding by the crm and undecapeptide initiates structural transitions ( dowd and tweten , 2012 ) , which are propagated between membrane - bound monomers ( hotze et al . , 2012 ) . circularization of the cdc oligomeric ring then triggers a dramatic conformational change in which helical segments from d3 convert to amphipathic hairpins and arrange into a transmembrane -barrel pore ( tilley et al . , 2005 ) . for a subclass of cdcs , of which ily is an archetypal member , control of regulating prepore transitions is transferred from the undecapeptide to cd59 binding ( dowd and tweten , 2012 ) . our structural and functional analysis of the ily - cd59 complex supports a model for these pores in which membrane - anchored cd59 recruits soluble ily monomers to the plasma membrane of human cells , orienting the crm and undecapeptide for insertion into cholesterol - containing lipid bilayers . through two distinct ily - binding interfaces on opposite sides of the molecule , cd59 brings together ily monomers in order to facilitate intermolecular interactions that drive the formation of an oligomeric prepore ring . relatively small alterations to the packing of our crystal lattice produces a 31-fold symmetric model for this early prepore state that agrees with a pneumolysin prepore model ( tilley et al . , 2005 ) . even smaller manipulations of our lattice produce alternate models for ily pores in line with the proposed stoichiometries observed for other cdcs by different biophysical techniques ( 31- to 44-fold ) ( czajkowsky et al . , 2004 ; tilley et al . , monomer - monomer interactions of cdcs have been shown to propagate early structural transitions required for pore formation ( hotze et al . , 2012 ) . specifically , the positioning of neighboring molecules has been suggested to allow the intermolecular pairing of 1 from d3 of one molecule with 4 from the equivalent domain of the next . analysis of the impact of applying curvature to our linear array shows that these contacts are indeed partially formed in our model ( movie s1 ) . in order to complete the zippering of the sheet - formation , 5 would need to swing out of the way . this transition is prevented in our crystal by the engineered disulfide bond in ily locking 5 to 4 , thus trapping the earliest prepore state . although ily and mac components ( c8 and c9 ) all bind cd59 , the result of this interaction has profoundly different outcomes on pore formation . although cd59 promotes the oligomerization of ily monomers ( lachapelle et al . , 2009 ) , the receptor inhibits mac formation by preventing membrane perforation and incorporation of multiple c9 molecules ( farkas et al . , 2002 ; meri et al . , 1990 ; rollins and sims , 1990 ) . ily directly competes for the mac binding site on cd59 ( lachapelle et al . , 2009 ; wickham et al . , 2011 ) , and a mutational analysis has confirmed that the ily and mac binding sites on the surface of cd59 overlap , at least in part ( bodian et al . , 1997 ; huang et al . , 2005 ; wickham et al . , 2011 ) . cd59-binding mac components lack the structural equivalent of ily d4 , therefore precluding further insight into how specific residues of c8 and c9 interact with the complement receptor . however , it is possible that cd59 engages the transmembrane segments of complement components after their transition to hairpins but prior to membrane insertion , similar to the interaction of cd59 with the hairpin extension of ily d4 . it is interesting to note that the residues on cd59 involved in mac binding map to the same two opposing faces observed in our ily interaction . cd59 inhibits mac assembly most efficiently when incorporated prior to the addition of c9 ( lehto and meri , 1993 ) . subsequently , it appears to lock a single c9 molecule into the complex in a conformation that prevents membrane perforation and further c9 recruitment ( lehto and meri , 1993 ; meri et al . , these observations , along with the ily - cd59 crystal structure , suggest a mechanism for mac inhibition whereby cd59 intercalates between c8 and c9 in an analogous manner to the ily nucleation , locking the mac in a nonfunctional state . in summary , our findings provide mechanistic insights into how ily pores form , revealing a dual role of cd59 in facilitating the assembly of the oligomeric prepore . our structural and functional data have defined two cd59-ily interaction interfaces , providing a framework for the development of new antibiotics and cd59-targeted therapeutics that could improve human health . recombinant soluble human cd59 with an additional c - terminal cysteine residue was expressed in e.coli , refolded , and purified from solubilized inclusion bodies as described previously ( leath et al . , 2007 ) . purified cd59 ( approximately 0.5 mm in phosphate - buffered saline [ ph7.3 ] pbs ) was treated for 16 hr at ambient temperature with two molar equivalents of tris-2-carboxyethyl phosphine ( tcep , sigma - aldrich ) for the generation of a free thiol at the carboxyl terminal cysteine of the protein . this was incubated for 2 hr at ambient temperature with a 3-fold molar excess of the cytotopic modification reagent bis - myristoyl lysyl sskkspskkddkkpgd ( s-2-thiopyridyl)-cysteine acid ( apt3146 , cambridge research biochemicals ) for the generation of cytotopic cd59 ( hill et al . , 2006 ) . the main protein peak fractions were pooled , buffer exchanged into pbs , and stored at 70c until use . an ily gene lacking cysteine residues ( ily ) and a disulfide - locked ily mutant containing the substitutions t346c and i361c ( ily ) were cloned into a ptrchisa vector and were generous gifts from r. tweten . this early prepore form of ily was characterized previously ( lachapelle et al . , 2009 ) and shown to be converted to functional pores by the addition of reducing agent . cells were grown to an optical density of 0.6 at 37c and induced with 0.5 mm iptg for 4 hr . cultures were pelleted and lysed in a buffer containing 1% triton , 200 mm nacl , 20 mm tris ( ph 6.8 ) , protease inhibitor mixture ( sigma - aldrich , 50 l per gram of cell pellet ) , 100 g of dnase i , and 100 g of rnase a ( sigma - aldrich ) . cleared lysates were loaded onto cobalt - chelated talon beads ( clontech ) and eluted with 500 mm imidazole . ily was further purified by size exclusion chromatography in 150 mm nacl and 20 mm tris ( ph 6.8 ) . ily and cd59 were incubated at room temperature in a 1:2 molar ratio , and the complex was separated on a superdex 200 hr 10/30 column ( pharmacia biotech ) . crystals were grown by vapor diffusion at room temperature in 96-well plates with 0.4 l droplets at 21c . equal volumes of preformed ily - cd59 complex ( 9.9 mg / ml in 150 mm nacl , and 20 mm tris [ ph 6.8 ] ) and precipitant ( 20% peg6000 , 0.1 m hepes [ ph 7 ] , and 0.2 m licl ) were mixed . crystals were cryo - protected with reservoir solution plus 20% glycerol before cryo - cooling in liquid nitrogen . data were collected at diamond light source ( harwell science and innovation ) at beamline i04 . diffraction data were processed with xia2 ( winter , 2010 ) in 3diir mode and initially assigned as p21 with a single copy of the complex per asymmetric unit ( indexing initially suggested c222 ( 1 ) , but this failed upon scaling , see the twinning discussion below ) . the structure of the ily - cd59 complex was solved by molecular replacement with the use of phaser ( mccoy et al . , 2007 ) . a solution was reached with the use of the known structures of ily ( pdb 1s3r ) ( polekhina et al . , 2005 ) and cd59 ( pdb 2uwr ) ( leath et al . , 2007 ) , with the ily split into two search models encompassing d1d3 and d4 . . , 2010 ) suggested that the data were twinned , and the structure was refined with phenix ( adams et al . , 2010 ) with the pseudomerohedral twinning operator ( h ,- k ,- analysis of the resultant maps showed poor density for large stretches of d1 with no extra density or residuals , and the rfree did not drop below 0.30 . therefore , we reprocessed the data in p1 ( xia2 , 3da mode ) and resolved the structure with two copies of the complex in the asymmetric unit . analysis of the p1 data set with xtriage suggested twinning with only one of the three possible pseudomerohedral twinning operators ( h ,- k - l ) significantly occupied ( twin fraction = 0.46 ) . although the data merge well to 3.5 in both p21 ( rpim = 0.077 , rmerge = 0.105 ) and p1 ( rpim = 0.098 , rmerge = 0.111 ) , refinement carried out in the p1 data set in phenix with the h ,- after rebuilding in coot ( emsley et al . , 2010 ) , the rwork / rfree converged to 0.212/0.266 ( compared to 0.248/0.300 for the same model in the p21 data ) . furthermore , the p1 maps showed clear features that differed between the two independent copies of the complex in particular , the engineered disulfide ( cys346-cys361 ) was clearer in one copy and the pattern of thermal motion factors over the two molecules reproducibly refined to different distributions . some stretches of d1 still had weak or no electron density associated with the model , although this varied between the two copies . however , refinement statistics worsen when these regions are removed , and molecular replacement of this domain placed it with z score > 15 in phaser . therefore , we decided to refine d1 in place and not rebuild in the density . the final refined p1 model gave good molprobity statistics for the resolution ( molprobity score = 2.28 , 99 percentile ) . running xtriage with the final refined model in p1 gave r versus r statistics ( lebedev et al . l operator of 0.132/0.373 ( robs / rcalc ) , consistent with twinning with rotational pseudosymmetry . a model for an early prepore was generated from the crystal lattice by bending a linear array containing 31 copies of the 1:1 ily - cd59 complex into a circle . in brief , the linear array was placed onto the deposited pneumolysin prepore model ( pdb 2bk2 ) by the superposition of d1d3 with secondary structure mapping ( lsqkab ) . then , a 15.3 rotation was applied to make the linear array tangential to the circular prepore density ( emdb 1106 ) . the linear array was then bent into the circular form by the application of a 0 , 0 , 11.6 rotation ( polar angles ) to a monomer . the cd59 n - linked glycan on asn18 and the gpi anchor were modeled with the coordinates described in rudd et al . , 1997 , and kindly supplied by m. wormald . 1,2-dioleoyl - sn - glycero-3-phospho - l - serine ( dops ) , 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine ( dope ) , 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc ) , and cholesterol ( supplied in chloroform from avanti polar lipids ) were mixed in molar ratios of 3:3:2:2 , respectively . chloroform was evaporated with a stream of nitrogen gas , leaving a thin lipid film . dried lipid was rehydrated with four freeze , thaw , and vortex cycles followed by extrusion through a 0.1 m pore - size membrane ( avanti ) for the formation of a homogeneous population of unilamellar liposomes , freshly prepared for each experiment . fluorescently - labeled liposomes were generated by in the inclusion of 50 mm calcein ( sigma - aldrich ) in the rehydration buffer . this initial calcein concentration of 50 mm is sufficient to cause almost complete self - quenching of its fluorescence ; if calcein is subsequently released from the liposomes , its concentration is reduced and an increase in fluorescence is observed . unencapsulated dye was removed by passage through a g-50 sephadex column ( sigma - aldrich ) run in a buffer containing 500 mm sucrose . lipids were tracked by the inclusion of phosphatidylethanolamine with lissamine rhodamine b - labeled head groups ( avanti ) in the lipid mixture described above at a final concentration of 2% ( w / w ) prior to the evaporation of chloroform . mg / ml were incubated with either soluble or cytotopic cd59 at a final concentration of 18 m . the liposome sample was adjusted to contain ficoll at a final concentration of 20% ( w / v ) and was overlaid with 10% ( w after centrifugation at 100,000 g at 20c for 45 min , gradients were fractionated from the top . fractions were analyzed for the presence of liposomes by the visualization of rhodamine and for the presence of cd59 by sds - page and coomassie staining . pore - formation assays were performed at 20c in a varian cary eclipse fluorescence spectrophotometer ( agilent technologies ) with excitation and emission wavelengths of 490 nm and 520 nm , respectively , and a slit width of 5 nm . the kinetics mode of the spectrophotometer was used with an average read time of 0.15 s , and measurements were made every minute . a peptide corresponding to the cd59-binding interface of ily ( residues 438452 ) was synthesized with bridging n- and c - terminal cysteines ( ceelghdadgyetirsc ) ( severn biotech ) . cytotopic cd59 at a final concentration of 0.7 m was incubated with 200 l of fluorescently - labeled liposomes in the presence or absence of peptide , and the average background fluorescence intensity was measured for 10 min . subsequently , ily at a final concentration of 0.7 m was added to the reaction and the fluorescence was measured for 45 min . liposomes were burst at the end of the experiment by the addition of 1 l 0.2 m c12e8 detergent ( sigma - aldrich ) , and the maximum fluorescence was found by monitoring the sample for an additional 10 min . the fluorescence measurements for each reaction were normalized according to the background and detergent readings . rates of calcein release ( which corresponds to pore formation ) were calculated by fitting the normalized fluorescence intensity data with a nonlinear fitting algorithm in originpro 8.6 . we incubated 0.75 mg ml liposomes with cytotopic cd59 and either ily or ily in equimolar ratios so that the final protein concentration was 1 mg / ml . we applied 2.5 l of the mixture to glow discharged holey carbon grids ( quantifoil r 2/2 ) and vitrified in liquid ethane with an fei vitrobot . images were acquired on a cm200 feg electron microscope ( fei ) operating at 200 kv . images were recorded with a defocus range of 2.5 to 3.5 microns underfocus at a magnification of 38,000 and an electron dose of 20 e / with a 4k 4k temcam - f415mp ccd camera ( tietz video and image processing systems ) .

summarypore - forming proteins containing the structurally conserved membrane attack complex / perforin fold play an important role in immunity and host - pathogen interactions . intermedilysin ( ily ) is an archetypal member of a cholesterol - dependent cytolysin subclass that hijacks the complement receptor cd59 to make cytotoxic pores in human cells . ily directly competes for the membrane attack complex binding site on cd59 , rendering cells susceptible to complement lysis . to understand how these bacterial pores form in lipid bilayers and the role cd59 plays in complement regulation , we determined the crystal structure of human cd59 bound to ily . here , we show the ily - cd59 complex at 3.5 resolution and identify two interfaces mediating this host - pathogen interaction . an ily - derived peptide based on the binding site inhibits pore formation in a cd59-containing liposome model system . these data provide insight into how cd59 coordinates ily monomers , nucleating an early prepore state , and suggest a potential mechanism of inhibition for the complement terminal pathway .

Introduction Results Discussion Experimental Procedures

PMC3184751

tooth whitening continues to be a rapidly growing area in esthetic dentistry because tooth color and brightness is so important for patients . dentists apply various treatment methods for this purpose such as microabrasion , macroabrasion and bleaching . nowadays , the whitening effects of these toothpastes are usually achieved by the incorporation of abrasives and bleaching components . an ideal toothpaste should remove unwanted surface deposits and stains with minimal influence on the enamel , dentine and restorations . thus , the effects of such products on properties of enamel and restorative materials are important . microhardness is one of the important properties of the materials which correlates with strength , proportional limit and wear resistance . many recent published studies have been conducted not only on the chemical stain removal and abrasive properties of the whitening products but also on their effects on the hardness of enamel , dentin and restorative materials [ 1,611 ] . in 2006 , joiner showed that whitening toothpastes are more effective in stain removal than non whitening dentifrices ; however , in 2007 , terezhalmy et al concluded that there is no significant difference between the efficacies of different whitening toothpastes in terms of removal of extrinsic stain . a few researches have been performed on the effects of whitening toothpastes on both enamel and composite resin stain [ 1214 ] . in 2005 , joiner et al showed that whitening toothpastes make no significant wear on the enamel and dentin . another study which was performed by joiner et al showed that there was no significant deference between enamel abrasivity of the whitening toothpaste and a standard silica dentifrice . zimmerman et al reported that whitening treatments could change the mechanical properties of the enamel . according to our search , there was no study to evaluate the effects of different whitening toothpastes on the microhardness of the enamel as well as composite resin . the main goal of this study was to assess the effect of two whitening toothpastes on the surface hardness of enamel and a microhybrid composite . in this in - vitro experimental study , there were two types of specimens made of enamel and composite . to prepare enamel specimens , 45 freshly extracted human insicors all teeth were examined under magnification 20 to ensure there are no microcracks and surface defects . to perform the test , the crowns were cut at the cement - enamel junction ( cej ) using a diamond saw ( microslice 2 , metals research ltd . , cambridge , uk ) using water coolant . the enamel slabs were embedded in acrylic mold ( gc pattern resin , gc co. , chicago , usa ) . in order to prevent dehydration of the teeth , for composite specimens cylindrical - shaped molds ( plexiglas mc ; rohm & haas , philadelphia , pa ) with disk - shaped specimen wells ( 2 mm thickness 6 mm diameter ) were used to make 45 specimens . the material used in this study was a microhybrid composite resin ( z-250 , 3 m co. , st.paul , mn , usa ) in a2 shade . initially , the molds were slightly overfilled with the material , covered with a plastic matrix strip ( universal strips ; extra dental , istanbul , turkey ) and pressed flat with a glass slab to extrude excess material . the composite resin specimens were light polymerized by astralis 7 ( vivadent , liechten stein , swiss ) for 60 seconds to ensure adequate polymerization . prior to the polymerization of each specimen , the intensity of the light source was fixed at 450 mw / cm2 , using a light meter ( apoza , apoza enterprise co. ltd . , all the specimens were polished by the same operator using medium , fine and superfine discs ( sof - lex , 3 m co. , st.paul , mn , usa ) and a slow - speed handpiece ( kavo electrotorque , kavo america , inc . , lakeside , il ) rotating in one direction . following each application , five strokes were made with each disk ( medium , fine , superfine ) in a sequence . the polished specimens were cleaned in distilled water in an ultrasonic cleaner ( sonica , soltec s.i.l . all specimens were then placed in 37c distilled water for 24 hours and then brushed ( table 1 ) . vickers hardness test was performed for all the specimens and values were recorded with a microhardness tester ( micrometer 1 , buehler , lake bluff , il , usa ) using a 300 gram load and a 15 second dwell time at room temperature . data were analyzed by one - way anova and tukey tests using spss version 13 at the significance level of = 0.05 . chi - square test , anova , multi - variant ordinal regression , and spearman correlation coefficience were employed for data analysis . mean and standard deviations were calculated for crestal bone loss measured after at least 2 years of implant insertion . the mean microhardness values , standard deviations for composite resin and enamel groups are given in table 3 . one - way anova showed statistically significant differences among three composite resin groups ( p<0.001 ) , but there were no significant differences among the three enamel groups ( p=0.132 ) . tukey 's test showed that there were significant differences between each of the two composite resin groups ( p < 0.05 ) . it is known that whitening dentifrices used with tooth brushing act to decrease plaque and surface deposits on teeth as well as helping in removing stains and discolorations . many commercially available ones contain ingredients that may have adverse effects on the surface of restorations and teeth . hardness is a surface property of a material that shows its resistance against permanent deformation . vickers hardness is a type of microhardness test which is commonly used to evaluate surface microhardness of brittle and restorative materials [ 518 ] . the composite resin filtek z250 as a hybrid composite which is applicable in anterior and posterior restoration was used in this study . this composite resin has good mechanical properties as well as hardness . in order to achieve an adequate polymerization , resin composite of a2 shade two whitening toothpastes were used to evaluate their influence on the surface hardness of enamel and composite resin . to minimize the possible effects of tooth brush on surface hardness of enamel and resin composite , the results of this study showed that significant difference in hardness was observed between composite control groups ( clc ) and composite treatment groups ( ctc and afc ) . this finding similar to the results of studies performed by garci et al and wang et al suggests that ingredients of the toothpastes used in the present study including a range of components such as sodium bicarbonate , hydrated silica , sodium tripolyphosphate and other ingredients which have the ability to influence substratum surfaces , could affect the surface characteristics of composite materials . comparison of the two composite treatment groups ( ctc and afc ) indicated that the hardness of composite resins exposed to crest toothpaste ( ctc ) decreased more than the hardness of those treated with aquafresh toothpaste ( afc ) and this decrease was statistically significant . the whitening ingredients in crest toothpaste are special silica abrasives that prevent formation of stains and remove stains from the surface . however , the whitening ingredient in aquafresh is sodium tripolyphosphate , a surfactant and chelator , which is effective against calcified stain . moreover , other factors such as particle size and shape , source and purity can affect agent abrasivity . the ph of these toothpastes was 7.62 for crest and 9.73 for aquafresh whitening toothpastes . a profilometric study revealed that dentifrices with a basic ph between 7.56 and 8.19 yielded enamel abrasion significantly lower compared with those with a neutral or acidic ph . while , the microhardness values that were obtained in our study were a result of complex factors and their alteration by ph could not be distinguished . in addition , the results showed no significant differences between enamel groups . because of high hardness of the enamel , different ingredients of these ( table 2 ) . on the other hand , because the ph of the whitening toothpastes used in the present study were in the reported range of previous studies and were not acidic ( ph>7 ) , they could not demineralize the enamel surface and decrease its hardness . minimal or non - significant reduction of enamel microhardness detected in our study may be related to safety abrasiveness of the studied toothpastes which are produced under the regulatory situation in the eu . since the whitening gradient of the tested toothpastes in the present study is different from whitening gels , our results concur taher 's study indicating a significant reduction of surface hardness values of composite resin after using bleaching agents . these results do conflict with nathoo et al s study , which reported no effect of a professional tooth whitening system on the microhardness of composite resins . this can be attributed to the kind of applied products and difference in the study methods . as solubility parameters of toothpastes future researches are recommended to compare the effect of various whitening toothpastes on others properties of composite resin materials and hard tissues of the teeth . this in - vitro study demonstrates that use of whitening toothpastes does not affect enamel hardness , but decreases the surface hardness of z-250 microhybrid composite resin .

introduction : whitening toothpastes which have been accepted in populations may affect properties of enamel and restorative materials . the aim of this study was to compare the microhardness of human enamel and z250 microhybrid composite resin after brushing with two whitening toothpastes.materials and methods : in this experimental study of enamel specimens , forty five freshly extracted human incisors were prepared and divided into three groups of control enamel ( cle ) , crest enamel ( cte ) and aquafresh enamel ( afe ) . for composite resin specimens , forty five cylindrical - shaped specimens of light - cured z250 composite were prepared and divided into three groups of control composite ( clc ) , crest composite ( ctc ) and aquafresh composite ( afc ) . the control groups were brushed without toothpaste . crest and aquafresh group specimens were brushed with crest and aquafresh whitening toothpastes , respectively . vickers microhardness test was performed for all groups . data were analyzed by one - way anova and tukey tests.results:microhardness values of cle , cte , afe , clc , ctc and afc groups were 332.99 26.59 , 313.99 20.56 , 323.57 27.96 , 137.1 3.16 , 122.95 3.27 and 130.36 4.8 , respectively . one - way anova showed no significant differences among three enamel groups but there was significant difference among composite groups ( p<0.01).conclusion : crest and aquafresh whitening toothpastes did not affect enamel hardness but reduced the microhardness value of z-250 composite resin . however , crest whitening toothpaste decreased the microhardness more than aquafresh .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

PMC4459120

residence in antarctica exposes humans working in the research bases to extremes of weather and photoperiod , in addition to geographic and social isolation . the annual average solar radiation decreases with increasing latitude , and this difference is marked during winter than in midsummer in the circumpolar regions . this alters temporal homeostasis and affects sleep wake cycles , mood , cognition and work performance of base personnel . the psychological disturbances in antarctica , referred to as the over winter syndrome , may also relate to change in the circadian rhythms of the hormones cortisol and melatonin [ 13 ] . photic stimuli are the primary synchronizer of the endogenous circadian pacemaker in the suprachiasmatic nucleus of the anterior hypothalamus . therefore temporal modulation of the ambient light settings over a 24 hour period in antarctica can promote better adjustment to the seasonal photoperiod . the endocrine control over the circadian pacemaker is reflected by the changes in serum levels of cortisol and the pineal indoleamine melatonin . melatonin , noradrenaline , and acetylcholine decrease with light activation , whereas cortisol , serotonin , gaba , and dopamine levels increase . in normal subjects , we expect a peak melatonin level at approximately 3 am , whereas cortisol peaks at approximately 9 am . in healthy individuals , the transition from low light to bright light in the morning results in a surge in cortisol levels and a decline in melatonin levels . since the photoperiod is the principal factor regulating seasonal adaptation , melatonin responses may mediate changes in endocrine , cognitive , psychological , and immune function . there is mounting evidence to suggest that long - term disruption of circadian rhythm correlates with disease states such as ischemic stroke , heart disease , metabolic dysfunction , and glucose intolerance . additional studies have also found that chronic shift work is significantly associated with an increased risk of colorectal , endometrial , and prostate cancers . disintegration of sleep wake cycles and poor sleep health is also associated with a range of neuronal diseases such as alzheimer 's or huntington 's disease [ 11 , 12 ] . psychological research in antarctica provides insight into how individuals ( and groups ) function in an extreme and isolated milieu . however , while interpreting and comparing published data , the racial , psychosocial , educational and cultural differences of expedition teams needs to be considered . sleep disturbances and psychological changes have been reported in overwintering teams , but only one study on 9 individuals has tested the effect of the changed diurnal rhythm in the antarctic on human cognition and performance . previous studies have reported that circadian cortisol levels have an important role in cognition in an age independent manner . studies demonstrated that frequent transmeridian flyers with a disrupted diurnal cycle had higher evening cortisol levels , which was associated with cognitive deficits and right temporal lobe atrophy . objective assessment of cognitive performance is important for comparison and reproducibility . computerized neurocognitive tests ( cnt ) allow accurate measurements of reaction times and test - retest reliability however , they may be less reliable than conventional tests when traits like verbal skills and personality are being tested . hence a combined approach is suitable in situations where comprehensive evaluation of personality and serial testing of cognitive skills is needed . the objective of this study was to ( i ) evaluate the seasonal variation in serum cortisol and melatonin diurnal rhythms during the polar summer and winter photoperiods , ( ii ) study mood and depression , and ( iii ) assess the change in objective neurocognitive performance of the base personnel over a period of one year in antarctica . twenty male members of the 30th indian scientific expedition to antarctica ( isea ) , certified to be in good mental and physical health and free of intrinsic sleep disorders following medical and psychiatric examination and acclimatization training , were recruited for this prospective study with written informed consent . the study was approved by an institutional ethics board of the national centre for antarctic and ocean research ( ncaor ) , india 's nodal agency for antarctic research and was conducted in accordance with the standards of the declaration of helsinki . the study was started in november 2010 soon after arrival of team members in antarctica and the group was followed up prospectively till the conclusion of the expedition in december 2011 . the over - wintering team consisted of 6 scientists and 14 technical support staff who are required to keep the base functional and the scientific experiments running . all the personnel are required to maintain the same sleep rhythm ( wake at 0700 hours utc ) and work schedule except for one person who is on night watch , which is a periodic duty once every ten days by rotation . one meteorologist also maintained a night shift duty for the greater part of the year . serum levels of melatonin and cortisol were measured in the subjects at 8 am , 3 pm , 8 pm , and 2 am in a single day using an indwelling antecubital catheter during 2 different photoperiods ; once during summer and the other during the polar winter . the samples reflecting the summer photoperiod were collected in january 2011 , a few weeks after arrival in antarctica , and processed to obtain serum which was stored at minus 25 celsius until assay was done . the samples were transported back to india on dry ice using monitored cold chain transport at 25c to 70c . melatonin was measured by radioimmunoassay ( ria ) , using i - melatonin ( labor diagnostika nord gmbh & co. kg , nordhorn , germany ) . the reference range , as stated by the manufacturer , was 30150 pg / ml . the sensitivity of the method was 2.3 pg / ml and the intra - assay variability less than 9.8% and inter - assay variability 8% . the antimelatonin antibody used had a cross reactivity of > 0.1% with melatonin related metabolites or precursors . serum levels of cortisol were assayed by gammacoat ria ( diasorin , stillwater , mn ) with a sensitivity of 0.2 g / dl and intra - assay coefficient of variation ( cv ) of 6.5% and inter - assay variability of 9% . the reference range was 725 g / dl for morning values and 29 g / dl for evening values . all subjects were required to maintain a sleep log over a 2 month period in summer and again in winter . the sleep log documented the sleep / wake times , caffeine and alcohol intake , night time awakenings , use of sleep aids such as sleeping pills , music , reading , total duration of sleep , and a subjective evaluation of restorative sleep . epworth sleepiness scale ( ess ) and morningness eveningness questionnaire ( meq ) were administered on arrival , during summer and during winter . the ess is a validated scale for measuring daytime sleepiness and the meq scores reveal individual adjustment to morning and evening photoperiods and the synchronization of the internal clock to work schedules . a combination of conventional psychological tests and a computerized neurocognitive test battery ( cnt ) were used to serially measure relatively mild degrees of neurocognitive impairment in the base personnel . the tests included visual and verbal memory , attention , visual motor speed , arithmetic and reaction times , a modification of the halstead reitan battery , to test domains likely to be relevant to wintering and working in a challenging environment like antarctica ( see table 1 ) . neurocognition tests : the battery of tests employed in this study included both conventional and computerized tests . the card sort test measures shifting attention , that is , the ability of the subject to focus on one set of instructions and then to shift to another set in response to the change in test response . the test involves 4 packs of cards which have different geometric shapes ( circle , star , triangle , and cross ) , different number of shapes ( 1 , 2 , 3 , and 4 ) and different colours ( red , yellow , green , and blue ) . then a card with a pattern having a specific colour and number of shapes is presented and the subject is instructed to match the card with one of the 4 packs by a rule which needs to be deduced from the test response . the rule is changed randomly ( matching of colour , number , or shape ) throughout the test and the subject is scored for the number of correct matches made in a fixed time . this is a modification of the wisconsin card sort test and measures attention and cognitive flexibility . random digits ( 0 through 9 ) are presented to the subject on the screen and simultaneously by computer generated voice in increasing sequence as long as correct responses are obtained . if the response is incorrect , the subject is tested with a shorter sequence which is then increased by one digit successively until the longest sequence of digits is correctly repeated . the test is continued for 20 trials and the mean number and longest sequence of correct responses is calculated . the subject is instructed to press the space bar when any alphabet is flashed on the screen except the alphabet the number of x foil stimuli is 25 and target alphabet stimuli are 300 , respectively . the test gives the accuracy rates for the targets and foil non targets and also the mean reaction times for correct and erroneous responses . the subject is presented with four colors / color words ( red , green , blue , yellow ) and is instructed to select either the colour or the word . at first 5 practice trials are allowed . then 20 random trials for congruent and incongruent colour are presented in a congruent color and word ( e.g. , word red in red font ) which generates a congruent reaction time and 15 test words are presented in an incongruent fashion ( e.g. , word red in green font ) , which generates a second reaction time . finger tapping test . the subject is asked to press the key x on the computer keyboard as many times as possible in 90 seconds . this simple test provides reliable and reproducible data about fine motor control , motor speed , and visual motor function . the visual memory test involves the presentation of a sequence of everyday object images with their names ( apple , pear , bus , bird , etc . ) on the screen . each image is flashed for 2 seconds in increasing number and the subject has to memorize the order of presentation . then a screen of nine figures arrayed in a grid is presented and the subject is instructed to click on each image in the order of presentation . the subject is presented with simple addition , subtraction , multiplication , and division equations ( up to 2 integers ) at first slowly and then with increasing frequency for a period of 3 minutes . the score is based on the accuracy and number of equations solved per minute . the subjects were instructed to click the left mouse button each time a triangle was flashed on the screen for ten trials . a similar test for auditory reaction time was used by presenting a tone stimulus by headphones . the subjects were presented an image of a geometric design ( circle , square , triangle , oval ) and another image at random immediately after . if the new image was identical to the one immediately before it , they were asked to click the right shift button and if not , the left shift button . the reaction times and accuracy was recorded over 1 minute for a sequence of random images . the instruments for assessing physical and mental health included demographic information on age , occupation , education , and subjective health complaints , and standardized scales such as depression , anxiety , and stress scale ( dass-42 ) and mini mental status examination ( mmse ) . dass-42 measures the behavioral manifestations of depression , anxiety , and stress in a self report interview to construct a score ( see table 2 ) . period differences in the circadian rhythm were tested by measuring serum cortisol and melatonin levels during summer and winter . the sampling periods were correlated with the ambient solar radiation over the station during the polar summer and winter photoperiods . this was measured by means of a sunlight photometer during the subjects ' sojourn in antarctica , and data was obtained with permission of the indian meteorological department ( imd ) . conventional methods , mean and standard deviation ( sd ) , student 's t - test , fisher 's t test , and pearson 's correlation coefficient were used to analyze the diurnal variation in cortisol and melatonin levels with the level of significance < 0.05 . the software spss for windows ver.13 ( spss inc ; chicago , il , usa ; http://www.spss.com/ ) was used for statistical analysis . the entire team was subjected to psychological testing prior to selection for the expedition and none of them suffered from depression or sleep disorders . there was a trend for an increased weight , bmi , and systolic blood pressure in older individuals . four ( 20% ) were hypertensive , requiring antihypertensive drugs in addition to lifestyle and diet modification . the maximum score of the dass-42 is 42 in each of the depression , anxiety , and stress subscales . the mean scores for depression and anxiety during midwinter were significantly higher than summer scores ( p < 0.005 ) suggesting the presence of over wintering . daytime sleepiness was excessive ( score 16 ) in just one subject as per the ess , but 4 subjects had borderline scores ( mean 11.4 1.1 ) during summer and 2 of them continued to report daytime sleepiness during midwinter . the work schedule during summer for the technical and maintenance staff involved reporting for duty at 0900 hrs and mild to moderate physical work for a period of 3 - 4 hours . post lunch ( 12301400 hrs ) there was another work period from 1500 to 1800 hrs . with the onset of the polar night , the evening shift was stopped due to adverse weather conditions and poor visibility . however during blizzards , special rotation duties for snow clearance and station maintenance resulted in a peak in physical activity . the scientists maintained the same work schedule in summer and winter , but participated in external work and blizzard duties . additionally , all team members participated in galley duties by rotation involving night watch every 810 days . the sleep logs showed a marked difference between summer and winter periods in 5 ( 25% ) subjects , all scientists . they reported a decreased latency , increased duration of sleep , fewer early morning awakenings , and restorative sleep as compared to summer . the sleep patterns of the technical support team varied only in the total duration of sleep during winter ( mean 8.42 1.18 hours ) as compared with summer ( mean 8.64 1.21 hours ) . in contrast , the total sleep duration in scientists was 7.33 0.98 hours in summer and 7.66 1.03 hours in winter ( see table 5 ) . the total duration of sleep was increased due to an increased frequency of afternoon napping which was related more to no work shift in the post lunch period rather than due to increased fatigue . sleep aids used by the subjects included listening to music , reading , and watching television . pharmacological therapy , including use of benzodiazepines and zolpidem , was needed in 7 ( 35% ) subjects at one or more times for sleep induction . the results of the diurnal and seasonal variation of cortisol and melatonin provide interesting biochemical correlates for our clinical data . as expected , the mean cortisol levels were highest at 8 am , with a progressive decline during the day , the nadir being reached at 2 am . the mean levels in winter were higher at 8 am and 3 pm but the difference was statistically equivalent ( see figure 1 ) . during midwinter , we noted a marked increase in the mean serum melatonin levels . figure 2 demonstrates the marked contrast in melatonin diurnal rhythm during the two seasonal photoperiods . while the mean levels of these hormones showed a physiological and predictable diurnal rhythm , we noted marked individual variation . this was more so for scientific staff involved in time bound data collection . the scores on meq provided clues to the degree of subjective acclimatization in relation to polar work and photoperiod . soon after arrival and during midsummer , 5 ( 25% ) subjects preferred starting work late between 10 and 11 am and up to 4 ( 20% ) persons responded that they were mentally and physically more active in the evening . definitely morning chronotype as opposed to a moderately morning 4 ( 20% ) or neither morning nor evening most reported a preference to start work earlier , between 8 and 9 am . we assessed whether cortisol and melatonin diurnal rhythms correlated with psychological tests and cognitive performance ( see tables 6 and 7 ) . in most cases , no clear trend was obtained for hormone levels and cognitive function . we did find that a higher evening cortisol negatively correlated with performance on tests for attention like digit span and card sort during summer . higher evening cortisol levels also correlated with increased dass 42 scores . during winter , elevated 8 am melatonin levels correlated significantly with better performance on tests for attention , digit span , and arithmetic during winter . although this may seem paradoxical , an increased 8 am melatonin level was also associated with better sleep quality , which may have improved performance status . the subjects were tested by both conventional methods and by computerized tests in the morning . those with a non morning chronotype were retested in the evening , but there was no significant change in the scores other than the continuous performance test ( cpt ) for attention . overall , the mean scores and reaction times of many tests were statistically equivalent in the summer and winter photoperiods . there was a significant increase in arithmetic computational accuracy and speed in winter . also subjects performed better on choice reaction time with lower mean rt and improved accuracy suggesting improvement in speed of processing . there was a marginal improvement in scores of the card sort test for shifting attention and digit span in winter . this was more so for individuals with higher 8 am melatonin levels ( p = 0.034 ) . subjects with higher dass-42 scores , scored lower on digit span ( p = 0.032 ) , shifting attention ( p = 0.045 ) , and cpt ( p = 0.012 ) . mean scores for simple reaction time , digit span , and visual memory were better in individuals aged < 30 years . higher ess scores did not correlate with poor performance on cpt , and no sleep attacks were noted during testing . adaptation to the polar environment involves synchronization to temporally relevant stimuli such as light , temperature , work , meal schedules and thus the internal clock gets aligned with the external solar time . rhythmic modulation of behavior and physiologic processes such as sleep , cardiovascular , cognitive performance results from the circadian control over diverse pathways . few studies have been done on circadian rhythm in antarctica and no study has ever been done on the impact of prolonged antarctic residence on circadian rhythm in indian expedition team members [ 13 , 26 , 27 ] . sleep schedules , activity , plasma melatonin , and core body temperature were documented in 9 japanese subjects at dome station ( 77s ) . they concluded that the sleep rhythm was largely governed by the work schedule while the circadian rhythm in plasma and core body temperature is influenced by photoperiod . adaptation of the melatonin rhythm in british antarctic personnel at halley station ( 75s ) has also been reported in night shift workers over short periods . another study also evaluated the usage of light intensity regulation at the stations to influence sleep behavior and mood disturbance issues . they concluded that further investigations of phase shifting techniques such as timed bright light and administration of melatonin are indicated to improve performance at work [ 3 , 13 ] . a recent study of salivary cortisol awakening response in 55 participants at 2 british antarctic stations rothera ( 6734s , 688w ) and halley ( 7535s , 2630w ) showed no difference in the diurnal cortisol response in the polar winter . we measured the serum levels of cortisol and melatonin during summer about a month after arrival in antarctica . the period of transition from one expedition team to the next involves adjustment to new routines , constant daylight and cold dry windy weather . but we found that the mean 8 am cortisol levels were higher in winter when compared with summer . an increased 8 am summer cortisol level correlated with a reduced reaction time ( p = 0.018 ) . subjects showing a higher 8 am melatonin level during winter also performed better in the neurocognition tests for immediate memory ( p = 0.034 ) , and arithmetic ( p = 0.041 ) , suggesting better adaptation . . the result may be best interpreted in the context of restorative sleep improving cognitive performance in winter . an elevated evening cortisol did not correlate with deficits in visual recognition memory , in contrast with previous studies . high evening cortisol levels may indicate chronic stress and poor sleep quality , which in turn impairs performance [ 28 , 29 ] . in our study , no clear association could be obtained for individual cortisol or melatonin values and the corresponding neurocognitive test results of the subjects . this is largely due to the smaller sample size , which is restricted by the number of team members in an over wintering expedition . so results need to be interpreted carefully , as multiple factors may affect an individual 's day to day performance . there was an improvement in sleep quality , duration , and decreased latency in winter . the maximum sleep disturbances occurred in the first one month after arrival , in the phase of adjustment to a 24 hour light photoperiod . the scientific staff had fewer total sleep hours than the logistic staff and also reported increased sleep latency . the sleep wake cycles of scientists were irregular , changing with night watch , galley duties , and timed data collection . subjects also reported better sleep quality , fewer night awakenings , and decreased latency during winter which correlated with higher melatonin levels . studies from other stations reported that poor sleep was associated with depressive symptoms . in our study , patients with higher dass-42 scores reported night interruptions and early awakening . winter was also associated with an increased volume and frequency of alcohol intake , but no changes in sleep parameters were associated with drinking . sleep aids are useful and pharmacological therapy is often needed in the early adjustment phase to avoid sleep disruption . the subtle changes in behavior , sleep , memory , and cognition during long duration expeditions may have long term health consequences on aging , cardiovascular , and cerebrovascular disease . in this study , we performed tests for domains like memory , attention , reaction time , and speed of processing which can be affected by extrinsic factors like drugs , disease states , and time of day testing . also , motivation , interest , mood , and vigilance affect individual scores . though the cognitive performance for the whole team averaged the same during summer and winter , we noted other outcomes . . this may reflect subtle changes in the level of alertness as a function of diurnal rhythm . despite advances of technology and upgraded facilities at scientific bases subjects have to perform energy consuming tasks including but not limited to snow clearance , prolonged driving , and maintenance of vehicles , heating systems , electrical generators , and so forth . these activities are prone to human error . in addition interpersonal relationships and closed group psychology and the sense of physical isolation affect performance [ 3032 ] . circadian rhythms are modulated by central pacemakers like melatonin and cortisol which synchronize to stimuli such as photoperiod and activity , and thus indirectly the internal clock becomes predictive of solar time . our study found that the diurnal rhythm for cortisol was comparable during the polar summer and winter . in contrast , circadian secretion of melatonin was affected during the polar winter and higher levels persisted throughout the day , peaking physiologically at 2 am in both seasons . again , higher levels of 8 am melatonin correlated significantly with better neurocognitive performances in tests for attention and immediate memory . we also report the effect of over wintering in our subjects reflected as higher depression , anxiety , and stress scale scores during the polar winter . the fact that the team size of wintering teams in the antarctic is kept to a minimum , pooling of data will be useful in making recommendations for future expeditions . timing of artificial light exposure and usage of melatonin supplements in improving sleep and cognitive performance in expedition teams are of future research interest .

background . altered circadian cortisol and melatonin rhythms in healthy subjects exposed to an extreme polar photoperiod results in changes in mood and sleep , which can influence cognitive performance . materials and methods . we assessed the circadian rhythm of 20 subjects who wintered over at maitri ( 70s , 11e ) , india 's permanent antarctic station , from november 2010 to december 2011 . serum cortisol and melatonin levels were measured by radioimmunoassay at 8 am , 3 pm , 8 pm , and 2 am in a single day , once each during the polar summer and winter photoperiods . conventional psychological tests , depression , anxiety , and stress scale ( dass-42 ) , epworth sleepiness scale ( ess ) , and a computerized neurocognitive test battery were used to measure mood , sleep , and cognitive performance . results . the mean scores for dass42 were higher during midwinter suggesting the presence of overwintering . mean diurnal cortisol levels during summer and winter were comparable , but the levels of melatonin were markedly higher during winter . higher 8 am melatonin levels were associated with better sleep quality , lower depression scores , and better performance in tasks like attention , visual memory , and arithmetic . conclusion . timing of artificial light exposure and usage of melatonin supplements in improving sleep and cognitive performance in expedition teams are of future research interest .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

PMC4268830

daily inactivity of young children is a major problem of societies all over the world . numerous studies proved a connection of children s activities with their future health status and the trend of progressive inactivity and obesity of children . inactivity or lack of proper physical activity in childhood can result in a failure to develop fundamental movement skills ( fms ) during preschool and primary school years and cause later failures in learning specialized movement skills . in contrast , mastering of these skills is a prerequisite for a successful introduction of specific sport activities . fms include locomotor skills ( e.g. , run , gallop , hop , leap , horizontal jump and slide ) ; object control skills ( e.g. catch , kick , overhand throw and dribble ) and body management skills ( e.g. , balance , climb and forward roll ) and provide a base for more advanced motor skills such as artistic gymnastics skills . moreover , the level of locomotor skills of children is positively correlated with the levels of participation in physical activities in adulthood , which implies that the development of fms has a great role in long - term health . as it is already known , fms do not improve only because of growth and maturation . children s daily activity , learning various skills , relevant feedback and encouragement , motor abilities and morphological characteristics , especially weight , play significant roles in development of fms . if the optimal age for mastering fms is between the ages of 2 and 7 and the optimal age to start with an organized gymnastics program is between the ages of 5 and 7 , then it is obviously important to find out how learning gymnastics skills reflects on the fms status of seven - year - old children . hypothetically , a positive transfer of skills from fms to gymnastics skills can be expected , according to the identical elements theory established by thorndike and later developed by osgood , meaning that similarities between the stimulus and response conditions of the two fundamental tasks are in the basis of positive transfer . also , a positive transfer could occur when practice conditions require learners to engage in problem - solving processes similar to those required by the criterion task according to the appropriate processing theory . delas kalinski , miletic and bozanic found out that gymnastics skills learned at the age of 6.5 are retained over time after a period without any practice which makes them suitable for pe classes . learning gymnastics skills in childhood can increase children s capacity for skill performance , improve their motor abilities , and , as we hypothesize in this investigation , to develop fms as well . if a positive transfer occurred , there is no reason why fms and basic gymnastics skills would not be developed simultaneously . the question is : will learning gymnastics skills develop all fms including locomotor , object control and body management skills equally ? the activities that develop versatile fms should be the major focus of primary school education . the purpose of this study was ( 1 ) to examine the influence of an 18-week basic gymnastics program on fms development in seven - year - old children ; ( 2 ) to determine correlations between daily activities and successful performance of fms and basic gymnastics skills . seventy five first grade pupils ( 30 boys and 45 girls ) of primary school from mostar , bosnia and herzegovina , participated in the study . they were all at the age of seven ( 6 months ) . all of them were chosen randomly , from a population of 270 children from three schools , and had written parental consent for their participation in this study . first grade pupils were chosen because of limited past experience with artistic gymnastics skills and because their fms development is still in progress . also , it is expected that their activity and inactivity outside of school would be similar . the artistic gymnastics program was conducted three times a week for 45 minutes ( for 18 weeks ) , by trained artistic gymnastics instructors and a pe teacher . the study procedure included initial and final a ) evaluation of the performance quality of artistic gymnastics skills , b ) measuring of the fms - polygon . eight artistic gymnastics skills ( forward roll , descended backward roll , handstand against wall , dominant frontal cartwheel , springboard jump with running start and landing , switching positions on the rings , front of the foot walking on a small beam , straight forward jump - off of small beam ) were chosen according to the following criteria : ( 1 ) they are teaching topics in the physical education curriculum ; ( 2 ) according to their abilities and pre - knowledge 7-year - old pupils can learn the same and similar artistic gymnastics skills ; 3 ) these skills represent basic skills in artistic gymnastics . the level of their performance was videotaped ( at the beginning and at the end of the program ) and evaluated according to standard procedure by independent assessment of five judges , on a five - point likert scale . evaluation was based on fig code of points , but adjusted in order to be suitable for the pe curriculum . the likert scale was used accordingly : ( 5 ) performance without technical and/or aesthetic mistakes ; ( 4 ) performance with small technical and/or aesthetic mistakes ; ( 3 ) performance with medium technical and/or aesthetic mistakes ; ( 2 ) performance with large technical and/or aesthetic mistakes ; ( 1 ) performance was not / could not be done at all . a new , quick and effective fms , norm - referenced test which can be easily administered for pe purposes was developed and used for this research . in this study we applied the fms assessment according to zuvela et al for three reasons : ( 1 ) considerably less time was needed for assessing a large number of participants ; ( 2 ) norm - referenced polygon fms assessment tool can be easily identified by pe teachers , ( 3 ) this norm - reference polygon fms test was validated with tgmd criterion - referenced test . after metric indicators had been precisely defined for a wider set of 24 fms tests , four tests ( tossing and catching the volleyball against a wall consecutively ; running across obstacles ; carrying the medicine balls ; and straight running ) with the highest factor scores were chosen to enter the final product : the fms - polygon . correlation with the test of gross motor development ( tgmd-2 ) revealed the coefficient of -0.82 , which indicates a proper validation for the fms - polygon . as it is considered a reliable and valid assessment tool for 8-year - old children , the authors assumed that it would be appropriate for seven - year - old children as well . the test itself is consisted of 4 tasks : tossing and catching the volleyball against a wall consecutively ; running across obstacles ; carrying the medicine balls ; and straight running . the participants task is : to stand on a starting line with the volleyball and begin the first task of tossing and catching a ball against the wall 6 times on the examiner s signal ; to leave the ball and run across three obstacles finally passing through the cones ; to lift and carry the first and the second medicine ball and put them on the swedish vault ; to run 20 meters until passing through the photocells . the final result of the test is the time needed to successfully accomplish the four mentioned tasks . the estimation of physical activity and inactivity was done by a proxy - questionnaire netherlands physical activity questionnaire [ npaq ] . the questionnaire is designed for parents , who need to assess activity of their children , and has two parts : ( 1 ) the estimation of children s activity which consists of seven statements , scaled from 1 to 5 , referring to children s activity habits and preferences ; ( 2 ) the estimation of children s inactivity , which consists of two questions , which refer to children s daily sedentary activity such as watching tv or playing on pc . the average value from all answers represents scores for activity while the sum of two answers represents the scores for inactivity . the analysis of data was performed by the statistica for windows 7.0 , package and the level of statistical significance was set at p<0.05 . t - test for dependent samples was used to determine the differences between participants performance at the beginning and at the end of the program . for analyzing : a ) correlations between gymnastics skills and the fms - polygon ( at the beginning and at the end of the program ) and b ) correlations between the fms - polygon and activity and non - activity variables , spearman boys averaged 132.05.5 cm in height and 31.45.8 kg in weight and had a bmi of 17.32.6 , while girls averaged 129.65.0 cm in height and 29.35.2 kg in weight , with a bmi of 16.82.9 . this study evaluated the effects of an 18-week artistic gymnastics program , provided three times a week for 45 minutes , on mastering gymnastics skills and on development of fms , on seven - year - old children . according to the results of mean values and standard deviations , determined at the beginning and at the end of the gymnastics program , presented in table 1 , significant differences were found in the fms - polygon and all gymnastic skills . the objectivity of judges of gymnastics skills was previously established by an intra - class correlation coefficient . their values ranged from 0.88 to 0.97 , which indicates high reliability for estimating the level of performance of basic gymnastic skills among beginners . the biggest differences between the means , between the initial and the final measurement point , were evaluated in descended backward roll ( 1.72 - 3.50 ) and forward roll ( 1.91 - 3.56 ) , while the smallest differences occurred in dominant frontal cartwheel ( 1.47 - 2.67 ) and switching positions on the rings ( 1.27 - 2.49 ) elements . significant correlations were established between gymnastics skills and the fms - polygon at the beginning and after 18 weeks of gymnastics program ( table 2 ) . results of fms - polygon and gymnastics skills before and after the gymnastics treatment correlation analysis between gymnastics skills and fms - polygon before and after 18-week gymnastics program the exception is the springboard jump with running start and landing which had no significant correlation at the beginning , but had a significant correlation with the fms - polygon at the end of the gymnastic program ( -0.63 ) . the highest value of correlation in the initial measuring point was found in the descended backward roll ( -0.47 ) and in the front of the foot walking on a small beam ( -0.44 ) , and in the final measuring point in all gymnastics skills that are pointed as basic gymnastics skills on different apparatus ( from -0.60 to -0.66 ) . the results gathered by a proxy - questionnaire ( npaq ) , which defines daily activities , show a mean value of 3.80 ( range from 2.29 to 5 ) , while sedentary activities were averagely 159.38 minutes daily . minimum daily time spent in sedentary activities was 25 minutes , and maximum was 420 minutes . the fms - polygon was significantly correlated with children s daily activity ( -0.38 ) , while the correlation with daily inactivity was not significant ( table 3 ) . the correlations between gymnastic skills and children s daily activity were all significant ( from 0.25 to 0.36 ) , with the exception of the handstand against wall ( 0.21 ) . results of correlation analysis between fms - polygon , final results of gymnastics skills and activity ( a ) and non activity ( n - a ) variables significant differences found in the fms - polygon and in all gymnastic skills , at the beginning and at the end of the 18-week gymnastic program , are a proof of an efficient motor learning process . pointed differences in the level of mastered gymnastics skills , between pre and post measuring , can be explained through complexity of those skills . forward and backward rolls are considered as simple gymnastics skills , while dominant frontal cartwheel is a complex gymnastics skill for seven - year - old children . since the applied program has prescribed the same learning time for all investigated gymnastics skills , we can presume that the complexity of skills affected the learning process . furthermore , the authors are aware that other factors such as style and learning strategy , fatigue , anxiety and lack of motivation can also affect the learning process and these questions still remain open . from the aspect of applying gymnastics skills in pe curricula and implementing the principle of progression in the learning process , it is important to point out simple and complex basic gymnastics skills . lower results of the switching positions on the rings in the final measurement point can be explained by insufficient strength of arms and shoulders that should be developed in progress . in conclusion , the period of 18 weeks and 39 school lessons is enough for appropriate acquisition of basic artistic gymnastics skills for seven - year - old children , and the complexity of skills should be considered in the pe curriculum design . according to the correlation analysis between gymnastics skills and the fms - polygon , at the beginning and at the end of the 18-weeks gymnastics program , all gymnastics skills were found to be in significant progress , confirming the thesis that basic gymnastics skills and fms could be developed simultaneously . significant progress in the fms - polygon during the 18 weeks period , according to the dependent samples t - test , higher values of gymnastic skills , and progressing correlation between gymnastics skills and the fms - polygon over time , suggests that a positive transfer occurred between gymnastics skills and fms . analyzing the described gymnastics skills and locomotor skills , we can conclude that the theory of positive transfer of similar skills , defined by osgood , has occurred . a lot of fms , such as skills for surmounting obstacles ( jumping , landing , vaulting , wriggling and climbing ) , represent basic gymnastics skills in their original or modified form ( e.g. , landing with or without running start , with or without swing , on one or both feet etc . ) . that is why it is not always possible to categorize a skill exclusively in a certain group of fms or as a gymnastics skill , and that is why artistic gymnastics is a basic sport suitable especially for young children . the adoption of complex motor skills after mastering simple motor skills is very important in gymnastics . when the level of difficulty of a task is not compatible with the current skills level of the learner , practice becomes futile and unsuccessful . mastering of basic gymnastic skills will provoke improvement of fms and finally be a prerequisite for successful introduction in learning more complex gymnastic skills . basically , gymnastics skills belong to the categories of discrete and closed motor skills , characterized by short and well defined action which includes moving from the beginning and/or rotating of the whole body around one or more axis . frequent practice of these skills naturally improves locomotor fms , and higher level of fms allows continuous learning of new skills as well as adjustment of dynamics of learning processes to individual characteristics and abilities . table 4 is designed to point out the differences between boys and girls in mastering the fms - polygon and to compare the results obtained here on seven - year - old children with results obtained by zuvela on eight - year - old children . when comparing the results of the fms - polygon separately by gender with those of children who are one year older ( table 4 ) , we can conclude that the fms - polygon is age sensitive which is one of the study limitations . new , wider investigations are necessary to establish age and gender sensitive categories of gross motor quotient for fms - polygon . also , one more study limitation arises . the question if learning gymnastics skills develops all fms skills equally ca nt be answered by this investigation simply because fms - polygon assesses the whole range of fms skills by a single number . to reach these answers , it can be predicted that gymnastics program does develop locomotor and object control skills because it was proven in previous research of preschool children , but it needs to be verified in future investigations . finally , a lack of assessment of other external factors such as style and learning strategy , fatigue , anxiety and lack of motivation that also affect skill acquisition present a certain study limitation , which needs to be represented in future investigation . comparison of fms - polygon gross motor quotient in this study with results obtained by zuvela ( 2009 ) on eight year old children in this study children s daily unorganized activities and inactivity were measured because of the possible influence on mastering gymnastics skills and development of fms . the significant correlations of gymnastics skills and fms - polygon imply that increased children s unorganized daily activities can improve the mastering of basic gymnastics skills , and simultaneous development of fms . in this way , this investigation is congruent with previous research and brings to a conclusion that inactivity among seven - year - old children can cause later failures in learning specialized movement skills . studies analyzing relations between organized learning and mastering of specific skills and fms development along with children s daily activity and inactivity are rare . it is not only organized gymnastics program that has effects on development of seven - year - old children s fms . daily activity of children affects both : the development of gymnastics skills and the development of fms . according to the results obtained by the fms - polygon , basic gymnastics program , in combination with proper daily activity of young children , for more exact results further research is needed with the possibility of separating locomotor , object control and body management fms . d. miletic : concept / design , critical revision of the manuscript , preparing the draft of the manuscript . kalinski : aquisition of data a. kezic : data analysis and interpretation , preparing the draft of the manuscript f. zuvela : aquisition of data all authors approved final version of the paper .

abstractobjectivethe objectives of this study were : a ) to examine the influence of an 18-week basic artistic gymnastics program on fundamental movement skills ( fms ) development in seven - year - old children ; b ) to determine correlations between children s daily activities and successful performance of fms and basic artistic gymnastics skills.methodsseventy five first grade primary school children took part in this study . a physical education teacher specialized in artistic gymnastics conducted a gymnastics program for 18 weeks , three times a week . the level of gymnastics skills and fms were identified at the beginning and at the end of the program . the level of gymnastics skills was evaluated by performance of eight artistic gymnastics skills , while fms were evaluated by the use of fms - polygon . physical activity and inactivity was evaluated by using a proxy - questionnaire netherlands physical activity questionnaire ( npaq).findingsaccording to the dependent samples t test , significant differences were found in the fms - polygon and all gymnastics skills before and after the 18-week gymnastics program . increasing correlations were established over time between gymnastics skills and the fms - polygon . unorganized daily activity of children significantly correlated with their mastering of gymnastics skills and fms . the presented findings confirm : ( 1 ) the thesis that basic artistic gymnastics skills and fms could be developed simultaneously , ( 2 ) the theory of positive transfer of similar skills between fms and artistic gymnastic skills.conclusionmastering basic artistic gymnastics skills will provoke improvement of fms and finally become a prerequisite for successful introduction of learning more complex gymnastics skills . the obtained results imply that an increase of children s unorganized daily activities can improve the mastering of basic gymnastics skills and simultaneously the development of fms .

Introduction Subjects and Methods Findings Discussion Conclusion Authors Contribution Conflict of Interest

PMC4719489

typologies have been delineated for body structure ( ectomorph , mesomorph , and endomorph ) , mental functioning ( multiple intelligences ) , emotional response ( levels of emotional reactivity or sensation- seeking ) , social relationships ( level of need for attachment ) , and behavioral tendencies ( level of stress reactivity ) . ayurveda , the traditional medical system of india , has delineated three categories of fundamental regulatory principles of the body , mind , and behavior . these three categories , called doshas , are named vata , pitta , and kapha . the category of vata dosha includes processes responsible for cell division and cell signaling , movement at all levels of the physiology , excretion of wastes , and also cognition . the category of kapha dosha includes processes responsible for anabolism , growth and maintenance of structure , storage and stability . the category of pitta dosha includes processes responsible for metabolism , thermo - regulation , energy homeostasis , pigmentation , vision , and attentional processes . the physical , mental , and behavioral characteristic of each dosha are summarized in table 1 . notice that the descriptions listed are primarily based on external observation of physiological characteristics and behavior [ table 1 ] . a body of research has grown , which explores patterns of blood chemistry , genetic expression , physiological states , and chronic diseases associated with each dosha type . triglycerides , total cholesterol , high low - density lipoprotein ( ldl ) , and low high - dl ( hdl ) concentrations - all common risk factors for cardiovascular disease were reported to be higher in kapha types compared to pitta and vata types . other research supports these findings that high levels of triglyceride , very ldl and ldl levels and lower levels of hdl cholesterol distinguish kapha types from other types . genetic expression also distinguishes dosha types . genes in the immune response pathways were up - regulated in pitta types , genes related to cell cycles were up - regulated in vata types , and genes in the immune signaling pathways were up - regulated in kapha types . inflammatory genes were up - regulated in vata types , whereas up - regulation of oxidative stress pathway genes were observed in pitta and kapha types . cd25 ( activated b cells ) and cd56 ( natural killer cells ) were higher in kapha dosha types . cyp2c19 genotypes , a family of genes that help in detoxification and metabolism of certain drugs were down - regulated in kapha types and up - regulated in pitta types . adenosine diphosphate - induced maximal platelet aggregation was the highest among vata / pitta types . in diabetic patients , there were significant decreases in systolic blood pressure in vata / pitta , pitta / kapha , and vata / kapha types after walking ( isotonic exercise ) . the vata / pitta types also showed significant decreases in mean diastolic blood pressure . in terms of biochemistry , kaphas had elevated digoxin levels , increased free radical production and reduced scavenging , increased tryptophan catabolites and reduced tyrosine catabolites , increased glycoconjugate levels , and increased cholesterol : phospholipid ratio of red blood cell membranes . a study of basic cardiovascular responses reported that heart rate variability and arterial blood pressure during specific postural changes , exercise , and cold pressor test did not vary with constitutional type . a more recent paper measuring cold pressor test , standing - to - lying ratio , and pupillary responses in light and dark reported that kapha types have higher parasympathetic activity and lower sympathetic activity in terms of cardiovascular reactivity as compared to pitta or vata types . an open question in this discussion is how these genetic , metabolic , and physiological factors express themselves in terms of more expressed lifestyle and behavioral characteristics . obviously , many genetic and metabolic factors are influenced by the nervous system , which not only controls metabolic and growth factors through the endocrine system but have an impact on the functioning of the immune , digestive , cardiovascular , and virtually every other system in the body . further , it controls all our mental , emotional , and behavioral systems including attention , learning , memory , and sleep . thus , the dosha types which are based on observable mental and physical behavior and characteristics , should also be based on different patterns of brain and nervous system functioning . for instance , vata dosha , which is highly variable in behavior and in response to the environment , would be associated with a greater range of functioning of the brain and nervous system . pitta dosha , which is characterized by dynamism , would be associated with fast , passionate responses of the brain and nervous system to challenges in the environment . kapha dosha , which is characterized by steadiness , would be associated with stabile activity patterns of the brain and nervous system . these three different types of brain functioning can be seen as different patterns of functioning of six major systems of the nervous system . patterns of brain functioning for vata , pitta , and kapha brain - types the first system is the frontal executive system of the brain , which includes the anterior cingulate gyrus ( attention switching and error detection ) , ventral medial ( emotional input ) , and the dorsal lateral prefrontal cortex ( decision making ) . the vata brain - type exhibits a high range of prefrontal functioning leading to the possibility of being easily overstimulated . the pitta brain - type reacts strongly to all challenges leading to purposeful and resolute actions . the kapha brain - type is slow and steady leading to methodical thinking and action . the second system is the reticular activating system ( ras ) of the brain , which is responsible for arousal level . the vata brain - type exhibits a high range of arousal levels leading to a sense of over - reacting to the world . the pitta brain - type becomes easily aroused and maintains high level of focused arousal to get a task accomplished . the third system is the autonomic nervous system that includes the sympathetic ( fight - or - flight ) and parasympathetic ( tend - and - befriend ) systems . ninety percent of our responses to the environment are governed by the autonomic nervous system . this system automatically maintains an optimal level of arousal to deal with any situation from watching a sunset to running after a taxi . the fight - and - flight response is easily turned on in vata brain - types and is variable in its level of response . their limbs are generally cold and have poor circulation since high sympathetic activation reduces periphery blood flow . the fight - and - flight response turns on to a high level in pitta brain - type and then return to resting levels again . autonomic response is tied to purposeful goal oriented behavior it turns on to reach the goal and then turns off . the fight - or - flight response the enteric nervous system interacts with the microbiome of the gut to modulate immune functioning and activity of the parasympathetic nervous system . the vata brain - type exhibits a high range of digestive power leading to an irregular appetite , irregular bowel movements , and frequent gas . they are always hunger and can eat at any time and , seemingly , any food . the kapha brain - type is not much affected by what or when they eat . the enteric nervous system interacts with satiety centers in the hypothalamus to govern feelings of hunger . it includes many nuclei around the center of the brain : the amygdala for survival and fear response , the hippocampus for anger and spatial awareness , the nucleus accumbens for pleasure , the insula for saliency of experience and tie bodily states to emotions , and hypothalamus that integrates the activity of the autonomic nervous system . the limbic system is highly sensitive to changes in the environment in vata brain - types . when over - activated , the vata brain - type can have excessive fear and phobias . the limbic system provides the fire for the pitta brain - type to react to the world . it automatically controls our responses to challenges , freeing us from considering hunger , thirst , and arousal levels . the output of the limbic system feeds into the hypothalamus , which then will activate the autonomic nervous systems as needed and even activate the prefrontal cortex . in vata brain - types , the hypothalamus is constantly changing the state of mind and body . they will experience bursts of activity and rest , and will frequently snack and drink . in pitta brain - types , the hypothalamus has a strong on and off switch . when turned on , the autonomic nervous system functions at its maximum to accomplish the goal . the hypothalamus maintains a higher core body temperature and dynamic mental and physical activity that leads to the preference for cool foods and drinks in this brain - type . in kapha brain - types , this table has been created based on the organizing principles of the three doshas in ayurveda . if these sub - categories of functioning of brain systems are valid , then other mental and physical typologies should also be explained by the patterns of nervous system functioning described in this table . the explanatory power of this proposed model is demonstrated in consideration of sensation - seeking . zuckerman defined the personality trait of sensation - seeking as the tendency to choose varied , complex , and intense sensations leading to increased risk . this trait is exhibited in a range of behaviors including substance use , gambling , and risky sexual practices . high sensation - seekers orient more strongly to novel stimuli and show larger cortical activation patterns . low sensation - seekers show defensive reactions to strong stimuli and show reduced or unresponsive cortical activation patterns to variations in stimulus intensity . sensation - seeking is tied to dopamine levels , with higher dopamine leading to more risky behaviors . dopamine appears to amplifier behavior when there is the chance for large gains , and decreases discrimination that the behavior may also yield large losses . this is seen in rats who press levels for food one yielding one pellet each time , the other leading 4 pellets from 12% to 80% of the time . this was also seen with humans who were given a dopamine agonist , cabergoline , which increases the effect of dopamine in the brain . during conditions with cabergoline compared to placebo , subjects selected the high - risk condition ( 60 - 40 ) when there was the chance for high gains . this effect in humans was only seen in subjects with low levels of sensation - seeking at baseline . high sensation - seeking is also tied to reduced attention to negative consequences of loss . a functional magnetic resonance imaging ( fmri ) study compared blood flow in high and low sensation - seekers during a wheel of fortune type test . both high and low sensation - seekers showed activation in bilateral insular and prefrontal cortical response to winning ( activation ) . however , the high sensation - seekers showed deactivation in these structures to negative outcomes compared to the low group . high sensation - seekers orient more strongly to novel stimuli and they tend to ignore negative consequences . high sensation - seeking shares many characteristics of the pitta brain - type with faster activation of the reticular activation that systemically increases arousal , activation of the amygdala that turns on the sympathetic nervous system to increase resource allocation , and activation of the anterior cingulate gyrus to turn your attention to the experience . low sensation - seekers have a defensive reaction to novel stimuli , cortical activation is lower , and they consider both positive and negative consequences of an action . low sensation - seekers shares many characteristics of the kapha brain - type with less reactivity overall - lower activation of the reticular activation , the amygdala , and the sympathetic nervous system . the vata brain - type may lead more to impulsivity rather than to sensation - seeking . impulsivity and sensation - seeking have different trajectories of development from age 12 to 24 . the vata brain - type is marked by variable response to external conditions that would lead to bursts in attention , in physical energy , and in autonomic response to situations . this type would probably be scored as neither as high nor as low sensation - seeker . an important consideration in this discussion is that the sensation - seeking trait develops during adolescence . a 12 years longitudinal study of 7640 individuals , age 12 years at baseline , reports substantial variation in the magnitudes of developmental change in sensation - seeking . some teenagers showing rapid changes in sensation - seeking as they matured and others maintaining relatively constant over the 12 years . these differences could reflect predominance of vata , pitta or kapha brain - types . a research program using physiological and cortical measures could explore the utility of dosha brain - types . for instance , resting state networks have become a recent focus in neural imaging research . resting state networks are large - scale cortical circuits that dynamically interact to process experiences a salience network , involving the anterior insula and cingulate gyrus , deactivates the default mode network and activates the central executive network . the salience network could be more variable in vata brain - types , less reactive in kapha brain - types , and function more decisively in pitta brain - types more quickly shutting down the default mode and turning on the central executive network . fmri could also be used to assess emotional responses to the international affective picture system . this research program could investigate sympathetic / parasympathetic balance as measured by heart rate variability , which is sensitive to anxiety levels and level of emotional stress . it could also measure cytokine and c - reactive proteins to assess immune functioning in the different brain - types . a focused analysis of brain - types would build the neural basis to understand dosha types [ table 3 ] . research program to explore physiological , neural , psychological , and behavioral correlates of dosha types zuckerman defined the personality trait of sensation - seeking as the tendency to choose varied , complex , and intense sensations leading to increased risk . this trait is exhibited in a range of behaviors including substance use , gambling , and risky sexual practices . high sensation - seekers orient more strongly to novel stimuli and show larger cortical activation patterns . low sensation - seekers show defensive reactions to strong stimuli and show reduced or unresponsive cortical activation patterns to variations in stimulus intensity . sensation - seeking is tied to dopamine levels , with higher dopamine leading to more risky behaviors . dopamine appears to amplifier behavior when there is the chance for large gains , and decreases discrimination that the behavior may also yield large losses . this is seen in rats who press levels for food one yielding one pellet each time , the other leading 4 pellets from 12% to 80% of the time . this was also seen with humans who were given a dopamine agonist , cabergoline , which increases the effect of dopamine in the brain . during conditions with cabergoline compared to placebo , subjects selected the high - risk condition ( 60 - 40 ) when there was the chance for high gains . this effect in humans was only seen in subjects with low levels of sensation - seeking at baseline . high sensation - seeking is also tied to reduced attention to negative consequences of loss . a functional magnetic resonance imaging ( fmri ) study compared blood flow in high and low sensation - seekers during a wheel of fortune type test . both high and low sensation - seekers showed activation in bilateral insular and prefrontal cortical response to winning ( activation ) . however , the high sensation - seekers showed deactivation in these structures to negative outcomes compared to the low group . high sensation - seekers orient more strongly to novel stimuli and they tend to ignore negative consequences . high sensation - seeking shares many characteristics of the pitta brain - type with faster activation of the reticular activation that systemically increases arousal , activation of the amygdala that turns on the sympathetic nervous system to increase resource allocation , and activation of the anterior cingulate gyrus to turn your attention to the experience . low sensation - seekers have a defensive reaction to novel stimuli , cortical activation is lower , and they consider both positive and negative consequences of an action . low sensation - seekers shares many characteristics of the kapha brain - type with less reactivity overall - lower activation of the reticular activation , the amygdala , and the sympathetic nervous system . the vata brain - type may lead more to impulsivity rather than to sensation - seeking . impulsivity and sensation - seeking have different trajectories of development from age 12 to 24 . the vata brain - type is marked by variable response to external conditions that would lead to bursts in attention , in physical energy , and in autonomic response to situations . this type would probably be scored as neither as high nor as low sensation - seeker . an important consideration in this discussion is that the sensation - seeking trait develops during adolescence . a 12 years longitudinal study of 7640 individuals , age 12 years at baseline , reports substantial variation in the magnitudes of developmental change in sensation - seeking . some teenagers showing rapid changes in sensation - seeking as they matured and others maintaining relatively constant over the 12 years . a research program using physiological and cortical measures could explore the utility of dosha brain - types . for instance , resting state networks have become a recent focus in neural imaging research . resting state networks are large - scale cortical circuits that dynamically interact to process experiences a salience network , involving the anterior insula and cingulate gyrus , deactivates the default mode network and activates the central executive network . the salience network could be more variable in vata brain - types , less reactive in kapha brain - types , and function more decisively in pitta brain - types more quickly shutting down the default mode and turning on the central executive network . fmri could also be used to assess emotional responses to the international affective picture system . this research program could investigate sympathetic / parasympathetic balance as measured by heart rate variability , which is sensitive to anxiety levels and level of emotional stress . it could also measure cytokine and c - reactive proteins to assess immune functioning in the different brain - types . a focused analysis of brain - types would build the neural basis to understand dosha types [ table 3 ] . research program to explore physiological , neural , psychological , and behavioral correlates of dosha types this brain - type model makes specific predictions about patterns of frontal executive functioning , ras activation , and patterns of autonomic and limbic functioning . future research will investigate the relation of dosha type to electroencephalographyeeg patterns , autonomic activation and autonomic balance , stress reactivity and behavior to support the proposed model . this line of research could help clarify variable response to drugs and lifestyle modifications in normal and clinical populations and so help target health promotion at all levels of life .

this paper explores brain patterns associated with the three categories of regulatory principles of the body , mind , and behavior in ayurveda , called vata , pitta , and kapha dosha . a growing body of research has reported patterns of blood chemistry , genetic expression , physiological states , and chronic diseases associated with each dosha type . since metabolic and growth factors are controlled by the nervous system , each dosha type should be associated with patterns of functioning of six major areas of the nervous system : the prefrontal cortex , the reticular activating system , the autonomic nervous system , the enteric nervous system , the limbic system , and the hypothalamus . for instance , the prefrontal cortex , which includes the anterior cingulate , ventral medial , and the dorsal lateral cortices , would exhibit a high range of functioning in the vata brain - type leading to the possibility of being easily overstimulated . the vata brain - type performs activity quickly . learns quickly and forgets quickly . their fast mind gives them an edge in creative problem solving . the pitta brain - type reacts strongly to all challenges leading to purposeful and resolute actions . they never give up and are very dynamic and goal oriented . the kapha brain - type is slow and steady leading to methodical thinking and action . they prefer routine and needs stimulation to get going . a model of dosha brain - types could provide a physiological foundation to understand individual differences . this model could help individualize treatment modalities to address different mental and physical dysfunctions . it also could explain differences in behavior seen in clinical as well as in normal populations .

INTRODUCTION PHYSIOLOGICAL PATTERNS SUPPORT THE TRIDOSHIC THEORY THEORY OF DOSHA BRAIN-TYPES Sensation-seeking in light of the three brain-types A research program to explore physiological correlates of dosha brain-types CONCLUSION Financial support and sponsorship Conflicts of interest

PMC4353033

-aminobutyric acid ( gaba ) is a major inhibitory neurotransmitter in the mammalian central nervous system . when gaba concentrations in the brain fall below a threshold level , convulsions occur . it has also been found that alzheimer s disease , huntington s chorea , parkinson s disease , and drug addiction are related to diminished gaba levels in the brain . gaba is produced by the -decarboxylation of the excitatory neurotransmitter l - glutamic acid , catalyzed by the pyridoxal 5-phosphate ( plp)-dependent enzyme glutamic acid decarboxylase ( gad , scheme 1 ) . gaba is metabolized by -aminobutyric acid aminotransferase ( gaba - at ) , also a plp - dependent enzyme , to succinic semialdehyde , which is further oxidized to succinic acid by succinic semialdehyde dehydrogenase ( ssdh ) . degradation of gaba to succinic semialdehyde converts the plp coenzyme to pyridoxamine 5-phosphate ( pmp ) ; -ketoglutarate is used in a second step to return pmp to plp with concomitant formation of l - glutamic acid . therefore , the concentrations of the inhibitory ( gaba ) and excitatory ( l - glu ) neurotransmitters are regulated by the appropriate balance between gad and gaba - at . when gaba is injected directly into the brain of a convulsing animal , the seizures are terminated . however , because gaba does not cross the blood brain barrier , peripheral gaba administration is not an effective approach for the treatment of epilepsy . an alternative approach to increase gaba levels in the brain is by inhibition of gaba - at , which blocks the degradation of gaba . a mechanism - based inactivator , an unreactive compound that initially acts as a substrate for the target enzyme and is converted to a species that causes inactivation of that enzyme , generally by covalent attachment , could increase gaba levels for an extended period of time . the design of these types of inhibitors requires insight into the mechanism of the target enzyme so that the catalytic chemistry can be utilized in the activation of the inactivator ; the mechanism of the first half of the ping - pong reaction catalyzed by gaba - at is shown in scheme 2 . incubation of gaba with gaba - at leads to the conversion of a lysine - plp aldimine ( 1 ) to a gaba - plp aldimine ( 2 ) . abstraction of the -proton by lys329 and tautomerization leads to a pmp aldimine ( 3 ) ; enzyme - catalyzed hydrolysis of this intermediate leads to succinic semialdehyde and pmp . 4-amino-5-hexenoic acid ( 4 , -vinyl - gaba ; vigabatrin ) is a mechanism - based inactivator of gaba - at that is an fda - approved drug ( sabril ) to treat infantile spasms and refractory partial seizures . however , because of a serious side effect , namely , retinal damage and blindness , a drug with more potency and less toxicity was desired . mechanistic studies performed by nanavati and silverman ( scheme 3 ) demonstrated that vigabatrin inactivates gaba - at via two mechanisms , a michael addition reaction and an enamine reaction , in a ratio of 70:30 . in the michael addition pathway ( pathway a ) , vigabatrin acts like substrate gaba and undergoes aldimine formation with plp , deprotonation , and tautomerization to a michael acceptor ( 5 ) , which undergoes michael addition by lys329 , the lysine residue to which plp is bound in the native enzyme , giving , after protonation , covalent adduct 6 . enzyme - catalyzed hydrolysis of 5 gives ,-unsaturated ketone 7 , which may be a possible cause for side effects of the drug . in the enamine pathway ( pathway b ) , an enamine intermediate ( 8) , formed by tautomerization through the alkene , attacks the enzyme - bound plp to inactivate gaba - at ( 9 ) , also by formation of a covalent adduct . hydrolysis of 8 gives a weak electrophile , which is unlikely to proceed through further reactions in the active site . to avoid the potentially toxic intermediate ( 7 ) , computer modeling based on the crystal structures of gaba - at and the vigabatrin plp complex in gaba - at was carried out to determine how to prevent the michael addition pathway . the model predicted that , in the energy - minimized structure , the alkene double bond pointed away from the nucleophilic lys329 ; for michael addition to occur , that bond had to rotate , allowing the alkene to be aligned with the side chain amine of lys329 . a conformationally rigid vigabatrin analogue was prepared ( 10 ) to prevent michael addition , and it was found that it inactivated gaba - at exclusively by the enamine pathway ; however , it was a weak inactivator . the second approach to prevent formation of reactive product 7 was to synthesize a vigabatrin analogue that would give rapid michael addition so that the equivalent of intermediate 5 would not be sufficiently stable to allow the enzyme - catalyzed hydrolysis leading to the equivalent of 7 . that approach required the synthesis of a compound with the alkene pointing toward the side chain amine of lys329 ( 11 ) . the obtained compound inactivated gaba - at , but only in the absence of the electrophilic trapping agent 2-mercaptoethanol . therefore , an undesirable reactive intermediate ( the corresponding ketoacid of 11 ) was formed prior to inactivation . to accelerate the inactivation rate , two electron - withdrawing fluorine atoms were attached to the alkene of 11 , and the obtained compound ( 12 ) inactivated gaba - at , even in the presence of 2-mercaptoethanol . further studies with 12 ( now called cpp-115 ) showed that it was at least 187 times more potent an inactivator of gaba - at than vigabatrin , was not active in the cerep panel of 111 pharmacological targets ( in vitro assays , carried out commercially by cerep , on cpp-115 that cover a broad range of targets , including receptors , ion channels , enzymes , transporters , and second messengers ) , does not bind to three human gaba transporters gat1 , gat2 , and gat3 , or to gabaa , gabab , or gabac receptors , does not bind to the herg channel , does not inhibit or induce cytochrome p450s , is not metabolized by hepatocytes , is rapidly and completely orally absorbed and eliminated , has no adverse effect on respiration , produces no mutations or chromosomal aberrations , and has a considerably lower retinal toxicity liability . it also is 300600 times more potent than vigabatrin in reducing cocaine - induced synaptic dopamine levels and in blocking expression of cocaine - induced conditioned place preference in freely moving rats . recently , it was shown to have no adverse effects in a phase i clinical trial . because of the clinical importance of this new drug candidate , we studied its inactivation mechanism to determine if it indeed favors the michael addition pathway for which it was designed . we have determined that cpp-115 inactivates gaba - at via an unexpected inactivation mechanism , namely , enzyme - catalyzed conversion of the difluoromethenyl group to a carboxylate , which causes a conformational change in the enzyme , to give a tightly bound complex . this inactivation mechanism presents a novel approach to inactivate gaba - at and other related enzyme targets using similar catalytic mechanisms . the turnover number for inhibition was determined by incubating gaba - at with various equivalents of cpp-115 . figure 1 shows the remaining enzyme activity versus time for 04 equiv of cpp-115 . to determine the turnover number , the data points at 24 h were replotted as a pseudo - first - order reaction using the remaining enzyme activity versus equivalents . however , as shown in the replot , nonpseudo - first - order inactivation kinetics was observed , possibly because of product inhibition . ( top ) ( bottom ) determination of the turnover number for the inactivation of gaba - at by cpp-115 . the data points at 24 h are replotted as enzyme activity vs equiv of cpp-115 . to test if the mechanism involves irreversible and/or reversible inhibition , the time - dependent reactivation of gaba - at was studied . first , the enzyme was treated with an excess of inactivator ; then , the mixture was dialyzed against bulk buffer . return of enzyme activity was observed , which displayed both time- and concentration - dependence ( see supporting information , figures s1 and s2 ) . the data also show that the amount of activity of the inhibited gaba - at that returned was dependent on the amount of cpp-115 used in the incubation and the incubation time . in figure s1 ( supporting information ) , up to 90% and 60% of enzyme activity was recovered when gaba - at was inactivated with 40 and 80 equiv of cpp-115 , respectively , for 3 h. however , when gaba - at was incubated for 24 h , even with 10 equiv of cpp-115 , only 50% of the activity returned ( figure s2 , supporting information ) . in the presence of 1000 equiv ( approximately 1 mm ) of cpp-115 in the incubation mixture , the activity of the enzyme returned to about 8% ; with 2000 equiv of cpp-115 , the activity return was only 1% , even after 50 h of dialysis . upon incubation of gaba - at with cpp-115 , fluoride ions were continually released , even after the enzyme s activity had fallen to almost zero ( figure s3 , supporting information ) . after normalization of the values with the controls , it was found that 66 3 equiv of fluoride ions were released slowly over a period of 27 h. however , when the same experiment was repeated with plp supplemented in the mixture , there were 127 5 equiv of fluoride ions released over time . because -ketoglutarate is essential to regenerate plp from pmp , if -ketoglutarate is omitted , the amount of fluoride ions released per single turnover of inhibition can be determined . the continual release of fluoride ions was not observed when there was no -ketoglutarate present , and only 4.3 0.5 equiv of fluoride ions were detected per enzyme dimer ( figure s4 , supporting information ) . however , when -ketoglutarate was added to the inactive enzyme , even without supplementation with plp , the fluoride ion concentration began to increase . a control experiment with no enzyme in the incubation mixture was performed , which confirmed that no fluoride ions were released from cpp-115 in the absence of the enzyme , even if plp is added . to determine the fate of the coenzyme by inactivation with cpp-115 the maximum recovered activity was 71.4% of the original , and the activity recovery plateaued after 5 h of the reactivation process ( table 1 ) , indicating that 29.6% of the enzyme remained inactive . [ h]plp - gaba - at was inactivated with cpp-115 , and the released radioactive compounds were analyzed using hplc ( figure s5 , supporting information ) . the experiment was performed with two controls : cpp-115 was omitted in a negative control ( all radioactivity should be labeled plp ) , and gaba replaced cpp-115 with -ketoglutarate omitted in a positive control ( all radioactivity should be pmp ) . the negative control released most of its radioactivity as plp ( but none as pmp ) , whereas a gaba - incubated control released both pmp and plp ( figure s6 , supporting information ) . given that the positive control with gaba should only produce pmp from active enzyme , the plp present in this sample represents that released from the inactive portion of gaba - at . after the background radioactivity from the control experiments was subtracted from the cpp-115 experiment , cpp-115-inactivated [ h]plp - reconstituted gaba aminotransferase was found to release 100% of its cofactor as [ h]pmp ( figure s7 , supporting information ) . the small radioactive peak at 33 min was confirmed to be degraded plp present in all of the samples . after inactivation of gaba - at by cpp-115 followed by denaturation and filtration , three metabolites were identified from the sample solution that were not present in the control samples : [ m / z ] 127.0389 , 171.0291 , and 401.0745 ( a , b , and c in figure s8 , supporting information , respectively ) , corresponding to 22 , 21 , and 20 , respectively , in scheme 5 ( see discussion ) . fragmentation data for m / z 127.0389 ( supporting information figure s9 ) confirmed the structure of 3-oxo-1-cyclopentanecarboxylic acid ( 22 ) . ms / ms for m / z 171.0291 and 401.0745 ( supporting information figures s10 and s11 , respectively ) confirmed the structures of 21 and 20 . since covalent modification of gaba - at was not detected using middle down proteomics ( see figures s12s14 in supporting information ) , we analyzed the intact gaba - at protein to see if any detectable mass shifts were present . the intact mass data showed multiple peaks , indicating that the gaba - at purified from pig brain was a mixture of gaba - at species with different n - termini . three samples were tested using lc / ms / ms : free enzyme ( negative control ) , vigabatrin - inactivated gaba - at ( positive control ) , and cpp-115-inactivated gaba - at ( figure s15 , supporting information ) . vigabatrin - inactivated gaba - at showed an added mass of 122 da from the mass of the native enzyme , which matches the covalent adduct proposed previously . however , cpp-115-inactivated gaba - at showed no significant peaks corresponding to any added mass . to stabilize any potential imine adducts throughout the lc / ms / ms process , the samples were reduced with sodium borohydride as described previously for crystallography studies . reduction resulted in stabilization of the plp cofactor on the enzyme , with an added mass of 236 from the original peak ( m ) . as expected , the peaks of vigabatrin - inactivated gaba - at ( figure s16 , supporting information ) were no different from those in figure s15 since the vigabatrin covalent adduct is stable . interestingly , for cpp-115-inactivated gaba - at , reduction had no effect on the resulting data and showed no added mass on the protein . to understand how time - dependent inactivation of gaba - at by cpp-115 could occur without covalent modification of the protein or cofactor , cpp-115-inactivated and dialyzed the crystal structures of native gaba - at from pig brain and inactivated enzyme were obtained at 1.63 and 2.19 resolution , respectively . the crystal structure for the native pig brain enzyme was very similar to that reported from pig liver by storici et al . the crystal structures of the native enzyme and the inactivated enzyme were compared to analyze the difference in overall structure ( figure 2 ) and in the active site ( figure 3 ) . the active site of the inactivated gaba - at was investigated to understand the ligand enzyme interactions ( figure 4 ) ; the omit map supports the ligand interpretation ( see supporting information , figure s17 ) . ribbon diagram of the superimposed native gaba - at ( yellow ) and gaba - at ( cyan ) bound to cpp-115 . superimposition of the crystal structures of native gaba - at ( pink ) and cpp-115-inactivated gaba - at ( green ) . stereoview of gaba bound by the cpp-115 adduct . the 2fo fc electron - density map is shown as light gray mesh at 1.1 level around the cpp-115 adduct . at the start of this investigation , we considered a variety of likely mechanisms for the inactivation of gaba - at by cpp-115 ( schemes 48 ) and then considered experiments to differentiate them . all of these inactivation mechanisms are initiated by the same steps shown in scheme 2 for substrate turnover or the two inactivation mechanisms for vigabatrin shown in scheme 3 . in mechanism 1 ( scheme 4 ) , following -proton abstraction of 13 , tautomerization leads to an ,-unsaturated intermediate ( 14 ) , which is attacked by the active site lysine residue or another base to covalently modify the enzyme . no fluorine is released , the enzyme is inactivated , and hydrolysis of 15 gives 16 with release of pmp as the cofactor . if intermediate 15 in scheme 4 ( where x is either the active site lysine residue or oh from attack by water ) eliminates one fluoride ion ( scheme 5 ) , the inactivator can form another reactive ,-unsaturated imine ( 17 ) , which can be attacked by a water molecule to release the other fluoride ion , forming 18 . depending on what x is , this may result in inactivation or turnover . if x is the active - site lysine residue ( 15a , pathway a , scheme 5 ) , the intermediate is a stabilized amide ; hydrolysis generates covalent adduct 19 with the release of pmp . however , if x is a hydroxyl group ( 15b , pathway b , scheme 5 ) formed by water molecule attack on reactive intermediate 14 ( scheme 4 ) , stable dicarboxylate intermediate 20 will form ; hydrolysis gives metabolite 21 and pmp , and subsequent decarboxylation of 21 gives 22 . the third potential mechanism involves allylic tautomerization of external aldimine 13 to form 23 ( scheme 6 ) . because 23 also is a reactive electrophile , it may undergo michael addition to form adduct 24 , which could be hydrolyzed to give 25 and release pmp . in the fourth potential mechanism ( scheme 7 ) , pathway a generates an enamine ( 26 ) , similar to that formed during vigabatrin inactivation ; this species might undergo enamine attack on the lys329-bound plp to form covalent adduct 27 , which could hydrolyze to 28 . in pathway b , one fluoride ion is released by nucleophilic substitution ( 29 ) before undergoing transimination to form enamine adduct 30 , which might undergo hydrolysis to 31 . pathway c from 30 involves elimination of a second fluoride ion ( 32 ) followed by hydrolysis to give 33 . the final potential mechanism involves fluoride ion elimination from enamine 26 to generate reactive michael acceptor 34 ( scheme 8) . attack by an active site nucleophile ( either lys329 or water ; pathway b ) gives either covalent adduct 36 or noncovalent adduct 37 , respectively . elimination of another fluoride ion forms 38 or 40 , each of which can be subsequently hydrolyzed to covalent adduct 39 or metabolite 41 ; further hydrolysis of 39 would also give 41 . in either case , this mechanism releases the cofactor as plp . all of these possible mechanisms can be differentiated by determination of the number of fluoride ions released , the fate of the cofactor , and the final metabolites or adducts formed ; these possibilities are summarized in table 2 . the partition ratio , the number of inactivator turnovers per inactivation event , was investigated by determination of the number of molecules of cpp-115 that were required to cause complete enzyme inactivation . time - dependent inhibition was observed for 14 equiv of cpp-115 , although pseudo - first - order kinetics was not observed ( figure 1 , top ) . a replot of these data with extrapolation of the first few data points gave a turnover number of 1.3 0.3 per active site ( figure 1 , bottom ) . the nonpseudo - first - order inactivation kinetics displayed indicated possible product inhibition and suggested possibly two concurrent mechanisms occurring at different rates , maybe one reversible and one irreversible . to test whether there was a reversible component to the inactivation , the titration experiment was repeated , first with 080 equiv of cpp-115 ( figure s1 , supporting information ) , then 02000 equiv of cpp-115 ( figure s2 , supporting information ) , followed by dialysis . aliquots removed during dialysis exhibited time - dependent reactivation of gaba - at over an extended period of time ; with increasing equiv of inactivator , less activity returned with maximal return by 24 h ( maximal return of activity occurred sooner with less inactivator ) . it required about 2000 equiv of cpp-115 before the enzyme reactivation was diminished to an insignificant level , supporting a reversible and irreversible component to the inactivation . during inactivation of gaba - at by 2 mm cpp-115 , it was found that 66 3 equiv of fluoride ions were released over a 26 h time period ; however , enzyme activity was lost within minutes ( figure s3 , supporting information ) . this result further supports the notion of two different mechanisms being involved , one reversible and one irreversible , but it does not resolve the question of how many fluoride ions are released per molecule . a single - turnover experiment was carried out to determine the number of fluoride ions per molecule released with each turnover . this experiment is possible because gaba - at converts substrate to product with concomitant conversion of its plp to pmp . the reaction cycle can not restart until -ketoglutarate binds and converts the pmp back to plp with concomitant formation of l - glutamic acid . by leaving out the -ketoglutarate , 4.3 0.5 equiv of fluoride ions was detected ( figure s4 , supporting information ) ; because gaba - at is a homodimer , two molecules of cpp-115 can be turned over per enzyme dimer . therefore , 4 equiv of fluoride ion indicates that both fluoride ions in cpp-115 are lost per enzyme turnover , and mechanisms 1 , 3 , and 4a , which release no fluoride ions during inactivation , and mechanism 4b , which releases one fluoride ion , can be excluded ( table 2 ) . the cofactor structure after inactivation by cpp-115 was determined using gaba - at containing [ h]plp ( figure s5 , supporting information ) . with the aid of two control experiments ( figure s6 , supporting information ) , it was found that all of the cofactor was converted to pmp after inactivation ( figure s7 , supporting information ) . therefore , mechanism 4a , which does not release any cofactor and mechanisms 4b , 4c , 5a , and 5b , which require the release of plp as a cofactor , can be excluded ( table 2 ) . of the mechanisms considered , high - resolution lc / ms identified parent ions with m / z 401.0745 , 171.0291 , and 127.0389 ( figure s8 , supporting information ) , which corresponds to 20 , 21 , and 22 , respectively . ms / ms fragmentation produced daughter ions ( figures s9s11 , supporting information ) consistent with these three products and with mechanism 2b . this mechanism appears to be responsible for the reversible component of the overall inactivation mechanism ; mechanism 2a would be expected to account for the irreversible component , so it was imperative to demonstrate covalent attachment to the enzyme ( 19 , scheme 5 ) for substantiation . mass spectrometry of the intact mass of cpp-115-inactivated gaba - at was carried out using lc / ms / ms on native enzyme as the negative control and vigabatrin - inactivated gaba - at as the positive control ( figure s15 , supporting information ) . compared with native enzyme , vigabatrin - inactivated gaba - at had an added m / z peak of 122 da , which corresponds to the expected added mass of the covalent adduct previously proposed . cpp-115-inactivated gaba - at showed no significant peaks corresponding to added mass . in case the potential covalent adduct with cpp-115 was an imine , the inactivated enzyme was reduced with sodium borohydride prior to lc / ms / ms ( figure s16 , supporting information ) . again , no added mass was detected with the cpp-115 inactivated enzyme , but the vigabatrin - inactivated enzyme had the expected added mass . these results were corroborated by results from peptide proteomics ( supporting information , figures s13 and s14 ) . the solution to this dilemma came from the x - ray crystal structure of cpp-115-inactivated pig brain gaba - at , which revealed that the inactivator was tightly bound to the protein noncovalently as 20 ( figures 3 and 4 ) . the inactivated species binds tightly ( stable to dialysis ) because of its covalent attachment to the cofactor and by two strong electrostatic interactions between the guanidinium groups of arg192 and arg445 and each of the two carboxylate groups of 20 . this unexpected phenomenon is the first time that arg445 has been observed interacting directly with a ligand and to be involved in the inactivation of gaba - at . on the basis of crystallography of gaba analogues , it is believed that gaba binds in the active site by schiff base formation with plp and an electrostatic interaction between the carboxylate of gaba and the guanidinium group of arg192 . arg445 is sequestered from the active site by an electrostatic interaction with glu270 . it has been proposed that this second salt bridge only disassociates during the second half of catalysis , the regeneration of plp from pmp , to aid in the binding of the second carboxylate of -ketoglutarate . a network of hydrogen bonds in the active site of gaba - at connects glu270 and arg445 to o3 of the cofactor through the side chain amide of gln301 . proposed that this network may be affected when the cofactor is in its pmp form , which weakens the interaction between glu270 and arg445 . to verify these hypotheses , many research groups have attempted to cocrystallize gaba - at with ligands such as -kg or glutamate that may directly show the involvement of this second arginine , but they have not yet been successful . our crystal structure of gaba - at inactivated by cpp-115 ( figure 3 ) clearly shows for the first time that the salt bridge between arg445 and glu270 has been broken , and glu270 is rotated away from its original position to accommodate a second guanidinium carboxylate electrostatic interaction between arg445 and the newly formed carboxylate group on 20 from enzyme - catalyzed hydrolysis of the difluoromethenyl group . the two guanidinium carboxylate interactions , the hydrophobic interactions of the pyridine ring , and the interactions of the phosphate group contribute to the stabilization of the metabolite for tight binding . therefore , cpp-115 appears to be , to the best of our knowledge , the first example of a mechanism - based inactivator that leads to a conformational change , forming a tightly bound inhibitor apparently , this conformational change occurs only about once with 2000 molecules of inactivator ; the remaining molecules of 2022 are slowly released as products . the ,-unsaturated intermediate ( 14 , scheme 4 ) generated during turnover is highly electrophilic , which should have been attacked by the nucleophilic lys329 residue and form a covalent adduct , as it does with vigabatrin . molecular modeling studies of the cpp-115plp aldimine were performed to analyze the effect of tautomerization during the inactivation of gaba - at by cpp-115 ( figure 5 ) . this model demonstrates that the pmp - imine form ( orange ) of the aldimine generated during enzyme - catalyzed tautomerization shifts the difluoromethenyl group ( fluorines are in green ) away from lys329 , thereby moving it too far for michael addition by this lysine residue . because of the lack of 1,5 steric interactions ( both carbons opposite the carboxylate group are sp - hybridized and thus planar ) , the difluoromethenyl group is able to release strain by moving closer to the carboxylate . as gaba - at normally catalyzes hydrolysis of the imine formed by tautomerization of gaba , the catalytic machinery is in place for water attack of the michael acceptor instead . this is consistent with the obtained crystal structure and explains why the inactivation mechanism did not follow that of vigabatrin and form a covalent adduct . molecular docking studies of cpp-115-plp aldimine in plp - form ( cyan ) vs cpp-115-pmp - form ( orange ) in the active site of gaba - at . on the basis of the evidence from fluoride release , cofactor release , metabolites formed , proteomics , and x - ray crystallography , the most consistent mechanism is shown in scheme 9 . although cpp-115 was rationally designed to inactivate gaba - at via a covalent michael addition mechanism , the results described here indicate that it inactivates gaba - at by mechanism - based formation of a metabolite that induces a conformational change and forms a tightly bound complex with the enzyme ( scheme 9 ) . the requirement of about 2000 equiv of cpp-115 for complete inactivation of gaba - at reflects the number of molecules of product that are released prior to interception by the enzyme in the correct state and conformation that lead to inactivation . the crystal structure of cpp-115-inactivated gaba - at revealed , for the first time , that the glu270-arg445 salt bridge in the active site can be disrupted , leading to the formation of a new binding pocket for the inactivator . our molecular modeling studies indicate a movement of the difluoromethenyl group upon tautomerization , which shifts that group too far from lys329 for nucleophilic attack . this leads to enzyme - catalyzed hydrolysis of the pmp - aldimine of cpp-115 and either release of products ( 2000 times ) or inducement of a conformational change to give a tightly bound pmp complex stabilized by the exposure of arg445 and formation of an electrostatic interaction with the newly formed carboxylate of the inactivator . once metabolite 20 is tightly bound to the enzyme , it is released from the active site very slowly , which accounts for the extended period of time that fluoride ions are released relative to the rate of inhibition of the enzyme . with our results revealing the possibility of additional binding with arg445 , future inactivators may be designed so that the newly formed carboxylate group more closely mimics the second carboxylate group of -ketoglutarate to encourage less turnover prior to conformational change - induced exposure of arg445 . gaba - at assays were recorded on a synergy h1 hybrid multimode microplate reader ( biotek , usa ) with transparent 96-well plates ( greiner bio - one , monroe , nc ) . measurements of ph were performed on a fisher scientific ap71 ph / mv/c meter with a ph / atc electrode . determinations of fluoride ion concentration were performed on the same meter with a thermo scientific 9609bn combination fluoride electrode . large - scale dialyses were performed with thermo scientific slide - a - lyzer dialysis cassettes ( molecular weight cutoff of 10 kda ) unless otherwise specified . small - scale dialyses were performed with emd chemicals d - tube mini dialyzer ( molecular weight cutoff of 1214 kda ) . radioactivity was determined with a packard tri - carb 2100tr liquid scintillation analyzer using perkinelmer ultima gold scintillation fluid . for centrifugations , a dupont sorvall rc 5b plus centrifuge was used with either an sla-3000 or an sa600 rotor . enzyme purification was carried out on a ge healthcare life sciences kta fplc system . the column media used were cm sepharose fast flow for weak cationic exchange mode , deae sepharose fast flow for weak anionic exchange mode , and hydroxylapatite ( ha ) for mixed mode . a prepacked hiprep sephacryl s-200 high - resolution column was used for size - exclusion mode . electrophoresis was carried out on a bio - rad mini - protean tetra cell using a vwr accupower model 300 power supply . lc / ms / ms was performed on thermo fisher q exactive mass spectrometer . mass spectrometry of intact mass of gaba - at was performed on a velos orbitrap elite ( thermofisher ) . crystallographic data were collected on beamline 23 id - b and 23 id - d of gm / ca of the advanced photon source ( aps ) using x - rays of 0.99 wavelength and a rayonix ( formerly mar - usa ) 4 4 tiled ccd detector . all reagents and materials were purchased from sigma - aldrich co. except the following : centrifugal filters ( molecular weight cutoff value of 10 kda and 30 kda ) were purchased form emd millipore ; dowex 50 and sodium dodecyl sulfate were purchased from bio - rad ; [ h]sodium borohydride was purchased from american radiolabeled chemicals , inc . ; pig brains were a generous gift from the park packing co. ( chicago , il ) ; all of the buffers and solvents used for fplc analyses were filtered through ge healthcare 0.45 m nylon membranes . gabase ( pseudomonas fluorescens ) and succinic semialdehyde were purchased from sigma - aldrich . gaba - at ( 2.65 mg / ml , specific activity 2.1 unit / mg ) was purified from pig brain by the procedure described previously . succinic semialdehyde dehydrogenase ( ssdh ) was obtained from gabase , a commercially available mixture of ssdh and gaba - at , using a known procedure . the final assay solution consisted of 11 mm gaba , 1.1 mm nadp , 5.3 mm -kg , 2 mm -mercaptoethanol , and excess ssdh in 50 mm potassium pyrophosphate buffer , ph 8.5 . the change in uv absorbance at a wavelength of 340 nm at 25 c caused by the conversion of nadp to nadph is proportional to the gaba - at activity . all enzyme assays were recorded with a perkinelmer lambda 10 uv / vis spectrophotometer . gaba - at ( 2 g ) was incubated with cpp-115 ( 0 , 0.2 , 0.4 , 0.6 , 0.8 , 1.0 , 1.2 , 1.4 , 1.6 , 1.8 , and 4 equiv ) at 25 c in 50 mm potassium pyrophosphate buffer solution , containing 2 mm -ketoglutarate and 1 mm -mercaptoethanol . aliquots ( 2 l ) were withdrawn at timed intervals and were added immediately to the assay solution ( 96 l ) containing excess ssdh ( 2 l ) . gaba - at ( 60 l ; final concentration 1 m ) was preincubated for 3 h with various amounts of cpp-115 ( 0 , 1 , 1.8 , 4 , 40 , and 80 equiv ) in 50 mm pyrophosphate buffer ( ph 8.5 ) containing 0.625 mm -ketoglutarate , in a total volume of 160 l at 25 c . in another experiment , it was preincubated for 24 h with various amounts of cpp-115 ( 0 , 1 , 2 , 4 , 40 , 80 , 100 , 1000 , and 2000 equiv ) . after preincubation , the enzyme solutions were transferred to d - tube mini dialyzers and exhaustively dialyzed against buffer ( 50 mm pyrophosphate buffer containing 0.1 mm -ketoglutarate and 0.1 mm plp , ph 8.5 ) at 4 c . the enzyme activity remaining in each of the solutions was assayed per time interval . gaba - at ( 150 l ) was incubated in 100 mm potassium pyrophosphate buffer at ph 8.5 , containing 2 mm cpp-115 and 2.5 mm -ketoglutarate in a total volume of 510 l . the incubation mixture was protected from light and was carried out at room temperature . the experiment was repeated with 2.5 mm plp supplemented in the incubation mixture . to construct a standard curve , the relative potential ( mv ) of sodium fluoride solutions in the range of 1 10 to 5 10 m in total ionic strength adjustment buffer ( tisab ; made from 5.8 g of sodium chloride and 5.7 ml of acetic acid in 100 ml of water , adjusted to ph 5.2 ) was measured . the measured potential was plotted against the concentration of sodium fluoride . at different incubation time ( 1 , 2 , 5 , 7 , 17 , 24 , 27 h ) , each aliquot was removed from the incubation samples and mixed with tisab to measure their relative potentials . using the formula obtained from the standard curve , the mv readings were converted to concentration of fluoride ions in the samples . the readings from the control sample were subtracted from the inactivated sample , and the concentration was divided by the concentration of gaba - at to obtain the equivalents of fluoride ion released per inactivation event . the similar experiment was run as above but without -ketoglutarate to test the amount of fluoride released during a one - turnover event . gaba - at ( 200 l ) was incubated in 100 mm potassium pyrophosphate buffer at ph 8.5 , containing 2 mm cpp-115 in a total volume of 510 l . when there is no -ketoglutarate in the mixture to regenerate plp , the reaction stops at one turnover per active site thirty drops of 1 m naoh were added to 1.8 ml of 0.5 m plp and cooled to 0 c in an ice bath . the mixture of sodium borohydride ( 5.8 mg , 0.15 mmol ) and [ h]-sodium borohydride ( 25 mci ) in 450 l of 0.1 m naoh was added to the plp solution and stirred for 1 h at 0 c . after being stirred , 120 l of concd hcl was added to the solution slowly ( became ph 4 ) . to the solution at ph 4 , 20 equiv of ground mno2 was added , and the mixture was stirred at room temperature for a total of 2 h. the mixture was brought to ph 8 with 1 m naoh , and the solution was centrifuged . the supernatants were collected and were loaded onto a gel filtration column packed with bio - rad ag1-x8 resin ( hydroxide form ) . the mobile phase used was water and 5 m acetic acid ( gradient of 90% water to 0% water ) , flowing at 1.5 ml / min for 300 min . fractions were collected every 10 ml and tested for its uv absorption and radioactivity . the lyophilized product was redissolved in 100 l of 1 mm plp and loaded onto an hplc with an econosil c18 column ( 10 mm 250 mm , 10 m ) . the mobile phase used was 0.1% aqueous tfa with 5% acetonitrile flowing at 0.5 ml / min for 40 min . under these conditions , fractions running with the plp peak were collected and counted for radioactivity using liquid scintillation counting . the product was collected and lyophilized . gaba - at that had been reconstituted with [ h]plp was incubated at room temperature and protected from light in 100 mm potassium phosphate buffer containing cpp-115 ( 2 mm ) , -ketoglutarate ( 3 mm ) , and -mercaptoethanol ( 3 mm ) , in a total volume of 100 l at ph 7.4 . a negative control was run under identical conditions as above , excluding the inactivator . a positive second control was run with 3 mm gaba in the absence of inactivator and -ketoglutarate . the first control should release the cofactor as plp , and the second control should release it as pmp . after incubation for 18 h , the activity of gaba - at was less than 1% of control , and the solutions were adjusted to ph 11 with 1 m koh and incubated for 1 h. trifluoroacetic acid ( tfa ) was added to quench the base and make the solution 10% v / v tfa . the resulting denatured enzyme solution was microcentrifuged for 5 min at 10 000 rpm after standing at room temperature for 10 min . a small amount of white solids was seen at the bottom of the tube . the supernatants were collected individually . to wash the pellets , 50 l of 10% tfa was added to each tube , vortexed , and microcentrifuged for another 5 min . cofactor analysis was carried out by dissolving the solids obtained from lyophilization with 100 l of a solution containing 2.5 mm plp and 2.5 mm pmp as standards and then injecting the samples into the hplc with an econosil c18 column ( 4.6 mm 150 mm , 10 m ) . the mobile phase used was 0.1% aqueous tfa flowing at 0.5 ml / min for 25 min . then the flow rate was increased to 1 ml / min from 25 to 30 min , and then a solvent gradient into 95% acetonitrile was run over the next 30 min . under these conditions , fractions were collected every minute , and the radioactivity was measured by liquid scintillation counting . gaba - at ( 0.3 nmol ) was incubated in 50 mm ammonium bicarbonate buffer ( ph 7.4 ) containing 0.1 mm cpp-115 , 1 mm -ketoglutarate , and 0.1 mm plp in a total volume of 70 l at room temperature in the dark for 12 h. a control containing everything except cpp-115 was also incubated . after 12 h , gaba - at in the inactivated sample was less than 1% active vs control . formic acid ( 1 l ) was added to each reaction mixture , and both were centrifuged in a 0.5 ml 30 kda mwco centrifuge tube ( millipore ) at 14000 g for 4 min or until most of the solution had passed through . an additional 20 l of 50 mm ammonium bicarbonate was added above the filter and centrifuged for 3 min . the flow - through ( 20 ul ) was injected onto a luna c18(2 ) column ( 100 a , 2 150 mm , 5 m , phenomenex ) . a 60 min gradient ( agilent 1100 hplc , solvent a = 5% acetonitrile and 0.1% formic acid ; solvent b = 0.1% formic acid in acetonitrile ) was run from 280% b over 40 min . the top five most abundant ions in negative ion mode were selected for fragmentation using mass normalized collision energies . commercially available 3-oxo-1-cyclopentanecarboxylic acid ( sigma ) was injected as a standard for comparison . three reaction samples were prepared as described previously with only gaba - at , including vigabatrin , or cpp-115 . after incubation with or without inhibitors , half of each reaction mixture was reduced with 15 mm sodium cyanoborohydride for 1 h. both reduced and nonreduced samples were buffer exchanged into 50 mm ammonium acetate using 30 kda mwco filters ( millipore ) . nano - lc / ms / ms runs were done on a 75 m i d 10 cm kinetex c8 ( phenomenex ) column connected to an autosampler ( dionex ultimate 3000 rslcnano system ) and a velos elite orbitrap ( thermofisher ) mass spectrometer . , gaba - at and cpp-115-inactivated gaba - at were exchanged into a buffer that contained 40 mm sodium acetate ( ph 5.5 ) . after the initial crystallization screening and optimization , the proteins were crystallized via the hanging drop method . hanging drops were prepared by mixing 1 l of 12 mg / ml native gaba - at protein solution and 1 l of the reservoir solution , containing 0.1 m ammonium acetate , 0.1 m bis - tris ( ph 5.5 ) , and 17% w / v peg 10 000 . crystals appeared within 24 h at 20 c and grew for 56 days before harvesting . crystals with good morphology and large sizes were transferred to cryoprotecting conditions , which contain 20% glycerol in addition to the original composition of the reservoir solution , before being frozen in liquid nitrogen . the same method of crystallization was applied for cpp-115-inactivated gaba - at ; however , before freezing in liquid nitrogen , the inactivated crystals were transferred to a cryoprotecting solution that contained 20% glycerol and 2 mm cpp-115 , in addition to the compounds of the reservoir solution . crystallographic data were collected on beamlines 23id - b and 23id - d of gm / ca@aps of the advanced photon source ( aps ) using x - rays of 0.99 wavelength and rayonix ( formerly mar - usa ) 4 4 tiled ccd detector with a 300 mm sensitive area . data collection and processing statistics are given in table s1 ( supporting information ) . molecular replacement for the native gaba - at was carried out using the program phaser from ccp4 software suite , using the previously reported coordinates of gaba - at from pig liver ( pdb i d : iohv ) as the starting search model . the initial rfree and r factor of the correct solution were 0.1904 and 0.1934 , respectively . manual adjustment and modification of the structure based on electron density maps were performed using the program coot . the rfree and r factor values of the final model were 0.1513 and 0.1766 , respectively . the data sets from the inactivated gaba - at crystals were isomorphous to those of native gaba - at . therefore , rigid body refinement could be used directly , placing the previously refined native gaba - at model into the asymmetric unit of inactivated gaba - at crystals . model building and refinements of the inactivated structures were carried out following the same protocol as the native structure . there were no ligand coordinates included in the refinement until the refinement converged . without ligands built in , the fo fc map shows a well - defined electron density supporting the existence of the bound ligand . the structure of the inactivator was made in the program chemdraw generating a mol file as the output . this output ligand structure was then regularized and its chemical restraints were generated in the program jligand . using coot , the inactivator was manually fit to the residual electron density in the difference ( fo fc ) map . the rfree and r factor for the inactivated structure were 0.1575 and 0.1872 , respectively . in essence , the ligands ( including the cofactor ) were docked into the active site of the prepared protein using autodock 4.2 , with lys329 being flexible . the best docked structures were then refined by molecular mechanics , using gromacs 4.5 . the sequence utilized started with energy minimization , continued with molecular dynamics ( 4 ns ) , and had a final energy minimization step .

-aminobutyric acid aminotransferase ( gaba - at ) is a pyridoxal 5-phosphate ( plp)-dependent enzyme that degrades gaba , the principal inhibitory neurotransmitter in mammalian cells . when the concentration of gaba falls below a threshold level , convulsions can occur . inhibition of gaba - at raises gaba levels in the brain , which can terminate seizures as well as have potential therapeutic applications in treating other neurological disorders , including drug addiction . among the analogues that we previously developed , ( 1s,3s)-3-amino-4-difluoromethylene-1-cyclopentanoic acid ( cpp-115 ) showed 187 times greater potency than that of vigabatrin , a known inactivator of gaba - at and approved drug ( sabril ) for the treatment of infantile spasms and refractory adult epilepsy . recently , cpp-115 was shown to have no adverse effects in a phase i clinical trial . here we report a novel inactivation mechanism for cpp-115 , a mechanism - based inactivator that undergoes gaba - at - catalyzed hydrolysis of the difluoromethylene group to a carboxylic acid with concomitant loss of two fluoride ions and coenzyme conversion to pyridoxamine 5-phosphate ( pmp ) . the partition ratio for cpp-115 with gaba - at is about 2000 , releasing cyclopentanone-2,4-dicarboxylate ( 22 ) and two other precursors of this compound ( 20 and 21 ) . time - dependent inactivation occurs by a conformational change induced by the formation of the aldimine of 4-aminocyclopentane-1,3-dicarboxylic acid and pmp ( 20 ) , which disrupts an electrostatic interaction between glu270 and arg445 to form an electrostatic interaction between arg445 and the newly formed carboxylate produced by hydrolysis of the difluoromethylene group in cpp-115 , resulting in a noncovalent , tightly bound complex . this represents a novel mechanism for inactivation of gaba - at and a new approach for the design of mechanism - based inactivators in general .

Introduction Results Discussion Conclusions Experimental Procedures

PMC3512314

human serum albumin ( hsa ) is the most abundant plasma protein in the human body and plays an important role in drug transport and metabolism . generally regarded as a nonspecific transport protein , additionally , the enzymatic activity of hsa on different substrates and drugs has also been studied and documented . nevertheless , the structural mechanism of this activity is yet unknown . to assess the structural basis of binding mechanisms , we evaluated the interaction between hsa and acetylsalicylic acid ( asa , c9h8o4 ) by means of molecular dynamics simulations ( md ) . starting from an experimentally resolved structure of the complex , we extracted the ligand by means of the application of an external force , under near quasistatic conditions , thus evaluating the work involved in breaking the interactions present in the protein - ligand complex , and hence obtaining an upper boundary for the free energy of binding of the complex . we quantified the force used to remove the asa from its specific site of binding to hsa and calculated the mechanical nonequilibrium external work done during this process . the aim of the present study is to calculate an upper boundary for the gibbs free energy difference associated to that process , through the average work obtained using the finite sampling estimator from the jarzynski equality . to evaluate the effect of the solvent ( treated explicitly ) this magnitude represents the work done to move the aspirin in the same trajectory through the solvent in absence of the protein . its principal sense follows from the fact that experimental techniques to obtain the protein - ligand affinity ( e.g. , quenching fluorescence , microcalorimetry ) do not take into account the contribution of the solvent to this out of equilibrium process ( since they analyze equilibrium states ) . finally , we compared our computational results with the experimental data available for the affinity constant of the hsa - asa complex , obtained by quenching fluorescence methods , showing reasonable agreement . we carried out all molecular dynamics ( md ) simulations using the gromacs 4.0.1 package in which the equations of motion are solved using a leap - frog integration step . we used gromos96 ( 43a1 ) force field for the minimization process , as well as for all the md simulation steps , and kept all protein bond lengths constrained using the lincs algorithm . water molecules were constrained using the settle algorithm . for the calculation of long - range coulombic interactions , we applied the reaction field method , with a 1.4 nm cut - off radius . likewise , we calculated lennard - jones interactions within a cut - off radius of 1.4 nm . for all the simulation runs , we have used a xeon - based , dual - processor cluster , running under gnu / linux , and for all plots and graphics ms windows or gnu / linux , using the reference visual molecular dynamics package , swiss pdb viewer , or xgrace software [ 911 ] . as starting configuration we used the human serum albumin - acetylsalicylic acid complex ( pdb i d : 2i30-resolution 2.9 ) that contains five myristic acid molecules ( myr , c14h28o2 ) forming part of the albumin structure . since the original structure was incomplete , we reconstructed both the n - terminus and the c - terminus . thus , asp1 , ala2 , and leu585 had to be added ad hoc to the experimental structure using swiss pdb viewer , using standard angles for the reconstructed peptidic planes . moreover , the experimental structure corresponds to the complex in the post reaction state ; that is , the protein was acetylated in residue lys199 and the aspirin hydrolyzed . thus , we removed the acetyl group from the protein and reconstructed the modified aspirin . having focused on the interaction ( and dissociation ) between hsa and its most internal ( and more tightly attached ) aspirin ligand , such process will be expected to occur in absence of the less tightly bonded ( and external ) aspirin ligand . the starting system consisted of a truncated dodecahedral simulation box of dimension parameter d = 12.9702 nm and a total volume of 1540.65 nm , containing one hsa molecule , one asa molecule , five myristic acid molecules , and 47.803 water molecules . we constructed the topologies for aspirin and myristic acid and added them to gromos96 43a1 force field residue data base , using all the corresponding atom types , charges , bonds , angles , proper , and improper dihedral angles from that database . the values used for all atom charges in both aspirin and myristic acid topologies are summarized in tables 1a and 1b . a detailed schematic representation for both molecules is additionally provided as supplementary material ( see supplementary material available online at doi:10.1155/2012/642745 ) . as previously validated in recent literature , myristic acid was modeled in the unprotonated state and described using parameters derived from the lipid force field . the carboxylic acid group was based on the parameters of glutamic acid , which were available from the corresponding gromos96 43a1 force field . we generated the topology of the total system , with standard protonation states for all amino acids ( ph 7 ) . in every step the production system was weakly coupled to a thermal and hydrostatic bath , in order to work in the isothermal - isobaric ensemble at t = 300 k and p = 1 bar . we minimized the energy of the system using firstly the steepest descent method , converging to machine precision . secondly , we applied the conjugated gradient method , converging in less than 20 cycles . the equilibration of the complete system proved to be nontrivial , because we had to complete the original structure and chemically modify it , and we had to perform a nonstandard series of computational steps to release the exogenous tensions introduced due to the structural additions and modifications mentioned previously . namely , we performed an md run in vacuo ( in absence of solvent ) , using a 0.0001 fs time step and for a total time of 2 ps lightly coupled to a 100 k temperature bath , after energy minimization in order to let the incorporated residues explore more stable configurations . during this run , we applied position restrains on all of the backbone atoms ( except for those incorporated ad hoc ) . in the same conditions , we subsequently performed a 10 ps run , using a 0.5 fs time step . next and for another 10 ps ( using the same time step ) , we incorporated the lincs algorithm to add restrictions in bond lengths and angles for all molecules belonging to the complex . once the previous procedure was completed , we gradually increased the time step up to 2 fs , likewise increasing temperature up to the final value of 300 k. at this point , we solvated the system adding 47.803 spc / e explicit water molecules . in these conditions , we allowed the solvated system to relax ( keeping all solute heavy atoms restrained to their corresponding crystallographic positions ) during 200 ps , at 300 k and 1bar . at the same temperature and pressure , we subsequently decreased the position restraints force constants during a 200 ps md run . finally , we released all position restraints and allowed the system to relax freely for a total simulation time of 20 ns , at 300 k and 1bar , after which we considered the system to be in conformational equilibrium . the stability of myr molecules in their binding sites throughout the total 20 ns of the equilibration run indicates that the conformational equilibrium was correctly achieved . together with this , we observed a relative stability of the hsa backbone ( evaluated by the convergence of hsa alpha - carbon root mean square displacement ( rmsd ) , ( see supplementary material available online at doi:10.1155/2012/642745 ) ) . these facts are in notable agreement with the widely known role of fatty acids in the stabilization of a hsa structure . once the system was effectively equilibrated , we performed 30 md quasistatic runs , applying an external force on the center of mass of the aspirin , in order to extract this molecule from its binding site . we coupled all 30 production simulation runs to a weak temperature and pressure bath of 300 k and 1 bar , in order to work in the isothermal - isobaric ensemble . we randomly generated the initial velocities , using a unique seed for every production run . the total simulation time of every production run was 500 ps , with a time step of 2 fs . the constant of the harmonical potential applied at the center of mass of the aspirin was of k = 1 , 000 kj/(molnm ) , and the pulling rate ( i.e. , the velocity used to move the free end of the spring in the pulling direction ) was v = 0.01 nm / ps . the pulling direction was established as the line connecting the center of mass of the ligand and the center of the triangle determined by the centers of mass of the r groups of residues glu 292 , his 440 , and cys 448 ( i.e. , the binding pocket threshold ) . to calculate the work done during this process , we plotted each component of the force ( fx , fy , fz ) as a function of the respective position component of the center of mass of the acetylsalicylic acid molecule ( xasa , yasa , zasa ) . the process of removing a ligand from the site of binding to its receptor can be done by applying an external force to the ligand , hence performing a corresponding external mechanical work . if this work is done quasistatically , the value obtained for the applied external work is equal to the free energy variation associated to that process . when the work done is not quasistatic , the jarzynski equality [ 19 , 20 ] gives a relationship between the variation in free energy values that describes the state changes in the system under study , and the correctly averaged ensemble of the measurements of the external work done to change that state in a finite time ( out of equilibrium ) . in the case of a system in contact to an external thermal bath ( in the canonical ensemble ) , the correct description of the state changes of the system is through helmholtz free energy , whereas for a system in the isothermal - isobaric ensemble like the one we are studying , the suitable descriptor of the equilibrium states is gibbs free energy . if the actual distribution is approximated by performing a finite sample of work performed through an applied external force , a superior value to the free energy variation can be obtained from the average work w of the finite sampling , taken as usual : ( 1)wa1nsi=1nswi , where ns is the number of measurements and wi the external work done in the ith process . we can think of wi as a random sampling from the distribution (w ) that satisfies the so - called jarzynski equality at any time ts : ( 2)ew / kbtw(w , ts)ew / kbtdw = eg / kbt , where t is the temperature , kb the boltzmann constant , ts is the time spent to do the described process , and g is the gibbs free energy variation . in this context , the expectation value of w , w , will give an upper boundary to the gibbs free energy variation : ( 3)wa=ww(w , ts)dwg . the jarzynski equality suggests that w is not the correct quantity to estimate g . a better upper boundary to this magnitude is given by the so - called finite sampling estimator of the average from the jarzynski equality : ( 4)wxkbtln[1nsi=1nsewi / kbt ] . for ns = 1 , w and w will be equal and the expectation value for both will be w , and for ns , w will tend to g , and w will tend to w. for intermediate values of ns , ( 5)gwxwa , is satisfied . the data obtained through nonequilibrium molecular dynamics pulling of a ligand may reasonably contain a nondesired contribution due to the work that is necessary to move a molecule through the solvent under such conditions , when compared to experimental data obtained in conditions of equilibrium . hence , we have undertaken the evaluation of the relevance of the so - called viscous work , in comparison with the total work . with this purpose , we prepared a system of the same shape and dimensions , consisting only of one acetylsalicylic acid molecule in the original position , solely surrounded by water molecules . firstly , we minimized the energy of this system as described in the previous section and , subsequently , performed short 200 ps stabilization . once temperature and pressure reached equilibrium values of 300 k and 1 bar , we coupled the system to a weak thermostatic bath and fixed the box dimensions . after stabilization , we proceeded as with the system in presence of hsa , performing 30 md runs with the same general setup . the data presented in figure 2 corresponds to the mean value of the magnitude of the force applied on the center of mass of the acetylsalicylic acid molecule , as a function of the magnitude of the displacement of the free end of the ideal spring , both for the system in presence ( black lines ) and absence ( red lines ) of the hsa complex . error bars show the standard deviation of those values . in order to obtain greater graphical clearness , only 80 of these bars are shown . in this figure , a marked difference between curves may be observed up to 2 nm . the duplication of this distance ( the point of error bar crossing , that is , 4 nm ) was considered as a reasonable criterion for convergence and was thus chosen as the limit value for production data . after this point , the magnitude of the applied force becomes comparable for both systems , which allows us to consider the acetylsalicylic acid molecule to be out of the binding site . in figure 3 , we present a histogram representation of the distribution of the performed mechanical work , both for the system in presence ( red bars ) and absence ( black bars ) of the hsa complex . from this study , we may interestingly observe that the studied samples of both systems are large enough to clearly distinguish between populations . the obtained values for w and w , using ( 1 ) and ( 4 ) , respectively , are ( 6)wa=(5.00.6)1019 j ( usual average),wx=(4.00.6)1019 j ( jarzynski 's average ) . from these results we can observe that w establishes a better highest value for g than w , in agreement with ( 5 ) . likewise , the average value of the viscous work obtained using the usual average is ( 7)waviscous=821020 j. and for the finite sampling estimator of the average value from jarzynski equality , ( 8)wxviscous=421020 j. for the present case , the obtained value of the viscous work falls within the order of magnitude of the standard deviation of the calculated binding work . viscous work may reasonably become a more important source of discrepancy with experimental observation , therefore rendering its evaluation mandatory . taking these into account , the obtained value for w would be ( 9)wxcorrected=(3.60.6)1019 j. regarding the ligand trajectory in this process , we can distinguish two regions : an interior region that corresponds to the movement within the binding pocket , where the pulling direction is univocally determined by the shortest path between the original position of the ligand and the binding pocket threshold and a second exterior one . in the first region , where the unbinding path is conformationally determined , we can assume that the contribution to the total average work is close to the optimal one , whereas in the exterior region it is expected that better path optimization may be achieved . the separation between both regions was established as the point in which the center of mass of the ligand crosses the plane formed by residues glu 292 , his 440 , and cys 448 ( i.e. , the binding pocket threshold ) . the first region represents 29.4% of the total trajectory , with a corresponding pulling coordinate of 1.47 nm , and its contribution to the corrected work is of ( 1.9 0.6 ) 10 j. the experimental affinity constant of the complex hsa - aspirin , obtained by bojko and coworkers from quenching fluorescence measurements , is ka = 18.79 10 m. from this value , we obtain the experimental g as ( 10)g = kbtlnka=4.071020 j. the upper boundary obtained using the finite sampling estimator of the average work gives a better value than that obtained from the usual average work . albeit more recent comments on jarzynski 's work , his response is clear and categorical , and this result comes in line with what he classically proposes . in the present study , we have succeeded in reconstructing , stabilizing , and equilibrating a protein ligand complex which proceeded from incomplete data provided from experiment . this achieved equilibrium state enabled us to study the hsa - asa - myr complex through nonequilibrium molecular dynamics , in near quasistatic conditions . the fact that complex biological structures , that are not totally resolved through experiment , may be eligible targets for molecular dynamic studies reinforces the importance and potentiality of this technique . we have likewise obtained an average upper boundary for the gibbs free energy of binding of the hsa - asa complex that is less than one order of magnitude above the experimental value . as regards the assessment of the relevance of the viscous work , the value obtained is low compared to the upper boundary for the gibbs free energy difference of the hsa - asa complex . nevertheless , its difference with the experimental free energy of binding is not negligible and should therefore be generally taken into account in such studies . moreover , the relevance of this effect , and the need for its correct evaluation may reasonably increase as computational methods are refined and sampling capacities increased . finally , the promising level of agreement of the data presented in this study with that available experimentally calls for further development of such studies as well as encourages the analysis of other similar systems .

in this work , we present a study of the interaction between human serum albumin ( hsa ) and acetylsalicylic acid ( asa , c9h8o4 ) by molecular dynamics simulations ( md ) . starting from an experimentally resolved structure of the complex , we performed the extraction of the ligand by means of the application of an external force . after stabilization of the system , we quantified the force used to remove the asa from its specific site of binding to hsa and calculated the mechanical nonequilibrium external work done during this process . we obtain a reasonable value for the upper boundary of the gibbs free energy difference ( an equilibrium thermodynamic potential ) between the complexed and noncomplexed states . to achieve this goal , we used the finite sampling estimator of the average work , calculated from the jarzynski equality . to evaluate the effect of the solvent , we calculated the so - called viscous work , that is , the work done to move the aspirin in the same trajectory through the solvent in absence of the protein , so as to assess the relevance of its contribution to the total work . the results are in good agreement with the available experimental data for the albumin affinity constant for aspirin , obtained through quenching fluorescence methods .

1. Introduction 2. General Procedure 3. Equilibration 4. AFM Pulling Simulation 5. The Jarzynski Relationship 6. The Viscous Work 7. Results and Discussion 8. Conclusion

PMC3389696

preterm neonate the classic high - risk neonate is one born before completion of week 37 of gestation [ 13 ] . medical and technological developments over the last few decades have increased the survival rates of infants born as preterm . yet many of the procedures that are a necessary part of their postnatal care can be , by nature , painful and stressful [ 4 , 5 ] . since preterm infants have incomplete physical development , their admission in neonatal intensive care units ( nicus ) provides them numerous stressors including painful stimuli , disruption of sleep , and excessive noise and light . sleeping and waking behaviors affect the development of preterm infants in various ways . according to also sleep and wakefulness may have direct effects on brain development and learning that continue after the infant has been discharged home . an important role of the neonatal nurse while providing care is to promote the preterm infant 's growth and development through the control of the nicu environment . since preterm infants experience numerous stressors in the nicu environment , identifying interventions that have immediate soothing effect is essential . touch is crucial for optimal growth and development of preterm infants and one of the most developed senses provided by health care staff to relax preterm infants . complementary therapies are attractive for nurses because they involve the whole human existence , and nurses are allowed to use them in their daily practice with no physician 's order . touch as a complementary therapy provides nurses an opportunity to get close to the patients specifically when they give care to the neonates . moreover therapeutic touch is a noninvasive treatment technique that requires no special equipment and technology . it can be easily combined with traditional medicine and thus decrease the cost of treatment , length of illness , and complications . thus , it is important for nurses to know the effects of touch on preterm infants in order to determine routine care and policies and to evaluate the potential of tactile stimulation as an effective intervention to promote overall growth and development . one type of tactile stimulation which has a relaxing effect on preterm infants is gentle touch [ 8 , 13 ] . in ght , one hand is placed on the infant 's head and the other hand is placed on the infant 's abdomen to provide a relaxing effect to the preterm infant . similar to ght , koreans have traditionally believed that they could relieve their sick children 's pain or discomfort by gently caressing their children on the aching body part . this caressing act is called yakson , where yak means medicine and son refers to hand . the yakson protocol for preterm infants includes the key aspects of traditional yakson which are appropriate for preterm infants , such as warm hands , gentle touching without pressure , and slow hand movement . in yakson , one hand is placed underneath the infant 's back and the other hand is placed on the infant 's abdomen and the abdomen is caressed . the work of showed that 40 preterm infants receiving yakson and ght twice a day for 15 days had significantly greater reduction in 24-hour urinary cortisol and norepinephrine on day 16 , compared to 20 other preterm infants receiving no touch therapy , and also after yakson or ght , the infants displayed an increased percentage of sleep states and a decreased percentage of awake and fussy states . in the other study , compared the effect of yakson and ght on behavioral patterns . in both methods , the infants exhibited an increased percentage of sleep states and a decreased percentage of awake and fussy states . literature review showed that ght and yakson have benefits for preterm infant . in the iranian context , no study was found to examine the effect of ght / or yackson on behavioral patterns . this study was conducted to compare the effect of these two methods on preterms ' behavioral patterns in nicu in south - east of iran . the sample of neonates in this study was selected from one hospital under the supervision of kerman university of medical science . ninety neonates ( yakson group , n = 30 ; ght group , n = 30 ; control group , n = 30 ) who were admitted to neonatal intensive care unit participated in this study in afzali pour hospital . the infants were randomly divided into 3 groups ( control , yakson , and ght ) following minimization approach . this allocation technique has been implemented considering baby 's weight at birth and gestational age . inclusion criteria were ( 1 ) a gestational age of 2634 weeks at birth , ( 2 ) no congenital anomalies , ( 3 ) not having undergone surgery , ( 4 ) apgar score more than 6 at 5 min , and ( 5 ) lack of medical condition contraindicating the running of yakson or ght such as symptomatic sepsis . the ght and yakson interventions were provided for each infant twice a day for 5 days . they stated that in previous studies , the time allocated to touch for preterm infants is between 15 and 30 minutes . it seems that infants become more familiar with stimuli and can better remember these stimuli if they exposed to touch intervention twice a day . the yakson protocol for preterm infants includes some of the key aspects of the traditional yakson ( warm hands , touching without pressure , and slow hand movement ) that are appropriate for preterm infants . before the initiation of study one of the researchers ( f. eshghi ) was trained for yakson protocol and at the same time one of the nicu nurses was trained for ght protocol . the protocol of yakson used in this study consisted of the following procedures . after wearing a clean inner gown , the researcher washed her hands and arms thoroughly with antimicrobial agents for 3 min . the researcher warmed both of her hands using a radiant warmer until the temperature of the palms reached 93.2 8f ( 34.0 8c ) . the researcher relaxed both arms and the muscles of both shoulders for 1 min and breathed deeply to concentrate ki energy on their palms . yakson lasted 15 min : hand resting ( 5 min ) , gentle caressing ( 5 min ) , and hand resting again ( 5 min ) . during the administration of yakson , the palms and all fingers of the researcher constantly maintained close contact with the infants up to the limit that preterm infants did not feel pressure . hand resting ( 5 min ) . while resting one hand on the chest and abdomen of a preterm infant while supporting the back of the preterm infant with the other hand , the researcher concentrated on her resting hands and envisioned that a healthy ki was passing to the preterm infant . at that time , the researcher breathed slowly to maintain a relaxed state.gentle caressing ( 5 min ) . in the same hand position , the researcher repeated caressing and resting for 5 min : caressing ( 1 min ) , resting ( 30 s ) , caressing ( 1 min ) , resting ( 30 s ) and caressing ( 2 min ) . the researcher caressed the infant 's chest and abdomen clockwise in a 1 cm diameter circular motion every 10 s.hand resting ( 5 min ) . hand resting ( 5 min ) . while resting one hand on the chest and abdomen of a preterm infant while supporting the back of the preterm infant with the other hand , the researcher concentrated on her resting hands and envisioned that a healthy ki was passing to the preterm infant . , the researcher repeated caressing and resting for 5 min : caressing ( 1 min ) , resting ( 30 s ) , caressing ( 1 min ) , resting ( 30 s ) and caressing ( 2 min ) . the researcher caressed the infant 's chest and abdomen clockwise in a 1 cm diameter circular motion every 10 s. hand resting ( 5 min ) . . the gentle human touch ( ght ) protocol was as follows : [ 8 , 16 ] . after wearing a clean inner gown , the nicu nurses trained washed both hands and arms with antimicrobial agents for 3 min . the nicu nurses trained warmed both of her hands using a radiant warmer until the temperature of the palms reached 93.2 8f . for 15 min , the nicu nurses trained placed the fingertips of one hand above the eyebrow line with the palm touching the preterm infant 's crown while the other hand was rested on the lower abdomen of the infant encompassing the waist and the hip . the person who was responsible for one intervention was not allowed to administer the other intervention . the interventions were provided for 5 consecutive days : 15 minutes in the morning and in the afternoon ( 911 a.m. and 35 p.m. ) . the other person who was nicu nurse was responsible for abss assessment . this person observed and assessed the infants ' behavioral states 2 minute before initiation of interventions and 2 minute after that . the behavioral state of preterm infants was evaluated by the anderson behavioral state scale ( abss ) . this scale determines the behavioral state of an infant based on observations of respiratory regularity , opening or closing of the eyes , limb and trunk activity , and the intensity of crying . this scale measures 12 infant 's behavioral states including 1 ; regular quiet sleep , 2 ; irregular quiet sleep , 3 ; active sleep , 4 ; very active sleep , 5 ; drowsy , 6 ; alert inactivity , 7 ; quiet awake , 8 ; active awake , 9 ; very active awake , 10 ; fussing , 11 ; crying and 12 ; hard crying . scores 68 indicate that the infant is awake and calm and in the most suitable state for nursing activity . scores from 9 to 12 indicate that the infant is in a state of restless activity or fussiness , which take substantial energy . sequelae for infants lasts 7 days , yakson and ght interventions began 7 days after birth . additionally , whenever one of the preterm infants was touched as part of any nursing care ( feeding or diaper changing ) , the ght or yakson treatments were postponed by 1 h. according to in case that infant indicated any sign of distress ( decreased heart rate or oxygen saturation levels ) during a ght or yakson intervention , the intervention was ended and nursing care was provided . for translation of abss scale from english into farsi , the standard forward - backward procedure was applied . translation of the items was independently performed by two professional translators and then temporary versions were provided . afterward they were back translated into english , and , after a careful cultural adaptation , the final versions were provided . there was an approval from the heads of nicu prior to the collection of data . the study proposal also was reviewed and approved by center 's office of research ethics in kerman medical university ( ethic code : k/90/342 ) . the consent form explained that participation was completely voluntary , and they can withdraw from the study at any time . they were informed about the purpose of study and procedure , both verbally and with written information . to secure confidentiality descriptive statistics was used to determine mean , sd , frequency , and percent of categorical variables . chi - square and anova tests were applied to compare distribution of demographic characteristics across two intervention and control groups . there were multiple measurements per subject ( 12 measures per subject ) ; therefore there is a degree of similarity or correlation between measurements of the same subject . this technique takes into account dependency of data and assesses trend in abss score over time and influence of independent variables on it . a p value less than 0.05 was considered as statistical significance level . a descriptive analysis of the background information ( table 1 ) indicated that the premature infants belonged to the gestational age 2634 weeks with a mean age of 31 weeks . about 67% of participants were born with c / s ( cesarean section ) method . three groups ' mean of birth weight was almost 1650 g. the mean score of apgar for three groups was 8 . analyses and anova showed that there were no significant differences among 3 groups in demographic variables ( table 1 ) . the results indicated no significant correlation between type of delivery / sex and abss scores . the mean ( se and p value ) of abss score in the infant with s / c type of delivery was 6.74 ( 0.11 , 0.7 ) . for infants with nvd type of delivery the mean ( se and p value ) of abss score for male infant was 6.87 ( 0.13 , 0.15 ) , and for female infant it was 6.67 ( 0.11 , 0.15 ) . the results of the study also demonstrated that birth weight ( p value = 0.35 ) and apgar score ( p value = 0.15 ) of infants have no significant effect on their abss scores . in the study , the infants ' behavioral reactions mixed model statistical test showed that infants ' abss scores are different between three groups . there was a correlation between the groups of infants and the change of abss scores . the mean ( se ) of abss score for yakson group , ght group , and control group was 4.83 ( 0.14 ) , 4.85 ( 0.14 ) , and 10.63 ( 0.14 ) . as indicated in table 2 , a significant difference was found between the effect of interventional groups and control group on infants ' sleep states ( p value < 0.001 ) . it means that infants ' sleep state increased after the interventions ( table 3 ) . no significant difference was found between yakson and ght groups in behavioral states ' scores ( p value = 1.00 ) ( table 2 ) . an increase was found in the infants ' sleep state after yakson and ght interventions ( figure 1 ) . a decrease was also found in the infants ' awake and fussy states after yakson and ght interventions ( figure 1 ) . as it is shown in figure 1 and table 3 , infants in control group experienced more fussy states compared to both interventional groups . in the analysis , the mean ( se ) of abss score in the first and second days of intervention was 8.97 ( 0.36 ) and 9.05 ( 0.33 ) . in the third day of intervention mean ( se ) of abss had a significant reduction 5.58 ( 0.18 ) . in the forth and fifth days of intervention the result of analytical analysis indicated no significant different in infants ' behavioral states between first and second days of intervention ( p value = 1.00 ) . a significant difference was found between behavioral states ' scores of first and second days with third , fourth , and fifth day of interventions ( p value < 0.001 ) . infants ' sleep states ' score increased in third , fourth , and fifth days of interventions . however no significant difference was found in infants ' behavioral states in days 3 , 4 , and 5 of interventions . the aim of this study was comparison of effect of yakson and ght on behavioral states among preterm infants . according to the results , both yakson and ght groups compared with control group indicated more sleep states after interventions compared to that before the intervention ( figure 1 ) . similarly , previous researchers [ 5 , 13 ] found that these interventions positively affect infant s ' sleep states . the work of also reported that these interventions decreased the secretion of stress hormones in preterm infants . in control group , the fussy state 's score of infants was more than that in both interventional groups . this result can be supported by early studies that showed infants in interventional group experienced a better behavioral state compared to those in control group [ 5 , 13 ] . it is well known that most of touch that preterm infants receive in the nicu is related with medical or nursing procedures . the infants in control group received no intervention but they routinely received procedural touch as a nursing care . so this type of touch as a routine manipulation could negatively affect on infants ' behavioral state . the findings also indicated no difference between effect of yakson and ght on infants ' sleep states after administration of these interventions ( table 2 and figure 1 ) . they found that calming effect of yakson intervention on infants is significantly greater than that of ght intervention . this difference could be related to the different sampling or sample size they used in their study . according to the results , after ght intervention sleep states were increased . this finding is consistent with previous researches [ 5 , 8 , 12 , 13 , 1820 ] where they found more sleep states during and after ght compared to those before ght . this finding therefore supports previous studies that reported positive effect of ght on preterm infants ' sleep state . according to the results , after ght and yakson interventions infants ' awake and fussy states decreased ( table 3 ) . this finding echoes the results of earlier studies that showed positive effect of ght and yakson on awake states after interventions [ 5 , 8 , 13 ] and on awake as well as fussy states after interventions [ 5 , 13 ] . this finding indicated that ght and yakson interventions promote infants ' comfort , diminish their stress , and help them to be calm during their hospitalization . according to the results , a significant difference was found between infants ' behavioral states in days 1 and 2 and days 3 , 4 , and 5 of intervention . the infants ' sleep states increased in days 3 , 4 , and 5 of intervention compared to days 1 and 2 . according to the results , no significant difference was found between infants ' sleep state in days 3 , 4 , and 5 . in this for example , in study , the intervention continued for 15 days . in these studies , there is no report about the day of study , for example , its relation with the intervention or its effect on infants ' sleep state . therefore , the difference found in this study between days of intervention on infants ' behavioral state can not be supported by and does not support previous studies . this finding can be explained by the fact that in days 1 and 2 the infants are not well familiar with these interventions . in days 3 , 4 and 5 of interventions , the infants become familiar with the interventions and interventions sufficiently affects infants ' behavioral states . the results of this study showed that yakson and ght increase infants ' sleep states and can reduce stress and energy expenditure and consequently decrease o2 dependency during the early weeks in the preterm infants hospitalized in the nicu . of course , further research needs to be done in order to examine the effect of yakson and ght on later developmental outcomes in childhood period . since touch is one of the first strong positive senses in neonate , possibly yakson and ght enhance sensory maturation and thereby promote more optimal behavioral organization at the time of hospital discharge . the study suggests that the appropriate effects of yakson and ght should be assessed in the different situations , for example painful procedures such as venipuncture , intubation , and suctioning . according to the findings of this study , yakson and ght could be one of the safe interventions that nurses use for preterm infants in order to enhance their development . nurses working in nicu and parents need to be educated on how to provide yakson or ght for preterm infants . all information about the risks and benefits as well as the best interventional strategies before this intervention should be included in this educational program . further study suggests to examine nicu nurses ' views about two types of touch therapy ( ght and yakson ) , their benefits , and their disadvantages as well . it also suggests a study to compare the effect of these two types of touch on preterm infants when they were administered by nicu nurses and parents .

objective . touch is one of the first strong positive senses that develop in neonate . therapeutic touch could be considered as a complementary treatment in neonate intensive care units ( nicu ) . methods . this quasi - experimental study was conducted to compare the effect of yakson and ght on behavioral reaction of preterm infants hospitalized in nicu in south - east of iran . 90 preterm infants participated in this study . they are randomly divided into 3 groups : ( 1 ) yakson group , n = 30 , ( 2 ) ght group , n = 30 , ( 3 ) control group , n = 30 . each infant received the ght and yakson interventions twice a day for 5 days . each session lasted 15 minutes . the control group received routine nursing care . results . in interventional group , an increase was found in sleep state score after the yakson and ght intervention . their awake and fussy states ' scores decreased after both interventions . no significant difference was found between yakson and ght group in their behavioral state scores . conclusion . the findings suggest that yakson and ght had soothing and calming effect on preterm infants and could be beneficial in nursing interventions .

1. Introduction 2. Method 3. Results 4. Discussion 5. Conclusion

PMC3270399

exploring patterns of phenotypic variation during ontogeny and phylogeny is fundamental to gaining insights into the processes of evolutionary diversification , including the mechanisms of speciation . the connection between development , evolutionary history , ecology , and morphology has intrigued evolutionary biologists for over the 150 years since darwin first published his ideas about natural selection . this is largely due to the idea that phenotypic evolution might be explained by changing or truncating the ancestral ontogeny , for which the characteristics can be inferred through phylogenetic analyses [ 24 ] . within a monophyletic group of closely related species , it is expected that shared evolutionary history is reflected by phenotypic similarity , due to a shared developmental basis inherited from a common ancestor . adaptive radiation and morphological divergence are usually attributed to differential selection acting upon geographical populations . in other words , ecological opportunity could lead to adaptive radiation [ 5 , 6 ] . when phenotypic divergence is paralleled in multiple complex phenotypic traits with separate developmental pathways , this is indicative of adaptation to differential environmental selection pressures [ 69 ] . this line of reasoning is especially strong in situations where phenotypic variation correlates with different ecological demands . the crested newts ( triturus cristatus superspecies ) are an example of an adaptive radiation . crested newts have been used as model organisms in various studies in evolutionary biology , including the processes and outcomes of speciation [ 1014 ] . the phenotypic and ecological divergence in crested newts , examined across different ontogenetic stages , provides an excellent model to explore the tradeoff between shared evolutionary history and divergent functional requirements within an adaptive radiation . the resulting framework can be used to evaluate how developmental and functional processes have impacted phenotypic evolution . crested newts belong to the group of the modern eurasian newts . crested newts have a biphasic life cycle with aquatic larvae that metamorphose into terrestrial juveniles and as adults return to the water annually to breed [ 34 , 35 ] . crested newts have low mobility , a small dispersal range , and strong philopatric behaviour , which promote genetic isolation . the crested newts form a well - supported monophyletic clade of closely related species for which the molecular phylogeny has been largely resolved [ 11 , 14 , 33 , 36 ] . according to current taxonomy , this group consists of six species : t. cristatus , t. dobrogicus , the closely related species t. carnifex and t. macedonicus , and two species that belong to the so - called t. karelinii group , t. karelinii and t. arntzeni . mitochondrial dna studies showing three distinct clades within the t. karelinii group [ 14 , 15 ] illustrate that the taxonomy of the t. karelinii clade is as yet unsettled . in an attempt to simplify the range of the crested newts spans most of europe and adjacent asia ( figure 1 ) . the nominotypical species ( t. cristatus ) is the most widely distributed over much of europe . t. dobrogicus is confined to the pannonian area and the danube delta and dobrugea plain . the other species have a more southern distribution , restricted to the apennine peninsula and the northeast balkan peninsula ( t. carnifex ) , the western balkan peninsula ( t. macedonicus ) and the eastern balkan peninsula , asia minor , crimea , caucasus , and southern shore of the caspian sea ( t. karelinii group ) . the species have a parapatric distribution and a potential to interbreed along the contact zones , especially in the balkan region [ 37 , 38 ] . generally , t. macedonicus , t. carnifex and the t. karelinii group are the more terrestrial species . the other crested newt species are associated with relatively small lentic ponds with variable hydroperiods [ 18 , 37 ] . the duration of the aquatic phase is directly related to their habitat and varies from a short , three months in the t. karelinii group up to six months in t. dobrogicus ( figure 2 ) . it is worth to note that the evolution of habitat preferences of crested newts still needs to be addressed properly . the most notable characteristic of the crested newts ' origin is that their evolutionary splitting occurred within a short - time span , which indicates that there was a burst of speciation rather than a prolonged process of speciation [ 11 , 14 , 36 ] . ecologically based , spatially heterogeneous selection , coupled with limited migration , can result in rapid phenotypic diversification [ 39 , 40 ] . such a scenario presumes the existence of divergent ecological conditions , as well as a low magnitude of phenotypic and genetic correlations . a high level of phenotypic divergence could be achieved even under substantial hybridisation and gene flow . the term body form refers to the robust morphological features of an organism 's external morphology and encompasses both size- and shape - related characteristics . body form can differ between species , but also between groups of species . in tailed amphibians , adaptation to an aquatic life is usually related to body elongation and limb reduction , which may increase swimming performance . a compact body and robust limbs are linked to a terrestrial life and a lack of passive buoyancy . crested newts show a range body forms from a slender body and short limbs in t. dobrogicus , via t. cristatus , and t. macedonicus and t. carnifex to a short body and long limbs in the t. karelinii group [ 37 , 42 ] . the ancestral phenotype , a large body with a short trunk and a wide head , characterises the t. karelinii group . the species t. carnifex and t. macedonicus have a large body and wide head accompanied by mild body elongation . the most derived phenotype includes body size reduction and more pronounced body elongation in t. cristatus and , especially , in t. dobrogicus ( figure 2 ) . body elongation in these newts is reflected in the modal number of rib - bearing vertebrae : 13 in t. karelinii group , 14 in t. macedonicus and t. carnifex , 15 for t. cristatus , and 16 or 17 in the most elongated t. dobrogicus [ 14 , 37 , 43 , 44 ] . the ontogenetic niche shift and transition between the aquatic and terrestrial habitats is coupled with metamorphosis and an overall change in the relationship between the individual and its environment . therefore , two different sets of adaptations and constraints during growth could shape the ontogenetic trajectories of crested newts and affect their phenotypic diversification . the analysis of ontogenetic shape changes and changes in developmental rate gives insight into the processes of the evolutionary diversification of the crested newts . the four analysed species of crested newts ( t. dobrogicus , t. cristatus , t. macedonicus , and t. arntzeni ( t. karelinii group ) , differ in size and shape as larvae , at least when the larval body form is fully developed ( i.e. , midlarval stage ) . the ontogenetic trajectories of larval shape diverge in both the direction and the rate of shape changes along species - specific trajectories . the species significantly differ in their developmental rate of larval shape , except for t. cristatus and t. dobrogicus , which are similar . t. dobrogicus clearly differed from the other species in having a higher and wider caudal fin , while t. arntzeni ( t. karelinii group ) has the most elongated larvae with the lowest tail fin ( figure 3 ) . based on the assumption that the shape of caudal fins is of high adaptive significance for larvae , it is tempting to hypothesise that the two larval shape types represent aquatic ecological adaptations of the two species groups of crested newt . contrary to clear discrimination between species in larval body shape , at the juvenile stage just after metamorphosis , the species converge on a similar body shape . the differences in body shape that we found may indicate that the body forms of larvae and adult individuals are subject to selection in both the aquatic and terrestrial environments , resulting in the same pattern of interspecific differences , despite the possibility of two distinct sets of constraints . a morphometric analysis of the limb skeleton of four crested newt species ( t. dobrogicus , t. cristatus , t. carnifex , and t. arntzeni ( t. karelinii group ) ) showed that although they differ in the size of skeletal elements ( stylopodium , zeugopodium , and third metapodial element ) , they all shared common allometric slopes . a similar relationship between limb skeleton size and body size could indicate a conservative direction of ontogeny . however , a lateral shift in the species - specific allometries of t. dobrogicus and t. cristatus indicates evolutionary changes in the allometric trajectories . moreover , the t. dobrogicus manus has a significantly lower ossification level and concomitant loose bone packaging compared with the other species ( figure 4 ) , which could be a result of the heterochronic changes [ 46 , 47 ] . the shared genetic factors ( e.g. , hox patterning genes ) are intrinsic to the covariation among the homologous structures within limbs ( e.g. , radius and tibia or humerus and femur ) and the overall morphological integration [ 48 , 49 ] . epigenetic factors , such as function , also could have an impact on limb integration . in empirical studies , the expression of functional and developmental interdependencies in the patterns of integration could be estimated . if covariation between the homologous parts of the fore- and hindlimbs is stronger than the covariation of skeletal elements within the limbs , then developmental constraints prevail over functional determinants [ 4951 ] . two opposing correlation patterns were observed in the more terrestrial species , the homologous limb elements were less correlated , and the within - limb elements were more correlated , whereas , in the aquatic species , the reverse pattern occurred ( figure 5 ) . all of these results indicate that function appears to be the covariance - generating factor that has shaped the patterns of morphological integration of crested newt limbs . the visualisation of the phylogeny superimposed in the morphospace , and the positions of the internal nodes in the phylomorphospace ( figure 6 ) indicate that most of the shape changes occurred along species - specific branches . t. dobrogicus markedly diverged in skull shape as reflected by a more slender and elongated skull ( figure 6 ) . the advantages of such morphology might be better locomotion in aquatic habitats due to a more streamline body shape but with the possible disadvantage of reduced abilities of suction feeding . the similarities in skull shape of t. macedonicus and t. arntzeni ( t. karelinii group ) probably reflect a symplesiomorphy . the analysis of the ontogenetic trajectories of skull shape changes between juveniles just after metamorphosis and adults indicate that t. dobrogicus has the highest rate of cranial shape change during postmetamorphic growth , as well as a distinctive ontogenetic allometric trajectory compared with the other three analysed species ( t cristatus , t. carnifex , and t. arntzeni ( t. karelinii group ) ) . to visualise the direction and amount of shape changes during crested newt skull shape ontogeny ( figure 7 ) , we performed an additional analysis using a larger sample of juveniles than available for the study of ontogenetic shape changes . the slope and the amount of ontogenetic shape change of t. dobrogicus clearly diverged from the other species . it is interesting to note that the changes in skull shape that clearly separate t. dobrogicus and t. cristatus from t. macedonicus and the t. karelinii group are shared in both analysed stages : the juvenile stage just after metamorphosis and the adult stage ( figure 7 ) . comparative study of crested newt development and early life history traits , such as egg characteristics , developmental rate , survival rate , and duration of the embryonic period [ 52 , 53 ] produced data valuable for understanding the forces shaping adaptation and evolutionary diversification . in vertebrates , vitellus size appears to be one of the key life - history traits reflecting maternal input and could affect the development rate and the size and stage of larvae at hatching . the vitellus size and thickness of the mucoid capsule that protects from injury , fungal infestation , and ultraviolet - b radiation were investigated in four crested newt species ( t. macedonicus , t. cristatus , t. dobrogicus , and t. arntzeni ( t. karelinii group ) ) . although the studied species shared a common allometric slope of the egg size versus body size relationship , the species differed in the egg size , which appeared to be a species - specific life - history trait with a cline - like distribution ; t. dobrogicus has the smallest eggs , the members of the t. karelinii group and t. macedonicus have the largest eggs , and t. cristatus has intermediate - sized eggs . the crested newts are similar with respect to basic developmental traits ( no differences in developmental sequences and survival rates ) . generally , the developmental rate highly depends on environmental factors , especially temperature . under experimental conditions , t. dobrogicus appears to be the outlier species , particularly in comparison to t. arntzeni ( t. karelinii group ) and t. macedonicus , which have the longest developmental period . also , there are differences in the pattern of correlation amongst life - history and developmental traits . the comparisons of phenotypic correlation matrices based on eleven life history and developmental traits revealed that t. dobrogicus have a correlation pattern similar to t. cristatus and t. macedonicus . t. arntzeni ( t. karelinii group ) has a similar correlation pattern to t. macedonicus , but there are no similarities in the matrix correlation pattern compared to the other two species . there are four criteria to detect an adaptive radiation ; common ancestry , rapid radiation , environmental correlation , and trait utility . in crested newts , common also , there is a perfect match between the patterns of interspecific differentiation in phenotypic traits and ecological preferences . however , no clear relationship between evolution of body form and locomotor function in newts was found . the onset of adaptive radiation often requires , in addition to the existence of a new habitat , the possession of a key innovation that allows rapid adaptation in novel ecological settings . the observed pattern of differences in the crested newts ' phenotypic characteristics shows that t. dobrogicus is in any respect the most derived species . t. dobrogicus has ( 1 ) the most elongated body and the largest number of rib - bearing vertebrae [ 14 , 42 ] ; ( 2 ) a significantly different size , ossification level , and pattern of morphological integration of limbs [ 13 , 30 ] ; ( 3 ) a marked difference in skull shape , including the direction and rate of ontogenetic shape changes ; ( 4 ) the smallest vitellus ; ( 5 ) peculiarities in life history traits ; ( 6 ) a distinct pattern of sexual dimorphism of morphometric traits ( the sexual dimorphism in body size is absent in t. dobrogicus , while in other species females are the larger sex ) . however , from this long list of diverged phenotypic traits that characterise t. dobrogicus , a key innovation can not yet be clearly recognised . external restrictions imposed by ecology had a strong influence on the crested newts ' phenotypic divergences , including development . in our view , the evolutionary diversification of phenotypic traits in crested newts was driven by ecological speciation . ( an additional separation between crested newts might occur through parapatric speciation , in which populations diverge with some gene flow . ) the pattern of divergence in developmental rate and correlation pattern between several early life - history and developmental traits [ 52 , 53 ] indicates that crested newt evolution seemed to be accompanied by a significant ecological diversification and by labile development patterning , including differences in developmental timing . heterochrony , differences in the sequence of developmental events , the timing and the rates of development , are often invoked as causes that underlie observed phenotypic evolutionary changes . the data collected so far suggest that heterochronic changes in early ontogeny can lead to the lateral transposition of the t. dobrogicus ontogenetic trajectories , as previously suggested for the cranial shape and allometric limb skeleton trajectories . natural selection related to shifts in ecology ( e.g. , invasion of new habitats ) can lead to extremely rapid divergence . the new , colonising populations are particularly likely to diverge , especially because they are usually small and likely to be genetically altered . recently , it has been proposed that new species generally emerge from single events ( e.g. , changes in environments ) , and that ecological adaptation promotes reproductive isolation and speciation [ 58 , 64 , 65 ] . also , a growing body of research demonstrates a link between rapid ecological divergence and speciation . we advocate the hypothesis that the phenotypic diversification in crested newts emerged due to an evolutionary switch in ecological preferences . the phenotypic characteristics of crested newts could have evolved over a short - time span during which the main crested newts phylogenetic lineages diverged in the central balkans [ 11 , 43 ] . the main ecological shift of t. dobrogicus ( and less apparently of t. cristatus ) could happen due to the availability of aquatic habitats on the central balkans [ 67 , 68 ] . the subsequent phenotypic evolution of t. dobrogicus could be amplified through ecological selection , when the species occupied extensive lowland floodplains along the present - day danube river and its tributaries which were covered with swamps and marshes . this event dated back to pliocene when the pannonian sea dried out , more than three million years after t. dobrogicus had separated from other crested newts ' species . similar changes might have occurred when t. cristatus spread across the european plains after pleistocene glaciations . if so , adaptive phenotypic radiation in crested newts appears as an extension of the initial process of speciation . for both t. dobrogicus and t. cristatus , the acquiring of new habitats by phenotypic diversification was not accompanied by additional lineage differentiation . most likely , the high connectivity of large lowland water bodies , distributed in areas affected by glaciations , prevented the long - term partitioning of populations and thus inhibited speciation [ 69 , 70 ] . in contrast , within the t. karelinii group , subdivision across a heterogeneous landscape , especially those in glacial refuges , promotes geographical isolation and thus speciation [ 15 , 69 ] . the morphologically and ecologically diversified species of crested newts exhibit a cline - like variation pattern in phenotypic traits , with t. dobrogicus and the t. karelinii group on the opposite poles and t. cristatus as an intermediate species , while t. carnifex and t. macedonicus are close to the t. karelinii group . this pattern matches the cline of the species ' ecological preferences indicating that phenotypic diversification in crested newts emerged most likely due to an evolutionary switch in ecological preferences . the patterns of variation also indicate that heterochronic changes underlie the observed phenotypic evolutionary changes .

the divergence in phenotype and habitat preference within the crested newt triturus cristatus superspecies , examined across different ontogenetic stages , provides an excellent setting to explore the pattern of adaptive radiation . the crested newts form a well - supported monophyletic clade for which at least the full mitochondrial dna phylogeny is resolved . here we summarise studies that explored the variation in morphological ( larval and adult body form , limb skeleton , and skull shape ) and other phenotypic traits ( early life history , developmental sequences , larval growth rate , and sexual dimorphism ) to infer the magnitude and direction of evolutionary changes in crested newts . the phenotypic traits show a high level of concordance in the pattern of variation ; there is a cline - like variation , from t. dobrogicus , via t. cristatus , t. carnifex , and t. macedonicus to the t. karelinii group . this pattern matches the cline of ecological preferences ; t. dobrogicus is relatively aquatic , followed by t. cristatus . t. macedonicus , t. carnifex , and the t. karelinii group are relatively terrestrial . the observed pattern indicates that phenotypic diversification in crested newts emerged due to an evolutionary switch in ecological preferences . furthermore , the pattern indicates that heterochronic changes , or changes in the timing and rate of development , underlie the observed phenotypic evolutionary diversification .

1. Introduction 2. About Crested Newts 3. Interspecific Variation in Phenotypic Traits 4. Adaptive Radiation Pattern 5. Possible Mechanism of the Crested Newts' Evolutionary Diversification in Phenotypic Traits 6. The Ecological Shift Drives the Evolution of Phenotypic Traits in the Crested Newts 7. Conclusions

PMC4485502

an increasingly sedentary lifestyle combined with rising consumption of caloric foods has transformed obesity into a global event . obesity is a low - level , chronic inflammatory disease with a multifactorial etiology that includes eating habits , a sedentary lifestyle , and genetic predisposition that promotes the excessive accumulation of body fat [ 2 , 3 ] . in addition to its roles as an energy reservoir and heat insulating material , adipose tissue also secretes cell - signaling molecules such as adipokines . adipose tissue secretes proinflammatory factors like interleukin 6 ( il-6 ) , tumor necrosis factor - alpha ( tnf- ) , and leptin and anti - inflammatory factors as and interleukin 10 ( il-10 ) and adiponectin . studies have demonstrated that the administration of a high - fat diet in rodents promotes metabolic changes , inducing the production of proinflammatory interleukins and a chronic inflammatory process [ 5 , 6 ] . the presence of large concentrations of endotoxins and saturated fatty acids in the diet promotes the activation of toll - like receptor 4 ( tlr-4 ) , which triggers the production of proinflammatory cytokines [ 4 , 5 ] . through receptors associated with transmembrane proteins , these cytokines interfere with the expression of other cytokines , thus affecting metabolic homeostasis [ 7 , 8 ] . among various implications that are closely correlated with obesity , menopause deserves particular attention because of the increase in life expectancy of the population . menopause is defined as the permanent cessation of menstruation due to loss of ovarian function [ 9 , 10 ] . the loss of ovarian function , particularly the decline in estrogen production , promotes adverse changes in the profile of lipoproteins , metabolism of glucose and insulin , distribution of body fat , clotting , and vascular endothelium . in combination with weight gain , these changes place women at a high risk of developing cardiovascular diseases , which are very common in this period of life [ 10 , 11 ] . the physiological actions of estrogens in the body are mediated by two distinct estrogen receptors ( ers ) : er and er ; these receptors are nuclear transcription factors involved in the regulation of several complex physiological processes [ 12 , 13 ] . according to benedusi and colleagues , the inflammatory processes that occur following the menopause result from a reduction in circulating levels of estrogens and their receptors ( ers ) , which have anti - inflammatory properties however , controversy persists as to whether the effects of estrogens are anti- or proinflammatory . studies in premenopausal women suggest a beneficial role for estrogen in the prevention of vascular inflammation and consequent atherosclerosis . estrogen exerts an anti - inflammatory effect on the vasculature , through antioxidant effects , generation of nitric oxide , prevention of apoptosis in vascular cells , suppression of proinflammatory cytokines , and modulation of the renin - angiotensin system . in vitro studies have also demonstrated that estrogen has an anti - inflammatory effect in several cell lineages . the hormonal changes that occur during the menopause are responsible for specific physical and metabolic remodeling that , when associated with weight gain and obesity , have a negative impact on women 's health . therefore , it is essential that both obesity and menopause , in particular the effects of estrogen on the inflammatory state , are investigated to improve women 's quality of life . in this study , we evaluated whether a high - fat diet with or without ovariectomy promoted the inflammatory process in rats by assessing the production of pro- and anti - inflammatory factors and their receptors and tlr-4 protein content . the experimental research committee of the so paulo federal university approved all procedures for the care of the animals used in this study . thirty - day - old female wistar rats were used in this study and were kept under controlled conditions of light ( 12 : 12 h light dark cycle with lights on at 07:00 ) and temperature ( 22 1c ) . during the experimental period the animals were maintained in collective cages and received water and the specific diet ad libitum . thirty - day - old rats were divided into two groups and fed for 4 weeks with one of the following diets : control diet ( c ) or hyperlipidic diet ( h ) . after this period the animals were divided into four groups : sham c : sham group treated with control diet;sham h : sham group treated with hyperlipidic diet;ovx c : ovariectomized group treated with control diet;ovx h : ovariectomized group treated with hyperlipidic diet . sham c : sham group treated with control diet ; sham h : sham group treated with hyperlipidic diet ; ovx c : ovariectomized group treated with control diet ; ovx h : ovariectomized group treated with hyperlipidic diet . the food intake and body weight were measured weekly at 09:00 . the control diet was prepared according to the recommendations of the american institute of nutrition ( ain-93 m ) and the hyperlipidic diet was by ain-93 m modified . the fat content in the control diet ( c ) was 4% containing 0.64 g of saturated fatty acids , and the hyperlipidic diet containing 20% of fat which 7.7 g was saturated fatty acids . the energy content of c diet was 4114.71 kj and of hc diet 6438.58 kj/100 g ( table 1 ) . at 60 days of age , the rats were anesthetized and received intraperitoneal injection of ketamine ( 70 mg / kg ) and xylazine ( 10 mg / kg ) . the ovx group was submitted to bilateral ovariectomy . for this , they were subjected to a muscular incision to open the peritoneal cavity for posterior connection of the uterine tubules and removal of the ovaries . all the animals received antibiotic ( penicillin ) immediately after the surgery and 0.1 ml / kg body weight of ibuprofen 50 mg for 2 days . the animals were euthanized by decapitation on the 90th day of life in the fasting state ( 10 h ) in the early morning to avoid chronobiological variations . the sham groups were in estrus phase ( phase when estrogen secretions exert their biggest influence ) . trunk blood was collected and immediately centrifuged at 4c and serum aliquots were taken and frozen at 80c to measure the concentrations of glucose , triacylglycerols , and total cholesterol using commercials kits from labtest diagnostic sa ( mg , brazil ) . the concentrations of insulin , leptin , and adiponectin were determined by elisa ( linco research inc . the retroperitoneal , parametrial ( par ) , and mesenteric ( mes ) adipose tissues , gastrocnemius muscle ( gast ) , and liver were dissected , weighed , frozen in liquid nitrogen , and stored at 80c until the protein extraction . for determination of carcass lipid and protein content lipid content was measured as described by stansbie et al . and standardized using the method described by oller do nascimento and williamson . briefly , the eviscerated carcass was autoclaved at 120c for 90 min and homogenized with double the mass of water . triplicate aliquots of this homogenate were weighed and digested in 3 ml of 30% koh and 3 ml of ethanol for at least 2 h at 70c in capped tubes . after cooling , 2 ml of 12 n h2so4 was added , and the sample was washed three times with petroleum ether for lipid extraction . results are expressed as grams of lipid/100 g of carcass . for protein measurements , aliquots of the same homogenate ( approximately 1 g ) were heated to 37c for 1 h in 0.6 n koh with constant shaking . after clarification by centrifugation , protein content was measured according to bradford ( bio - rad , hercules , ca ) . adipose tissue depots , gastrocnemius muscle , and liver ( 0.150.3 g ) were homogenized in ice - cold solubilization and total protein extraction buffer ( 100 mm tris , ph 7.5 , 100 mm sodium fluoride , 10 mm sodium orthovanadate , 2 mm phenylmethylsulfonyl fluoride , 10 mm sodium pyrophosphate , and 0.1 mg / ml aprotinin ) . homogenates were centrifuged at 19283 g for 40 min at 4c . the supernatants were saved and the protein concentrations were determined using a bradford assay ( bio - rad , hercules , ca ) with bovine serum albumin as a reference . fifty micrograms of the specific tissue total protein was loaded onto a sodium dodecyl sulfate - polyacrylamide gel ( 5% stacking gel ; 10% running gel ) , separated by electrophoresis , and then electroblotted onto nitrocellulose membranes ( hybond - c extra , amersham ) using a wet electroblotter ( bio - rad , ca , usa ) . after blotting , the membranes were blocked in tris - buffered saline- ( tbs- ) tween buffer , ph 7.5 ( 20 mm tris/500 mm nacl/0.05% tween-20 ) , containing 1% bovine serum albumin and then exposed to specific antibodies diluted in tbs - tween buffer ( ph 7.5 ) containing 1% bsa for 2 h. the membranes then were washed and incubated with anti - rabbit ig or anti - mouse ig conjugated to horseradish peroxidase and diluted to 1/1000 in the same buffer for 1 h. after a series of washes in tbs - tween buffer , the bands were visualized with enhanced chemiluminescence scanned at uvitec ( cambridge ) after adding the ecl reagent ( ge healthcare bio - sciences ab , uk ) . the antibodies against anti - tlr4 ( sc-99183 ) , anti - tnf - r1 ( sc-7895 ) , anti - il-6 r ( sc-660 ) , anti - myd88 ( sc-8197 ) , and anti--tubulin ( sc-58667 ) were obtained from santa cruz biotechnology ( santa cruz , ca , usa ) , and the anti - rabbit ig and anti - mouse ig conjugated to horseradish peroxidase were obtained from sigma ( usa ) . the results were analyzed using two - way analysis of variance followed by tukey 's test . the initial body , liver , gast , and par weights were similar in all groups . in the ovx h group , the ret weight was higher than in the sham c group and the mes weight was higher than in the sham h group . administration of a hyperlipidic diet in the sham group did not modify these parameters compared with the administration of a control diet . however , the association of a hyperlipidic diet with ovariectomy ( ovx h ) caused a significant increase in final body weight . carcass protein content was greater in the ovx c group than both sham groups . however , carcass lipid content in the ovx h group was higher than that in the sham h group ( table 2 ) . the initial 4 weeks of hyperlipidic diet administration did not modify body weight gain and food efficiency ( figures 1(a ) and 1(b ) ) . however , the combination of ovariectomy and administration of a hyperlipidic diet ( ovx h ) increased body weight gain accompanied by high food efficiency ( figures 1(c ) and 1(d ) ) . the serum concentrations of total cholesterol , hdl ( high - density lipoprotein ) cholesterol , glucose , adiponectin , and estradiol were similar in all groups . serum levels of triacylglycerol ( tg ) were decreased in the sham h group compared with the sham c group . serum insulin concentration was lower in the sham h , ovx c , and ovx h groups than in the sham c group . ovariectomy combined with the administration of a hyperlipidic diet caused an increase in serum leptin levels compared with all other groups ( table 3 ) . the cytokines content was similar in the sham c , sham h , ovx c , and ovx h groups and in the liver and gast ( figures 2 and 3 ) . however , administration of a hyperlipidic diet caused a decrease in il-6 and il-10 levels in mes adipose tissues . furthermore , administration of a hyperlipidic diet associated with ovariectomy decreased the tnf- content of mes ( figure 4 ) . the il-6 content was decreased by the hyperlipidic diet ; however , in combination with ovariectomy , this effect was reversed in ret adipose tissues ( figure 5 ) . the hyperlipidic diet caused a decrease in il-10 levels , but ovariectomy had the opposite effect , and the tnf- content of ret adipose tissues was high following ovariectomy ( figure 5 ) . the il-6 receptor , tnf receptor , tlr-4 , and myd88 protein content did not differ between groups in the liver ( figure 6 ) , gast ( figure 7 ) , and mes adipose tissues ( figure 8) . in this study , we demonstrated that administration of a hyperlipidic diet for 8 weeks combined with short - term ovariectomy promoted an increase in body weight gain and a decrease in metabolic efficiency , accompanied by hyperleptinemia . the cytokines content of the liver and gast was unchanged ; however , levels of all cytokines analyzed were decreased in mes adipose tissues . also , we demonstrated that 4 weeks of ovariectomy ( ovx c group ) per se did not modify these parameters compared with the sham c group . however , ryou et al . demonstrated that 8 weeks of ovariectomy in rats caused an increase in body weight and food efficiency with elevated plasma leptin . these results suggest that the time period after ovariectomy may influence body weight gain and food efficiency . previously , jen et al . showed that high - fat diet fed male rats were heavier than chow - fed male rats by the sixth week of a high - fat diet , whereas female rats were heavier by the ninth week . consistent with this , we demonstrated that the administration of a hyperlipidic diet for 8 weeks compared with a control diet did not cause an increase in body weight gain in female rats . . showed that ingestion of a high - fat diet for 8 weeks did not modify body or tissue weight . however , the combination of a high - fat diet and ovariectomy caused an increase in body weight gain accompanied by an increase in ret and mes weight , similar to our findings . this excessive weight gain is primarily due to fat deposition , reflected by increased serum leptin levels , which rise in direct proportion to fat mass . carcass lipid content in the ovx h group was higher than in the sham h and sham c groups . diets rich in saturated fatty acids decrease uncoupling protein-1 ( ucp-1 ) activity in brown adipose tissue liu et al . showed that low estrogens levels , characteristic of ovariectomy , increase ucp-1 expression in subcutaneous adipose tissue from 4 weeks until 12 weeks of ovariectomy . in contrast , ko et al . indicated that a high - fat diet combined with ovariectomy decreased ucp-1 levels relative to controls . also , it has been reported that ovariectomy causes a decrease in the lipolytic responsiveness to norepinephrine of fat cells and an increase in adipose tissue lipoprotein lipase ( lpl ) . lpl hydrolyzes tg from chylomicrons and very - low - density lipoproteins to free fatty acids and monoacylglycerols . a hyperlipidic diet was shown to cause an increase in lpl activity in white adipose tissue . by these mechanisms , ovariectomy promotes increased adiposity , particularly in rats receiving a hyperlipidic diet . taken together , these findings imply that a high - fat diet combined with ovariectomy may cause a decrease in energy expenditure and an increase in adipose tissue deposits , probably via enhanced uptake of tg from chylomicrons . also , in the present study , it was observed that serum tg was lower in sham h group compared with sham c group . studies have reported that low - fat , high - carbohydrate diets increase the liver de novo lipogenesis and cause hypertriglyceridemia compared to high - fat diets [ 29 , 30 ] . . showed that the administration of estrogen promoted a slight decrease in body weight without entailing a significant reduction in adiposity index or in ret weight in obese rats . these data suggest that this treatment is insufficient to restore these parameters , although there is a 77% reduction in the expression of lpl , which would reduce fat deposition by decreasing fatty acid uptake from the circulation . however , this is inconsistent with studies in which estrogen replacement managed to recover both body weight and adiposity index in female control rats . this discrepancy may be related to the structural form of estrogen used , in addition to the dosage and duration of estrogen replacement and the age at which the female rats were ovariectomized . increase in serum leptin concentration was observed only in ovx h group , suggesting that the decrease in estrogen could potentiate the effect of high - fat diet on leptin secretion . hyperleptinemia may arise as a result of the increased body weight of rats in the ovx h group . showed that animals fed a high - fat diet were hyperleptinemic and overweight compared with animals receiving a control diet . it is established that serum leptin levels rise with increasing adiposity and are directly proportional to fat mass . loss of efficacy of endogenous or exogenous leptin in the therapeutic maintenance of body weight is attributable to the development of resistance to leptin , a common occurrence in obese individuals . possible explanations for this include a lack of leptin at central sites resulting from defective transport , for instance , through the blood and the brain barrier , decreased production of leptin by the hypothalamus , or interruption of signal transduction between leptin and receptors in the hypothalamus . saravanan et al . showed that animals fed a high - fat diet had a state of hyperleptinemia accompanied by overweight when compared to animals that received control diet . it is understood that ovariectomy promotes hyperphagia and weight gain , and although this hyperphagia is transitional , the gain in body weight is more durable . estrogen replacement has been shown to decrease food intake and restore weight in ovariectomized rats . these facts suggest that estrogen is involved in the modulation of energy homeostasis by direct action on the appetite and central pathways regulating energy and by controlling the secretion of hormonal signals peripherals such as leptin , which in turn regulates effector pathways central and essential to the integration of the hypothalamus in energy homeostasis . content , administration of a hyperlipidic diet in this study caused a decrease in il-6 and il-10 content in both ret and mes adipose tissues . previously , our group showed that administration of a lard diet for 8 weeks promoted a decrease in il-10 in ret and mes adipose tissues of male mice , with no effect on il-6 or tnf-. cani et al . observed an increase in lps , accompanied by elevated mrna concentrations of pai-1 , il-1 , tnf- , and f4/80 in subcutaneous adipose depots and pai-1 , il-1 , and f4/80 in mes adipose tissues , of high - fat diet fed male mice relative to control diet fed mice . these results suggest that the presence of female hormones could modify the response to a high - fat diet in terms of il-6 adipose tissue content , especially in ret adipose tissues . previously , lafontan and berlan reported that the physiology , metabolism , and function of white adipose tissue vary in a depot - specific manner . several factors could contribute to this , such as difference in the amount of hormones receptors , blood flow , hormone , cytokines , and polypeptides production . for instance , it has been demonstrated by yamashita et al . that aerobic training was effective in reducing adipokines levels related to inflammation in mesenteric adipose tissue but not in retroperitoneal adipose tissue . in this study , the rats were euthanized , between 9:00 and 12:00 h , in the estrus phase , a period when estrogen secretions exert their biggest influence . in fact , in vitro studies demonstrated an inhibitory effect of estradiol on il-6 production in several cell types in humans and rodents , including macrophages , monocytes , osteoblasts , and bone stromal cells [ 3941 ] . conversely , ovariectomy combined with a hyperlipidic diet promoted a decrease in the tnf- , il-6 , and il-10 content of mes adipose tissues . riant et al . demonstrated that estrogens , particularly chronic e2 administration to ovariectomized mice , enhance the expression of inflammatory factors , such as il-6 and tnf- , in mice fed with a high - fat diet for 4 or 12 weeks , indicative of a proinflammatory effect of e2 , at least in visceral adipose tissues . accordingly , in this study , we observed a decrease in tnf- in mes adipose tissues in the ovx h group compared with the sham h group , supporting the idea of a possible in vivo proinflammatory effect of estrogen on white adipose tissue , in special visceral one . both hyperlipidic diet and estrogen have been reported to influence the circadian rhythm modifying the expression of clock genes involved in the regulation of metabolism and protein expression [ 43 , 44 ] . in this sense , cano et al . demonstrated that high - fat diet disrupts the daily variations of circulating of several adipocytokines , particularly by a reduction in plasma concentration of tnf- from 9:00 to 13:00 h , compared to control diet treated rats . it could be speculated that the results found in the present study , related to the effect of diet and ovariectomy on adipose tissues cytokines content , could be related to the disruption on the clock genes , since the presence of circadian clock genes has been reported in fat . content in white adipose tissue by the administration of a hyperlipidic diet and/or ovariectomy , protein levels of il-6r , tnfr1 , tlr-4 , and myd88 were similar between groups in all tissues studied ( figures 6 , 7 , and 8) . myd88 knockout mice showed no response to the tlr-4 ligand lps in terms of inflammatory mediator production by macrophages , b cell proliferation , or endotoxin shock [ 47 , 48 ] . however , in the case of tlr-4 stimulation , lps - induced activation of nf-b and c - jun n - terminal kinases ( jnk ) was observed with delayed kinetics , even in myd88 knockout cells , although these cells did not produce any inflammatory cytokines in response to lps . taken together , it is possible to speculate that the modification in cytokines content in white adipose tissue observed in this study may have been stimulated by another mechanism , such as mapk dependent pathway . in conclusion , our results demonstrate that administration of a hyperlipidic diet for 8 weeks combined with short - term ovariectomy did not alter the cytokine content of the liver and gastrocnemius muscle but caused a decrease in all analyzed cytokines in mesenteric adipose tissue . however , ovariectomy in rats fed a hyperlipidic diet caused hyperleptinemia and increased body weight gain , food efficiency , and carcass lipid content , which , in combination with long - term ovariectomy , may contribute to the induction of proinflammatory processes , particularly those associated with a high - fat diet . these results emphasize that , after the menopause , women must decrease their consumption of fat .

four - week - old female wistar rats were divided into two groups and fed a control diet ( c ) or a hyperlipidic diet ( h ) for 4 weeks . rats from each group underwent ovariectomy ( ovx ) or sham surgery ( sham ) . they received c or h for the next four weeks . the body weight gain ( bw ) , food efficiency ( fe ) , and carcass lipid content were higher in the ovx h than in the sham h. the ovx h exhibited a higher serum leptin level than other groups . il-6 , tnf- , and il-10 content of mesenteric ( mes ) adipose tissue was lower in the ovx h than in the ovx c. il-6 , tnf- , and il-10 content of retroperitoneal ( ret ) adipose tissue was lower in the sham h than in the sham c. the sham h showed decreased tg relative to the sham c. similar results were obtained in relation to il-6r , tnfr1 , tlr-4 , and myd88 contents in the mes and ret white adipose tissue among the groups . a hyperlipidic diet for 8 weeks combined with short - term ovariectomy decreases the cytokine content of mes adipose tissues but increases bw , enhancing fe and elevating serum leptin levels . these suggest that the absence of estrogens promotes metabolic changes that may contribute to installation of a proinflammatory process induced by a hyperlipidic diet .

1. Introduction 2. Material and Methods 3. Results 4. Discussion

PMC3603493

prolonged imbalance of caloric intake and energy expenditure leads to complex metabolic disorder of obesity . it is associated with most common and chronic human diseases including type 2 diabetes , heart diseases , hypertension , and cancer . angiogenesis , the formation of new blood vessels from preexisting ones , is tightly linked with adipogenesis and is considered as an essential component in development and expansion of adipose tissue . since expansion of adipose tissue ( increasing cell size and number ) creates adipose tissue hypoxia , it can lead to stabilization of the transcription factor hypoxia inducible factor1 ( hif-1 ) [ 4 , 5 ] that induces an angiogenic response . ghrelin is a gastrointestinal endocrine peptide and is identified as an endogenous ligand for the growth hormone secretagogue receptor type 1a ( ghs - r ia ) ; however , it also regulates food intake and is associated with obesity . ghrelin and its receptors are expressed in endothelial cells and stimulate endothelial cell proliferation , migration , and angiogenesis . recently , the impact of ghrelin on cardiovascular system has been reported including a decrease of peripheral vascular resistance in consequence an increase in cardiac index and stroke volume , improvement of ventricular remodeling , protection of myocytes from apoptosis , decrease of cardiac injury induced by ischemia / reperfusion ( i / r ) injury , and reduction of the infarct size ( l ) . it also improves endothelial dysfunction , reduces vasoconstrictor effect of endothelin-1 , and decreases blood pressure . the main objective of this study was to investigate the effect of ghrelin administration on serum biomarkers of angiogenesis including leptin , nitric oxide ( no ) , vascular endothelial growth factor ( vegf ) , and its soluble receptor ( vegf receptor 1 or sflt-1 ) in control and obese mice . male c57bl/6 mice ( 5 weeks old , n = 24 ) were purchased from pasteur institute ( tehran , iran ) , and three or four animals were housed together in one cage in controlled environment under a light - dark cycle ( lights on at 19:00 and off at 07:00 ) . the experimental procedures followed the guiding principles for the care and use of animals and were approved by the isfahan university of medical sciences . all mice were randomly divided into four groups : normal diet ( nd ) or control , nd + ghrelin , high - fat - diet ( hfd ) or obese and hfd + ghrelin ( n = 6/group ) . mice were rendered obese by the hfd ( bio - serv research diets , nj , usa ; cat # f3282 ) contained with 59% from fat , 14% from protein , and 27% from carbohydrate ( of total calories ) starting at 5 weeks of age for 15 weeks . after 15 weeks , the ghrelin ( tocris co. , bristol , uk ) was administered subcutaneously 100 g / kg twice daily for 10 days [ 15 , 16 ] . blood glucose was measured by glucometer ( acon lab inc san diego , ca , usa ) elisa kits were used for determination of mice serum insulin ( mercodia , uppsala , sweden ) , vegf and sflt-1 ( r&d systems , minneapolis , usa ) , leptin ( invitrogen , camarillo , ca 93012 ) and nitrite , the main metabolite of no ( promega corp , usa ) concentrations . one - way anova was used to compare data between groups using lsd post - hoc test . figure 1 illustrates that administration of ghrelin for 10 days did not significantly change body weight in obese and control mice ( p > 0.05 ) . as shown in figure 2 , there was a significant difference in blood glucose level between obese and control groups ( p < 0.05 ) . administration of ghrelin did not significantly change blood glucose in obese and control mice ( p > 0.05 ) . serum insulin concentration in obese mice was significantly higher than that of control ( p < 0.05 ) . ghrelin administration did not alter serum insulin concentration in control groups ( p > 0.05 ) , while significantly reduced it in obese group ( p > 0.05 ) ( figure 2 ) . the results indicated no significant differences in serum vegf and sflt-1 between obese and control animals ( p < 0.05 ) ; however , serum no concentration in obese mice was higher than that of control ( p < 0.05 ) . ghrelin administration increased serum vegf and reduced serum no level in obese mice and had no effect on sflt-1 concentration ( figure 3 ) . serum leptin level in obese mice was higher than that of control ( p < 0.05 ) , and ghrelin significantly reduced it in obese group ( p < 0.05 ) ( figure 4 ) . the main finding of this study is that the obese mice had higher serum insulin , no , and leptin concentrations compared to control without changes in serum vegf and sflt-1 levels . ghrelin administration reduced serum no , and leptin and increased serum vegf concentrations in obese mice . higher blood glucose and insulin levels in hfd group indicate the insulin resistance in these animals . we demonstrated that although ghrelin treatment could not alter blood glucose level , it reduced serum insulin concentration in obese mice . ghrelin may also act on cellular glucose uptake and may involve in control of glucose metabolism and insulin sensitivity . ghrelin stimulates insulin release ; however , leptin inhibits insulin . perhaps , only ten days ghrelin treatment was the reason for unchanging of blood glucose level in the present study . modulation of vascular tissue and angiogenesis in adipose tissue is a strategy to affect obesity . adipose tissue endothelial cells produce several angiogenic factors including leptin , no , vegf , fgf , hgf , and other growth factors . no is an endothelium - derived relaxing factor which has antiatherosclerotic effects through different mechanisms . it is suggested that at the initial stage of obesity , a compensatory increase in no production occurs due to upregulation of no synthase . on the other hand , adipogenesis increases upregulation of inos which increases no synthesis due to chronic low - grade inflammation during obesity . these data are in line with the results of the present study that we showed higher serum no concentration in obese mice . leptin is an adipocyte - derived hormone that not only directly promotes angiogenesis and endothelial cell migration but also upregulates vegf expression . as we expected , in the present study , the obese animals had higher serum leptin level than that of control . although some studies indicated higher serum vegf level in obese subjects , a recent study showed that hfd did not affect plasma concentration of vegf . sflt-1 leads to anti- or proangiogenic signaling and inhibits angiogenic signaling through sequestration of vegf ligands [ 28 , 29 ] . in the present study , hfd did not change serum concentration of sflt-1 . in our study , ghrelin administration reduced serum no and leptin and increased serum vegf concentrations in obese mice . ghrelin and leptin circulate in the blood and have a role in regulation of body weight and energy homeostasis . study in human showed that plasma ghrelin inversely correlated to degree of obesity and in this study , ghrelin reduced serum leptin level in obese mice . thus , it seems that ghrelin has a protective mechanism including leptin resistance in setting obesity . an in vitro studies indicated that ghrelin activates no - dependent vasorelaxation in patients with metabolic syndrome . thus , we expected that in the present study , ghrelin administration increased serum no concentration . one explanation for this discrepancy is that ghrelin and leptin have mutually antagonistic effects on inflammatory cytokine expression in obesity and reduced leptin after ghrelin administration may involve in reduction of serum no level . recently , yuan m.j . showed that in a rat model of myocardial infarction , chronic ghrelin treatment increased vegf expression in peri - infarct zone and they suggested that ghrelin may induce angiogenesis after mi . we also found that ghrelin altered serum biomarkers of angiogenesis and it seems that it may mediate angiogenesis through different mechanisms . taken together , our results suggested that ghrelin administration changes the serum biomarkers of angiogenesis and can be involved during states with abnormal angiogenesis .

introduction . ghrelin is a gastrointestinal endocrine peptide that was initially identified as the endogenous ligand of growth hormone secretagogue receptor ; however , recently , the cardiovascular effect of this peptide has been indicated . in this study , we investigated the effect of ghrelin administration on serum biomarkers of angiogenesis including leptin , nitric oxide ( no ) , vascular endothelial growth factor ( vegf ) , and its soluble receptor ( vegf receptor 1 or sflt-1 ) in control- and diet - induced obese mice . methods . male c57bl/6 mice were randomly divided into four groups , normal diet ( nd ) or control , nd + ghrelin , high - fat - diet ( hfd ) or obese and hfd + ghrelin ( n = 6/group ) . obese and control groups received either hfd or nd for 15 weeks . then , the ghrelin was injected subcutaneously 100 g / kg twice daily for 10 days . at the end of experiment , blood samples were collected for blood glucose , serum insulin , vegf , sflt-1 , no , and leptin measurements . results . the obese animals had higher serum no and leptin concentrations without changes in serum vegf and sflt-1 levels compared to control . administration of ghrelin significantly increased serum vegf and decreased serum leptin and no concentrations in hfd group . conclusion . since ghrelin changes serum biomarkers of angiogenesis , it seems that it gets involved during states with abnormal angiogenesis .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

PMC4175264

although it is difficult to define a high altitude , it is generally accepted as being over 3,000 m above sea level where clinical , physiological , anatomical , and biochemical changes can be substantial1 . at high altitude , the human body experiences various physiological responses , such as an increase in breathing capacity , an increase in cardiac output during submaximal exercises , and an increase in maximum aerobic power2 . in addition , it is well known that decreases in atmospheric pressure at high altitude result in pulmonary function efficiently carrying oxygen due to a decrease in the partial pressure of oxygen in alveolar and arterial blood3 . these characteristics of high altitude can be utilized to strengthen physiological function in relation to pulmonary function4 . to breathe , oxygen is inhaled and transferred to the bloodstream and carbon dioxide is expelled by exhalation after the oxygen has been used by the body . although korea has many mountains , the number of mountains exceeding 1,500 m is relatively few so hardly any koreans live at high altitude . due to this environment in korea , many athletes spend much money to participate in high - altitude training overseas . despite equivocal findings about the benefits at high - altitude training , current theory dictates that the best approach is to spend several weeks living at 2,500 m and train near sea level5 . for these reasons , various methods have recently been attempted to gain the advantages of high altitude while overcoming its disadvantages . for example , a training program called living high - training low ( lhtl ) uses low - oxygen tents , intermittent exposure to low oxygen , and low - oxygen sleep to gain the advantages of high - altitude training6 . in addition , recent studies have reported that improvement in pulmonary function can be achieved by living alternately at 850 m above sea level , and at low altitude . daily living at high altitude can improve the pulmonary function while alternate daily living between high - altitude and low - altitude places can elicit similar effects7 . according to roh s study7 , which argues that pulmonary function can be improved by alternate high and low - altitude living , it is appropriate to find a method for strengthening pulmonary function using the environment at high - altitude location . if daily living at high altitude could improve the pulmonary function , the high costs of training overseas could be reduced , and a new method for improving the skills of athletes whose pulmonary function is important to their athletic performance could be established . in addition , not only athletes but also the public would benefit from advancements in methods for improving pulmonary function or respiratory - related difficulties through environmental conditions . accordingly , the present study conducted a comparison of respiratory function and oxygen saturation levels between female students living at high altitude and low altitudes . the present study investigated the differences in pulmonary function of university students living at high and low altitudes . to achieve this objective , the pulmonary function of female university students attending k university , located approximately 850 m above sea level , and y university , located at low altitude , were determined . the study s objective and procedure the ethical committee of kangwon national university hospital s institutional review board approved the study . students of k university , which is located 850 m above sea level , take classes for approximately 20 hours every week for 15 weeks in a single semester , commuting to and from a dormitory located at 200 m above sea level . during school holidays , they return to their homes located in metropolitan cities , which are at low altitudes . thus , k university students experience daily living at both high and low altitudes . on the other hand , y university y university and its dormitory are both located at a low altitude . in this study , students of k university are henceforth referred to as high altitude living students while students of y university are referred to as low - altitude living students . the oxygen saturation levels , hr , and pulmonary function of the female students of k and y universities were measured at a dormitory located at a low altitude at the same time between 5 pm and 6 pm . the experiment was conducted on october 225 , 2013 . in order to eliminate measurement errors between the two universities , we had two measurers from both universities to ensure the same measurement method was used . after training , three measurers performed the test measurements and the intra - class correlation coefficient of inter - tester reliability was found to be 0.93 . the study subjects were chosen based on a history of no respiratory - related diseases , autonomic nervous system disability or symptoms , as well as no smoking history . in addition , for the two months prior to the measurements , they did not exercise for more than 20 minutes a day , which can affect cardiovascular function . the subjects attending k university included 25 first - year students ( average age : 18.920.57 years , average height : 163.044.25 cm ) , 25 second - year students ( average age : 19.960.54 years , average height : 161.525.45 cm ) , 25 third - year students ( average age : 21.040.35 years , average height : 159.844.40 cm ) , and 25 fourth - year students ( average age : 22.120.67 years , average height : 160.804.17 cm ) totaling 100 students . the average age and height of the study subjects from k university were 20.51 years and 161.30 cm . subjects attending y university included 28 first - year students ( average age : 18.140.36 years , average height : 161.865.27 cm ) , 25 second - year students ( average age : 19.120.44 years , average height : 162.564.27 cm ) , 25 third - year students ( average age : 20.040.46 years , average height : 159.885.04 cm ) , and 26 fourth - year students ( average age : 21.120.59 years , average height : 162.005.08 cm ) a total of 104 students . the average age and height of the study subjects from y university were 20.66 years and 161.59 cm . oxygen saturation and hr ( beats per minute : bpm ) levels were measured using an instrument that calculates heart rate and arterial blood oxygen saturation levels ( pulse oximeter , mp110p , mek - ics co. , korea ) . the pulse oximeter was attached to the subject s middle fingers and the levels were measured in a stable state 68 times within five minutes . pulse oximetry is a non - invasive method that uses an instrument to monitor oxygen saturation levels easily and quickly , and displays the measured levels as spo2 . in this study , forced vital capacity ( fvc ) , forced expiration volume of 1 second ( fev1 ) , peak forced expiratory flow ( pef ) , and forced vital capacity of 1 second were measured using cardiopulmonary measuring instruments ( microplus spirometer , carefusion co. , uk ) . when subjects arrived at the measurement location , they were instructed to rest for more than five minutes to ensure that their condition was normal . then , oxygen saturation and hr levels were measured using the right index finger . demonstrations and explanations about the required posture were provided before measuring cardiopulmonary function , which was done three times . vital capacity was measured using the method recommended for chronic obstructive pulmonary disease patients by referring to the standard guidelines8 for the implementation and reading of pulmonary function tests published by the american thoracic society and the european respiratory society . when pulmonary function was measured , the subjects were instructed to look forward , while standing in an upright position . the subjects were instructed to maximally inhale and then exhale quickly and strongly for the longest time possible , while firmly holding the nose and properly biting the mouthpiece . to obtain better measurement results , the subjects were encouraged by a researcher saying , more , more , more , hoo . the subject was instructed to rest for approximately two minutes after each measurement so as not to record rebound measurements . when measured levels could not be properly obtained because the subject coughed or otherwise , the subject was instructed to rest , after which the measurement was repeated . the data were analyzed using spss 12.0 for windows and the paired t - test to determine respiratory function and oxygen saturation levels according to grade . this study was conducted to compare the cardiopulmonary functions of female university students living at high altitude and low altitudes . oxygen saturation and cardiopulmonary function levels of the students attending the college located at low altitude and those attending the college at high altitude were determined . hr , pef , and fev1/fvc levels of the students living at high altitude were lower while their oxygen saturation , fev1 , and fvc levels were higher than the levels of the students living at low altitude . there were significant differences between the two groups of students in all the measured item ( p<0.01 ) ( table 1table 1.comparison of pulmonary functions of female university students at low and high altitudehr * * ( bpm)oxygen saturation * * ( % ) fev * * ( ml)fvc * * ( ml)pef * * ( ml)fev1/fv**high altitude77.7310.3498.261.022.650.382.870.46318.1071.780.930.77low altitude82.159.0997.681.502.390.462.450.49363.2074.630.980.04**p<0.01 , hr : heart rate , fev1 : forced expiratory volume of one sec , fvc : expiratory flow rate obtained by fastest expiration after maximal inhalation , pef : peak expiratory flow , fev1/fvc : the ratio of forced expiration volume for 1 sec to forced vital capacity . ) . that is , the hr of students living at high altitude was slower while their oxygen saturation , lung capacity , and inspiratory capacity levels were higher than the levels of students living at low altitude . * * p<0.01 , hr : heart rate , fev1 : forced expiratory volume of one sec , fvc : expiratory flow rate obtained by fastest expiration after maximal inhalation , pef : peak expiratory flow , fev1/fvc : the ratio of forced expiration volume for 1 sec to forced vital capacity . differences in oxygen saturation , bpm , and respiratory function levels of students living at low and high altitudes were determined according to grade . first - year students showed statistically significant differences in all items , except for bpm and fev , between students living at low and high altitudes ( p<0.01 ) . second - year students showed statistically significant differences in all items , except for oxygen saturation levels , between students living at low and high altitudes ( p<0.05 , p<0.01 ) . fourth - year students showed a significant difference only in fvc ( p<0.05 ) . hr and pef levels of students living at high altitude were lower while their oxygen saturation , fev1 , and fvc levels were higher than the levels of students living in a low - altitude place . however , first- and second - year students living at high altitude had lower fev1/fvc levels than students living at low altitude . therefore , it can be said that living daily at high altitude affects respiratory function ( table 2table 2.comparison of pulmonary function of female university students by grade levelhr(bpm)oxygensaturation ( % ) fev1(ml)fvc(ml)pef(ml)fev1/fvc(%)freshmenh78.0410.2398.440.77**2.620.462.960.47**279.8482.26**0.880.02l82.768.8897.321.952.380.462.410.47374.2472.290.990.02sophomoresh76.8411.85 * 98.320.802.690.40**2.980.53**307.0462.84**0.910.08**l83.649.5697.961.022.280.322.310.32371.8176.020.990.02juniorsh75.927.6398.321.182.570.352.660.39326.2762.730.970.54l78.969.9797.601.662.450.352.540.3432571.770.970.06seniorsh80.1211.3197.961.242.700.302.860.38**356.1557.560.950.06l83.327.5097.881.202.460.632.530.71378.7265.170.970.05*p < 0.05 , * * p < 0.01 , h : high altitude , l : low altitude , hr : heart rate , fev1 : forced expiratory volume of one sec , fvc : expiratory flow rate obtained by fastest expiration after maximal inhalation , pef : peak expiratory flow , fev1/fvc : the ratio of forced expiration volume for 1 sec to forced vital capacity . ) . * p < 0.05 , * * p < 0.01 , h : high altitude , l : low altitude , hr : heart rate , fev1 : forced expiratory volume of one sec , fvc : expiratory flow rate obtained by fastest expiration after maximal inhalation , pef : peak expiratory flow , fev1/fvc : the ratio of forced expiration volume for 1 sec to forced vital capacity . this study aimed to determine whether living at high altitude place can change pulmonary function by comparing the pulmonary functions of female university students living at high and low altitudes . it is generally accepted that hrs at high altitude are higher while oxygen saturation levels are lower than at low altitude . in contrast , maximum oxygen uptake and endurance levels are lower at high altitude than at low altitude9 . in this study , students living at high altitude had lower hr and higher oxygen saturation levels than students living at low altitude . the reason for this is that the students living at high altitude had adapted to the high - altitude environment and their hr and oxygen saturation levels were similar to those seen in individuals living in a high - altitude environment . therefore , their hr levels were lower while their oxygen saturation levels were higher than the levels of students living at low altitude when the measurement was done at low altitude . changes in the respiratory system at high altitude are related to lung capacity , diffusion capacity , the perfusion and ventilation relationship , and changes in lung ventilation capacity10 . in order to reduce the excessive ventilation of the lungs , indigenous people at high altitude have a larger lung capacity and 2128% lower residual capacity than those living at low altitude11 . in this study , students living at high altitude had higher fev1 and fvc and lower pef levels than those of the students living at low altitude a result , which is consistent with previous studies1 , 10 , 11 . therefore , it can be said that living daily at high altitude affects pulmonary function . hr and pef levels of students living at high altitude were lower while oxygen saturation , fev1 , and fvc levels were higher than those of students living at low altitude . first - year students showed statistically significant differences in all items , except for bpm and fev , between living at low and high altitudes and second - year students showed differences in all items , except for oxygen saturation levels . the main items showing physiological changes at high altitude are cardiac output , hr , and pulmonary artery pressure10 . in particular , the first- and second - year students who lacked the ability to adapt to daily living at high altitude , had lower fev1/fvc levels than those of students living at low altitude . third - year students living at high altitude showed no significant differences when compared with students living at low altitude , while fourth - year students showed different fvc levels . these results indicate that the pulmonary function of third- and fourth - year students had adapted to living at high altitude through living there for a long period . our results also showed that cardiac output increased in those taking a rest while living at low altitude as well as those exercising at an earlier time while living at high altitude . however , cardiac output returned to the value seen at sea level once the subjects had adapted to living at high altitude10 . this is explained by the fact that people living at low altitude increase pulmonary perfusion and lung capacity in response to the low atmospheric pressure and low oxygen partial pressure when temporarily living at high altitude11 . also , the amount of ventilation experienced while resting does not recover to the value seen at sea level even if complete adaptation is achieved . when living at high altitude for a certain period of time , the human body acclimatizes by increasing pulmonary perfusion and lung capacity , increasing the oxygen - binding capacity of blood and peripheral tissues , and increasing the amount of red blood cells in order to endure the low atmospheric pressure and low partial pressure of oxygen12 therefore , third- and fourth - year students had increased lung capacity and pulmonary perfusion because of adaptation to daily living at high altitude . our results suggest that , if an individual is not accustomed to high altitudes of between 1,500 m or 3,000 m above sea level , living at high altitude would mainly elicit physiological changes in oxygen saturation and pulmonary function . however , this study did not identify how long a daily living period at high altitude is needed to influence pulmonary function enough that it can improve the performance of athletes . in addition , the students living at high altitude did not stay at high altitude all the year round , only for 30 weeks of the year . thus , the time spent at school at high altitude would be much shorter than 30 weeks . nonetheless , our study results show that there were changes in oxygen saturation and pulmonary function levels , even under such conditions . therefore , it would be a good idea to search for measures to utilize high - altitude locations to improve the pulmonary function of athletes . that is , changes in pulmonary function can be obtained through training that alternates between low- and high - altitude locations . it would be more advantageous to open a training field at high altitude and have athletes stay there for a certain period rather than travel overseas to training facilities at a high altitude locations , in terms of finances and the administration of athletes . thus , an in - depth study should be conducted of pulmonary function and physiological changes at high altitude . also , future research of male subjects and individuals of various ages is needed .

[ purpose ] this study compared the respiratory function and oxygen saturation levels of university students living at high altitude , to present a new approach for improving respiratory function using high altitudes above sea level . [ subjects and methods ] the subjects were 100 female students attending a university located approximately 850 m above sea level and 104 female students attending a university located at low altitude . oxygen saturation , heart rate ( hr ) , and respiratory function levels were measured . [ results ] for the students living at high altitude , hr , pef , and fev1/fvc levels were low . in contrast , their oxygen saturation , fev1 , and fvc levels were higher than the levels found in students living at low altitude . differences in respiratory function were revealed in first- and second - year students living at high and low altitudes . on the other hand , no significant differences in respiratory function were found between third- and fourth - year students . [ conclusion ] university students living at high altitude had a slower hr and higher oxygen saturation levels as well as higher lung and inspiratory capacity levels . thus , physiological improvement in oxygen saturation levels and pulmonary function were seen in the individuals living at high altitude .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4972912

the use of emerging technologies such as smartphones and tablets offers researchers and coaches opportunities to undertake physical performance assessments in the field , rather than the sports science laboratory . current smartphone technology includes advanced computing capacity , inertial sensors , a global positioning system , and high speed video capacity . recently developed smartphone applications ( apps ) for sport have capitalised on these advances and are shown to be valid and reliable tools to assess lower limb functional performance during countermovement jump [ 2 , 3 ] , drop jump , balance [ 4 , 5 ] , and maximal strength tests . however , while the assessments of jumping performance and muscular strength are important characteristics of human performance , sprint speed is critical for success in a range of activities including football and netball . yet unlike other physical performance characteristics , the assessment of sprinting capacity has received relatively less attention in the peer - reviewed literature . although fully automated timing systems using pressure sensitive starting blocks or photo finish recording systems remain the gold standard for the assessment of sprint running performance , such systems are rarely found outside athletic venues . they lack portability and are cost prohibitive to many teams , coaches , and athletes . dual beam photocell systems are increasingly used due to their lower cost , portability , and capability to wirelessly send data to a handheld receiver . dual beam systems exhibit greater accuracy compared to single - beam systems and have been recommended over single - beam systems . recent developments in error detection algorithms to address some of the shortcomings associated with single - beam systems have shown promising results . for example , the smartspeed ( fusion sport , coopers plains , australia ) , single - beam system with error detection , has been shown to comply with australia 's national sport science quality assurance standards , which require sprint testing systems to achieve a maximal typical error of 0.05 seconds over 30 m. d'auria and colleagues showed that the smartspeed system achieved a typical error of 0.03 seconds at distances of 5 , 10 , and 20 m. studies also show that video analysis of running performance has been shown to closely match fully automated timing systems and dual beam photocell systems , even when recorded at 50 or 100 hz . therefore , even low - speed video capture may represent a low - cost and portable option to assess running performance . recent innovations in smartphone technology have seen video capture speeds up to 240 hz on iphone 6 . coupled with improved image detection algorithms and computing power , smartphone apps have been developed to allow coaches and researchers to determine running speed using these popular and low - cost tools . speedclock uses the ios device camera to detect and record motion and , with the user input of a reference distance , calculates speed . speedclock can be used on a single standalone device , with the reference frame defined by the edges of the image detection field , or connected via bluetooth with a second ios device to record time over longer distances . although apps such as speedclock are purported to accurately record sprint performance , to the best of our knowledge , there are currently no studies which demonstrate the validity of speedclock . such studies are important in confirming the usefulness of emerging technologies and are of great interest to athletes , coaches , and researchers . therefore , the purpose of the present study is to examine the validity of the speedclock app using timing lights as a reference . participants were a convenience sample of 24 recreationally active females ( > 18 years ) recruited via personal discussion with investigators . an information sheet outlining the purpose , risks , and benefits of participation prior to inclusion in the study , participants were screened for musculoskeletal , neurological , or cardiorespiratory concerns which would contraindicate the performance of maximal sprinting , using stage one of the adult preexercise screening system . written informed consent was obtained from all participants prior to the commencement of data collection . participants stature was measured using a seca 213 portable stadiometer ( seca gmbh , hamburg ) and weight determined using a seca robusta 813 portable scale ( seca gmbh , hamburg ) . all participants underwent a standardised warm - up comprising five minutes of light jogging , static and dynamic stretching , and a series of submaximal sprints . participants then performed four maximal effort 20 m sprints , separated by five minutes of passive rest . participants started on a line between the first set of timing lights and commenced the sprint in their own time . standardised instructions were provided regarding maximal effort and to decelerate only when past the final set of timing lights . all sprints were performed in an indoor sports stadium ( ~26c , 50% rh ) on a suspended timber floor . sprint times were recorded using smartspeed pro timing lights ( fusion sport , coopers plains , australia ) , with gates at zero , 10 , and 20 metres . this system uses a single - beam design to improve battery life and ease of setup , however , incorporates novel error detection algorithms to reduce false triggers . in the event of multiple triggers , sprint times were converted to mean sprint velocity ( m / s ) for 010 m , 1020 m , and 020 m using a standard linear motion equation ( v = d / t ) . to examine the validity of the speedclock ios application , the app was installed on an iphone 5c running ios version 9.2.1 ( apple corporation , cupertino , ca ) , which records video at 60 frames per second . in speedm ( motion ) mode , the app incorporates motion detection zones at each edge of the image which trigger and terminate data collection ( figure 1 ) . for the present study , to ensure repeatability of the method and to minimise movement of the device , iphone was mounted in a plastic tripod mount on a velbon ex 330 tripod ( velbon corporation , tokyo , japan ) , with the camera lens 1.0 m from the ground . the tripod was positioned 10.5 m perpendicular to the midpoint between the timing lights at 10 and 20 m , such that the motion detection zones of the application were aligned with the 10 and 20 m timing lights . the tracking sensitivity on the app was set to 0.92 ( arbitrary units ) to avoid false triggers from background movement . in this manner , as the participant ran through the timing lights at 10 m , motion detection triggered data acquisition . similarly , as the participant ran through the timing lights at 20 m , motion detection terminated data acquisition . mean velocity ( m / s ) for the flying 10 m sprint was displayed on - screen and manually recorded for later analysis . all four flying 10 m sprints recorded simultaneously using timing lights and the speedclock application were used for analysis . independent sample t - tests ( two - tailed ) were used to identify differences in average velocity obtained from the timing lights and speedclock app . between - device agreement was examined using intraclass correlation coefficients ( icc 2,1 ) with 95% confidence intervals ( 95% ci ) and interpreted according to munro . bland - altman plots were then constructed using microsoft excel ( microsoft corporation , redmond , usa ) to visualise the level of agreement between average velocity obtained from the timing lights and the speedclock app . with the exception of bland - altman plots , all statistical analyses were performed using statistical package for the social sciences ( spss ) , version 22 ( ibm corporation , chicago , ill ) . twenty - four recreationally active females ( mean age 26.6 5.4 years , mean body mass index 25.0 3.1 kgm ) provided informed consent and participated in the study . independent samples t - test showed no statistically significant difference in mean flying 10 m sprint velocity between data from the timing lights ( 6.47 0.49 sec ) and the speedclock app ( 6.31 0.48 sec ) ( t(190 ) = 1.83 , p = 0.07 ) . intraclass correlations ( icc 2,1 ) showed excellent agreement in mean flying 10 m sprint velocity between data from the timing lights and the speedclock app ( icc ( 2,1 ) = 0.93 , p = 0.00 , 95% ci 0.640.97 ) . bland - altman plots to visualise the difference between mean flying 10 m sprint velocity determined by timing lights and speedclock app are shown in figure 3 . a small systematic bias ( mean difference = 0.13 seconds ) shows the app consistently gave slightly lower values compared to the timing lights . to the best of our knowledge , no other studies comparing app - based measures of sprint running speed have been published in the peer - reviewed literature . as such this study makes an important contribution to the exercise and sports science domain . the findings from the present study suggest that the speedclock app installed on an iphone 5c recording video at 60 frames per second is a valid measure of average flying 10 m sprint velocity in recreationally active females . a number of other studies have examined the use of video technology in determining sprint running performance . for example , haugen and colleagues reported no systematic variation between brower dual beam infrared timing and dartfish - based video analysis of 40 m sprint times in national level male and female track athletes . in another study , harrison and colleagues reported the validity of video recorded at 50 hz and at 100 hz , to determine mean sprint velocity over 3 m. no statistically significant differences and excellent iccs were observed between a laser sports measurement system and video recorded at either frame rate . however , at medium to fast sprinting velocities , video recorded at 100 hz compared more favorably with laser - derived measures than video recorded at 50 hz , due to the higher sampling rate . in the present study , mean flying 10 m sprit velocity recorded using the speedclock app was not significantly different to that recorded using dual beam timing lights . moreover , iccs showed excellent agreement with bland - altman plots indicating only a small degree of systematic bias , with a mean difference of 0.13 seconds ( 1.97% ) . although this level of mean difference may be significant with respect to changes in sprint performance over time , these values are likely within expected test - retest reliability of sprinting performance , and researchers should examine the smallest worthwhile change in performance to determine if a real change has occurred . one notable difference between the timing lights and the speedclock app is that the timing lights report data to three decimal places , while the speedclock app reports data to two decimal places . therefore , we reran our icc analysis using velocities based on timing light data under similar conditions , firstly truncated and then rounded to two decimal places . the resultant analyses were not substantially different from our initial findings ( icc ( 2,1 ) = 0.92 , p = 0.00 , 95% ci 0.680.97 , and 0.93 , p = 0.00 , 95% ci 0.640.97 , resp . ) and do not change our interpretation of the validity of this app . emerging technologies such as the use of smartphone applications in sport have the potential to provide athletes , coaches , and researchers with additional data not otherwise available with a single laboratory based system . for example , when using speedclock , an image of the participant can be captured midway through the image capture field . this may provide valuable data to assess running technique , and , since the image is stamped with the mean velocity , it serves as a permanent record of the attempt . unlike gps - based system , speedclock is video - based and therefore can be used indoors . finally , unlike manual stopwatch timing , the data is not affected by parallax error when standing at the start or finish line , and in the case of speedclock , two devices can be used together in a manner not unlike timing gates . in general , smartphone - based apps for sprint performance assessment offer low - cost , portability , and ease of use , not otherwise available with laboratory based systems , and their widespread applicability to training , rehabilitation , and research warrants further investigation . a strength of the present study is the use of bland - altman plots to examine systematic bias between methods used to determine sprint velocity . however , a potential limitation is the use of the iphone 5c , which records video at 60 frames per second . future studies should examine the use of high speed video on newer ios devices as the increased resolution afforded by the higher frame rate may improve accuracy and reduce systematic bias . future studies should also examine other speed recording apps on android operating systems , since , at the present time , speedclock is only available on ios and , although ios devices are widely utilised , they are not the sole software platform . finally , future studies should examine the validity of the speedclock or similar apps over shorter and longer distances and in clinical populations . speedclock for ios devices is a low - cost , valid tool for the assessment of mean flying 10 m sprint velocity in recreationally active females . further studies are required in different populations and settings to generalise the findings from the present study .

recent innovations in smartphone technology have led to the development of a number of applications for the valid and reliable measurement of physical performance . smartphone applications offer a number of advantages over laboratory based testing including cost , portability , and absence of postprocessing . however , smartphone applications for the measurement of running speed have not yet been validated . in the present study , the ios smartphone application , speedclock , was compared to conventional timing lights during flying 10 m sprints in recreationally active women . independent samples t - test showed no statistically significant difference between speedclock and timing lights ( t(190 ) = 1.83 , p = 0.07 ) , while intraclass correlations showed excellent agreement between speedclock and timing lights ( icc ( 2,1 ) = 0.93 , p = 0.00 , 95% ci 0.640.97 ) . bland - altman plots showed a small systematic bias ( mean difference = 0.13 seconds ) with speedclock giving slightly lower values compared to the timing lights . our findings suggest speedclock for ios devices is a low - cost , valid tool for the assessment of mean flying 10 m sprint velocity in recreationally active females . systematic bias should be considered when interpreting the results from speedclock .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions

PMC3020601

mass deaths occurred at a large rearing facility for north american bullfrogs ( rana catesbeiana ) , 46 km from montevideo , uruguay . the farm is a commercially managed operation , with trained employees under permanent , technical supervision . mortality rates during the days before the outbreak were approximately 0.5% per week , which is considered normal for this type of farm . frog growth rates were also within normal parameters for these conditions ( 10 ) . at this farm , spawning occurs in 50-l tanks from october to february ( spring and summer ) , tadpoles grow until march , and metamorphosis occurs from march to may ( fall season ) . newly metamorphosed frogs ( 58 g ) are harvested from tadpole ponds and stocked in tanks with circulating water until resorption of the tail is complete . frogs are then transferred to large growing tanks , in which the frogs are reared at a density of 500 per square meter while standing in shallow water with only their bodies immersed ( 10 ) . permanently running water frogs are fed with trout chow ( purina aquamax carnivorous ) and earthworms ( eisenia foetida ) cultured at the farm . tanks are cleaned every day to eliminate excess feed and feces , and animals are sorted to maintain homogeneous sizes in each tank . earlier in the same week , a tank containing 5,000 recently metamorphosed frogs had been emptied , and the population placed in three other tanks along with other frogs that had metamorphosed during the same week . the following day , frogs started to die in unusually high numbers ( 300 deaths on day 1 ) in these three tanks . to reduce the risk of bacterial infections , poly - hexamethylene - biguanide - hydrochloride ( vantocil ib , zeneca biocides , blackley , manchester , uk ) the deaths continued and spread to other tanks ; 15,000 frogs died during the first week . an antifungal therapy ( benzalkonium chloride 2 ppm baths , farmazul 1% solution , lab . farmaco - uruguayo , acua de figueroa , montevideo ) ( 11 ) was used , but this treatment was also ineffective and almost 95% of the farm s total population of recently metamorphosed frogs died within 30 days . mortality rates in individual tanks were extremely high , with affected tanks losing 10% of the population on day 1 and usually > 90% of the population by day 3 or 4 . by day 10 , all frogs in the three index tanks were dead , and the disease had spread to metamorphs in other tanks on the farm . the epidemic continued for 26 days ( until may 27 ) , when only 2,000 of the original 30,000 metamorphs remained . none of the broodstock was affected , even though these animals were in close spatial association with the affected tanks . although nonspecific , these signs , the high mortality rate , and the age of affected frogs are consistent with those reported for chytridiomycosis ( 2 ) . many of the frogs had agonal convulsions and extension of hind limbs ; death occurred 1224 h later . reddening of ventral skin was found in some cases a few hours before death , usually in the pelvic and femoral areas . routine bacteriologic tests ( culture on blood agar for 24 h and 48 h at 37c ) were performed on liver , kidney , and heart blood collected from a sample of euthanized frogs , but these tests did not implicate causative pathogens . necropsies were performed systematically on 30 frogs , but no gross abnormalities of the viscera were found . internal organs ( lung , liver , kidney , heart , and spleen ) from 10 of these frogs were examined by histologic testing , but no lesions were detected . examination of skin smears ( wet - mounted , stained with a 1:1 mixture of cotton blue [ parker ink ] and 10% aq . koh ) , and histologic and ultrastructural analyses of fixed skin indicated subspherical developing sporangia and flask - shaped mature sporangia within the superficial layer of the epidermis ( figure ) . septa , characteristic of b. dendrobatidis , were observed in many developing sporangia ( figure ) . slight hyperplasia of the keratinized cells of the stratum corneum and stratum granulosum was observed in some areas where chytrid developmental stages were present . specific examinations for viruses were not performed , as facilities to test for viruses were unavailable . characteristic sporangia ( s ) containing zoospores ( z ) are visible in the epidermis ( asterisk , superficial epidermis ; arrow , septum within an empty sporangium ; bars , 10 m . c , skin smear from infected frog , stained with 1:1 cotton blue and 10% aqueous potassium hydroxide ( aq koh ) ( d , developing stages of batrachochytrium dendrobatidis ; arrow , septum within a sporangium ; bar , 10 m . d , electron micrograph of an empty sporangium showing diagnostic septum ( arrow ) ( bar , 2 m ) . in a basic transmission experiment , two groups of five apparently healthy frogs from affected tanks were placed in separate aquaria , one contained five frogs from an unaffected farm in uruguay and the other empty . deaths began on day 4 , with death occurring 2448 h after first signs of disease . within 8 days , after the incident , death rates returned to levels before the event . as a result of this event , monthly surveillance for cutaneous chytrid infection was instigated ; ventral hind limbs , feet , and digits are examined from five healthy frogs and from any sick or dead frogs . b. dendrobatidis has been repeatedly observed in skin smears of healthy frogs from the farm at which the outbreak occurred . similar results have since been obtained from six other bullfrog farms in uruguay ( r. mazzoni , unpub . data ) . histologic examination of infected frogs on which necropsies were performed since the 1999 epidemic shows focal mild hyperplasia of the superficial epidermis associated with sporangia of b. dendrobatidis . cutaneous chytrid infection was detected during an investigation into mass deaths in farmed north american bullfrogs . although chytridiomycosis has been reported as an important cause of mass deaths in wild amphibians , whether chytrid infection was the cause of deaths in the disease outbreak described in this paper is unclear . first , while no examinations for b. dendrobatidis had been performed before the deaths , this organism has been repeatedly observed in skin examinations of healthy frogs in follow - up studies . second , although not proved efficacious against cutaneous chytridiomycosis , the use of benzalkonium chloride , a recognized therapy for infections with nonhyphal fungi , failed to reduce mortality rates . possible causative agents include an infectious pathogen , which could not be detected by the examinations used , such as a ranavirus ( 2 ) or adverse environmental factors . alternatively , chytridiomycosis could be the cause of the outbreak if the current chytrid is a different strain from that causing the outbreak or became attenuated , if the farmed bullfrogs had immunity to the pathogen , or if a change in the environment before the die - off allowed a normally benign infection to become pathogenic . for example , increased temperatures reversibly inhibit both growth of b. dendrobatidis in culture ( 3 ) and the progress of disease outbreaks ( d. nichols , smithsonian institute , pers . the deaths we describe occurred at the beginning of winter and may have been precipitated by lowered environmental temperatures . the rearing of bullfrogs is a growth industry in south america , particularly brazil , uruguay , and argentina . anecdotal reports of similar loss of stock in a number of farms in uruguay ( r. mazzoni , unpub . data ) suggest that disease is a major economic threat to this industry in south america . this situation is exacerbated by a lack of veterinary diagnostic capacity , management protocols , and treatment or prophylaxis . regardless of the cause of the deaths , the north american bullfrog may be relatively resistant to chytridiomycosis , at least under the environmental conditions in the current study . unpublished data suggest that metamorph bullfrogs can be experimentally infected by b. dendrobatidis without developing signs of disease ( 12 ) , and others have noted differential susceptibility to this disease among amphibian species ( 1,2 ) . nonclinical ( silent ) infections in farmed bullfrogs suggest that this species may act as a carrier of chytridiomycosis , which has serious implications for conservation of biodiversity . b. dendrobatidis has been implicated in the complete removal of multiple species amphibian populations over large geographic areas in the wild ( 1,2 ) . farmed bullfrogs originate in north america and have been introduced to south america during the last few decades for the lucrative restaurant trade . live , farmed south american frogs are exported to other south american countries and to the united states . the trade in bullfrogs is large in scale and global in scope . according to the u.s . fish and wildlife service , over 1 million bullfrogs are imported into the united states each year from south america alone ( 9 ) , with others shipped in from asia . in the united states , recently , guidelines for amphibian translocations have been published that include specific quarantine and testing recommendations for chytridiomycosis ( 13 ) . these procedures , however , are intended for animal movements for international union for conservation of nature and natural resources programs and are not legally binding . in the united states , the u.s . department of agriculture ( usda ) may be the most suitable regulatory body for the bullfrog trade since these animals are a commercial agricultural product . the farm we studied was opened in october 1998 with stock that originated from brazil . the brazilian farmers originally imported 300 pairs of frogs from canada to set up the first south american farms approximately 30 years ago . the pathogen has been present in bullfrogs in north america since at least the 1970s ( 14 ) . b. dendrobatidis in this outbreak may have been imported with breeding stock from north america . the uruguay farm in our study is enclosed by concrete walls , but outdoor ponds have open access to wildlife , and wild amphibians ( tree frogs ) have been observed in the greenhouses where indoor rearing tanks are located . whatever the origins of b. dendrobatidis in farmed bullfrogs in uruguay , such open - plan farms are likely sources of infection and disease for wild amphibian species . in view of this risk , surveillance for chytrid infection and for unusual levels of deaths in endemic amphibian species should be established in the vicinity of frog farms in uruguay and elsewhere in the world . global trade and commerce are regularly cited as drivers of disease emergence in humans , domestic animals , wildlife , and plants ( 6,1518 ) . for wildlife emerging infectious diseases , quantitative analyses demonstrate that this process is the most important driving factor ( 8) . for example , the ( unknown ) causative agent of the bullfrog deaths we describe may be spread by such trade . also , the identification of b. dendrobatidis in an international food animal trade has implications for amphibian conservation and for disease emergence in general . our study adds to reports of chytridiomycosis in the international pet trade ( 19 ) , amphibians for outdoor pond stocking in the united states ( 2,20 ) , importation for zoo collections ( 5 ) , trade in laboratory animals ( 21 ) , and species known to have recently been introduced into new geographic regions ( e.g. , cane toads in australia ) ( 1 ) . with a continued rise in the international air transport volume ( 22 ) , we predict a growing impact of trade and commerce on disease emergence within all populations , including wildlife . for this reason , we urge a revision of national and international veterinary guidelines for the inspection and quarantine of imported animals and animal products .

chytridiomycosis is an emerging disease responsible for global decline and extinction of amphibians . we report the causative agent , batrachochytrium dendrobatidis , in north american bullfrogs ( rana catesbeiana ) farmed for the international restaurant trade . our findings suggest that international trade may play a key role in the global dissemination of this and other emerging infectious diseases in wildlife .

The Study Conclusions

PMC4948571

a 30-year - old caucasian man presented to the pulmonary function laboratory for a pulmonary function test ( pft ) and methacholine challenge test . measured height and weight on the day of testing indicated a body mass index of 28.1 kg / m . baseline pft data are summarized in table 1 , the flow - volume loop and volume - time curve from spirometry are shown in figure 1 . all testing satisfied american thoracic society ( ats)/european respiratory society acceptability and repeatability quality standards ( 1 ) . baseline spirometry suggested a restrictive ventilatory defect , which was confirmed by lung volume measurements via whole body plethysmography . following baseline testing , a methacholine challenge test was performed using the five - breath dosimeter technique ( 2 ) . following inhalation of the final dose of methacholine ( 20 mg / ml ) , the forced expiratory volume in 1 s ( fev1 ) was 8% below baseline . negative methacholine challenge test ; however , the patient complained of chest tightness and dyspnea . the patient s symptoms in the absence of a significant decline in fev1 prompted repeat measurement of specific airway conductance ( sgaw ) as an alternative method to assess the airway response to methacholine inhalation . the sgaw was found to be 73% below baseline , indicating significant airway narrowing compatible with marked airway hyper - responsiveness . following bronchodilator administration ( 2.5 mg albuterol via small volume nebulizer ) both the fev1 and sgaw values were larger than those recorded during baseline testing , suggesting a degree of pretest bronchoconstriction ( table 2 , figure 2 ) . the 1999 ats guideline for methacholine challenge testing states that fev1 should be the primary outcome measure for the discovery of airway hyper - responsiveness ( 2 ) . the guideline recognizes that alternative measures of pulmonary function ( eg , sgaw , impulse oscillometry ) may be used during a methacholine challenge test ; however , these measures are recommended only when the patient can not produce high - quality spirometry data . a whole - body plethysmograph ( often called a body box ) the patient is asked to breathe small volumes at a rate of 1.5 breaths / s to 2.5 breaths / s . flow at the mouth is plotted against the pressure inside the plethysmograph ( ie , box pressure ) . after the collection of several breaths , a shutter or valve is closed so that mouth ( alveolar ) pressure can be plotted against box pressure . this is accomplished by having the patient continue shallow breathing at a rate of approximately 1 breath / s . following open and closed shutter breathing , mouth pressure can be divided by flow to calculate airway resistance . sgaw is considered to be a more useful value because it represents the conductance adjusted to the lung volume where the measurement was recorded . in other words , the ats guideline recommends that a larger reduction in sgaw ( eg , 45% ) be used as a cut - off point for a khalid et al ( 3 ) evaluated sgaw and fev1 in 138 patients undergoing a methacholine challenge test . the researchers found that a 51% to 52% reduction in sgaw was a more appropriate cut - off point for a positive methacholine challenge test than the 45% reduction suggested by the ats . a remarkable finding was that 32 patients with an fev1 decline < 20% exhibited a reduction in sgaw > 50% . in a similar study , parker and mccool ( 4 ) measured fev1 and sgaw following methacholine challenge testing in 248 consecutive patients with asthma - like symptoms . forty patients showed a response to methacholine as assessed by sgaw ( 40% reduction ) without a significant decline in fev1 ( < 20% ) . the obvious question that arises from these observations is whether using fev1 as the sole outcome measure during methacholine challenge testing results in false - negative tests in some patients . in other words , does the positive sgaw / negative fev1 response indicate asthma or an expected response in some nonasthmatic patients or both ? parker and mccool ( 4 ) found that subjects with this response had a higher baseline sgaw and forced expiratory flow between 25% and 75% of the forced vital capacity ( fef2575 ) to forced vital capacity ratio , suggesting that dysanapsis ( large tracheobronchial tree compared with lung size ) may be causal . the authors offered other possible mechanisms for the positive sgaw / negative fev1 response including disproportionate proximal airway narrowing , differences in airway compliance and airway smooth muscle distribution . both studies used the five - breath dosimeter technique with inhalation of aerosol to total lung capacity . cockcroft and davis ( 5 ) showed that this technique results in false - negative methacholine challenge tests as judged by fev1 in patients with mild asthma . it is not clear whether sgaw and fev1 are equally affected by the bronchodilatory and bronchoprotective effects of deep inhalation . in addition to nonspecific airway challenge testing ( eg , methacholine , histamine ) , larbanois et al ( 6 ) reported that 13% of patients undergoing specific inhalation challenge had a 50% decline in sgaw without a 20% decline in fev1 . because sgaw is believed to be more reflective of large airway function ( 7 ) , the authors speculated that this pattern may , in part , be related to the site of aerosol deposition . given the complexity of pulmonary structure and function in the realm of airway hyper - responsiveness ( 8) , the etiology of the positive sgaw / negative fev1 response is likely multifactorial . whether one chooses to follow fev1 , sgaw or both , the inherent limitations of bronchial challenge tests must be appreciated to avoid misinterpretation and misdiagnosis . methacholine challenge tests are not perfect , the sensitivity and specificity can be < 60% and 70% , respectively ( 9 ) . methacholine challenge tests simply determine the presence of airway hyper - responsiveness to a nonspecific agent . the presence of airway hyper - responsiveness only increases the probability of asthma in patients with an intermediate or high pretest probability of asthma ( 10 ) . it would also be incorrect to assume that every patient with a positive sgaw / negative fev1 response to methacholine challenge testing has asthma . this would be especially suspect in patients with a low pretest probability of asthma . in the present case , however , the patient had an intermediate pretest probability of asthma due to his asthma - like symptoms and family history . the fact that the large reduction in sgaw during methacholine challenge testing was accompanied by familiar symptoms that he had experienced after exercise increases the post - test probability of asthma , even in the absence of a 20% reduction in fev1 .

methacholine challenge tests only determine airway hyper - responsiveness to a nonspecific agent , and can be associated with relatively low sensitivity and specificity ; therefore , they should not be used in isolation to diagnose asthma . accordingly , alternative adjunctive measures of pulmonary function have been recommended in cases in which an individual can not produce high - quality spirometry data . this report describes a case involving a 30-year - old man with a primary complaint of dyspnea with exercise . the ensuing discussion briefly reviews these alternative measures and the inherent limitations of the methacholine challenge test .

CASE PRESENTATION DISCUSSION

PMC5288636

discoid meniscus is a well - documented knee pathology , and there are many cases of medial or lateral discoid meniscus reported in the literature . however , ipsilateral concurrent medial and lateral discoid meniscus is very rare , and only a few cases have been reported . a 27-year - old japanese man complained of pain on medial joint space in his right knee that was diagnosed as a complete medial and lateral discoid meniscus . in magnetic resonance imaging , although the lateral discoid meniscus had no tear , the medial discoid meniscus had a horizontal tear . arthroscopic examination of his right knee similarly revealed that the medial discoid meniscus had a horizontal tear . in addition , the discoid medial meniscus also had an anomalous insertion to the anterior cruciate ligament , and there was also mild fibrillation of the medial tibial cartilage surface . we performed arthroscopic partial meniscectomy for the torn medial discoid meniscus but not for the asymptomatic lateral discoid meniscus . we report a rare case of ipsilateral medial and lateral discoid meniscus with medial meniscus tear . the medial discoid meniscus with tear was treated with partial meniscectomy , whereas the lateral discoid meniscus without tear was only followed up . discoid meniscus is not only at peripheral attachments of the tibial plateau but it is also at the central sites . it is reported that discoid meniscus causes meniscus tear due to poor blood flow at the central sites , direct compression , and an unstable connection with posterior capsule . according to previous reports , discoid lateral meniscus is sometimes observed , but concurrent ipsilateral medial and lateral discoid meniscus is rare [ 1 , 2 ] . we report a case of concurrent ipsilateral medial and lateral discoid meniscus with medial meniscus tear . a 27-year - old male patient complained of pain on medial joint space in his right knee after running on the road for few years . he subsequently consulted a nearby clinic and was diagnosed with medial and lateral discoid meniscus in his right knee with medial meniscus tear on magnetic resonance imaging ( mri ) . physical findings for his right knee showed that although the range of motion was not limited , there were swelling and pain on the medial joint space . in addition , the mcmurray test revealed pain and clicking on the medial joint line with external rotation . . t2-weighted coronal and sagittal mri of his right knee revealed medial and lateral complete discoid meniscus , which was confirmed by arthroscopic examination of his right knee . the medial meniscus had a complete discoid conformation with a horizontal tear , whereas the lateral meniscus had no tear ( fig . arthroscopic examination of his right knee similarly revealed that the lateral discoid meniscus had no tear , and the medial discoid meniscus had a horizontal tear . in addition , the discoid medial meniscus also had an anomalous insertion to the anterior cruciate ligament , and there was also mild fibrillation of the medial tibial cartilage surface ( fig . 2 ) . t2-weighted coronal and sagittal magnetic resonance imaging ( mri ) of his right knee . ( a ) at lateral side , the lateral meniscus had a complete discoid conformation without tear . hence , we did not perform surgery , ( b and c ) at medial side , the medial meniscus had a complete discoid conformation with a horizontal tear , ( d ) we performed arthroscopic partial meniscectomy of the medial discoid meniscus . we performed no surgical procedure for the lateral discoid meniscus because the patient had no clinical symptoms , and the lateral discoid meniscus had no tear . we also checked the stability of the connection between the meniscus and the posterior soft tissue both medially and laterally ( fig . three months later , the patient had no pain or symptoms in his right knee and resumed playing sports . at the last follow - up , 18-month post - surgery , he had no limitation of motion , could play sports with full effort , and was satisfied with the result . the medial joint space in his right knee was observed clearly on mri , and the lateral meniscus had no tear ( fig . the medial joint space was observed clearly , and the lateral meniscus had no tear . yaniv and blumberg and ikeuchi reported that lateral discoid meniscus is more common in asian countries than in other countries . the reported incident of discoid meniscus ranges from 0.4% to 17% for the lateral [ 1 , 3 , 4 ] , 0.06 - 0.3% for the medial , and < 0.012% for the medial and lateral in same knee . the incidence of bilateral lateral discoid meniscus is up to 20% of the lateral discoid meniscus cases , whereas bilateral medial meniscus ranges from 0.01% to 0.03% [ 5 , 6 ] . however , cases of ipsilateral concurrent medial and lateral discoid meniscus are rare . to the best of our knowledge , only five cases have been reported [ 4 , 5 , 7 , 8 ] . although the developmental formation process of the normal meniscus is unclear , the normal meniscus is clearly defined at the 8 week of gestation and assumes mature anatomical shape at the 14 week . smillie reported that in many cases , the fetal meniscus has an oval shape and covers the central zone of the tibial plateau . the shape of a normal meniscus is the result of gradual absorption of the central zone by stimulation of the knee joint during the latter half of fetal life . differences in the occurrence rate of the error result in differences in the incidence rate between medial and lateral discoid meniscus . recently , fukazawa et al . reported a difference in collagen deposits between the medial and lateral meniscus during fetal life . histology of the meniscus in human fetuses , at the 14 week of gestation , showed spindle - shaped cells aligned in parallel in the lateral meniscus but randomly arranged cells in the medial meniscus . collagen deposits formed wavy bundles - like structure in the lateral meniscus , whereas those in the medial meniscus were distributed relatively randomly . it was suggested that the strength of the medial meniscus is weaker than that of the medial meniscus in human fetuses . in response to this report , yujiro et al . suggested that the medial meniscus likely forms due to absorption of the central zone as the result of kinematic load at the knee joint ; therefore , medial discoid meniscus is not likely to occur because of the difference in collagen deposits between the medial and lateral meniscus . however , we did not investigate collagen deposits in the current case , so further studies are needed to confirm this . the treatment of discoid meniscus is controversial . in adults , many discoid meniscus cases , both medial and lateral , early surgery is important when symptoms are clear . because total meniscectomy has a high risk of osteoarthritis , partial meniscectomy or meniscoplasty should be performed . in younger patients , it is best to perform auxiliary meniscorrhaphy whenever possible . in a case of lateral discoid meniscus in a child under 8 years old , stilli et al . reported that subtotal meniscectomies are preferable when the meniscal tissue is degenerated as some adaptation of the knee to stress activity may occur . in children reported clinical results of four cases of medial discoid meniscus and showed that meniscoplasty provided satisfactory but imperfect results in all cases while avoiding total meniscectomy . they also reported that residual pain and knee snapping may persist after treatment ; thus , careful follow - up is needed . both kim and lubis and choi et al . reported clinical results of ipsilateral concurrent medial and lateral discoid meniscus , the same as the present case . they performed arthroscopic partial meniscectomy for the torn discoid meniscus and no surgical procedure for the asymptomatic discoid meniscus , reporting good clinical results for each case . our patient also had no complaints and has been satisfied with the result of his surgery . however , a longer follow - up period is needed because discoid meniscus and partial meniscectomy have been reported as risk factors for articular cartilage lesions , so we will continue to follow the patient for a long time . we report a rare case of ipsilateral concurrent medial and lateral discoid meniscus with a medial meniscus tear . we performed arthroscopic partial meniscectomy for his torn medial discoid meniscus and no surgical procedure for the asymptomatic lateral discoid meniscus . for 18-month post - surgery , the patient has had no limitation of the motion and is satisfied with the result . although medial or ipsilateral medial and lateral discoid are rare cases , we should know this presence . if there is torn discoid meniscus , we should perform arthroscopic partial meniscectomy . however , if there is asymptomatic discoid meniscus without tear , we should continue long time follow - up .

introduction : discoid meniscus is a well - documented knee pathology , and there are many cases of medial or lateral discoid meniscus reported in the literature . however , ipsilateral concurrent medial and lateral discoid meniscus is very rare , and only a few cases have been reported . herein , we report a case of concurrent medial and lateral discoid meniscus.case report : a 27-year - old japanese man complained of pain on medial joint space in his right knee that was diagnosed as a complete medial and lateral discoid meniscus . in magnetic resonance imaging , although the lateral discoid meniscus had no tear , the medial discoid meniscus had a horizontal tear . arthroscopic examination of his right knee similarly revealed that the medial discoid meniscus had a horizontal tear . in addition , the discoid medial meniscus also had an anomalous insertion to the anterior cruciate ligament , and there was also mild fibrillation of the medial tibial cartilage surface . we performed arthroscopic partial meniscectomy for the torn medial discoid meniscus but not for the asymptomatic lateral discoid meniscus . the latest follow - up at 18 months indicated satisfactory results.conclusion:we report a rare case of ipsilateral medial and lateral discoid meniscus with medial meniscus tear . the medial discoid meniscus with tear was treated with partial meniscectomy , whereas the lateral discoid meniscus without tear was only followed up .

Introduction: Case Report: Conclusion: Introduction Case Report Discussion Conclusion

PMC3821401

annually , over 4 million patients within the 27 european union member states acquire a nosocomial infection , of whom approximately 37 000 will die as a result . surgical site infections ( ssis ) are the third most common form of healthcare - associated infection ( hai ) , behind urinary tract ( 27% ) and lower respiratory tract ( 24% ) infections , comprising 17% of cases . the economic impact of ssis is considerable , with an estimated burden on european healthcare providers of 7 billion in 2008 . costs are driven predominantly by increased length of hospital stay , estimated to be an average 98-day increase compared to patients not experiencing a ssi . in france , it has been estimated that the crude incidence of ssis in france , between 1996 and 2006 , was 154% in patients undergoing either elective or emergency surgery . the incidence varied depending on the surgical intervention , from 049% for knee prosthesis to 924% for colon surgery . despite an increase in the number of patients undergoing surgical procedures , in particular patients deemed to be at high risk of ssi , such as elderly patients , analysis of temporal trends has shown that the incidence of nosocomial infections in france has decreased in recent years [ 57 ] . the reduction in ssi incidence has been attributed , in part , to the successful introduction of active surveillance programmes such as the infection du site opratoire rseau alerte investigation surveillance des infections ( iso - raisin ) and incidence des infections du site opratoire ( inciso ) , and the utilization of infection control strategies that have accompanied surveillance [ 4 , 8 ] . the majority of ssis are preventable ; with a recent usa - based study finding that up to 55% of cases could be avoided . iso - raisin data from 2007 showed that the most common infectious causative agents of ssis were escherichia coli ( 25% ) , staphylococcus aureus ( 19% ) and pseudomonas aeruginosa ( 10% ) . of the identified strains , 10% of e. coli isolates were cefotaxime- or ceftriaxone - resistant , 19% of s. aureus were methicillin - resistant and 25% of p. aeruginosa were ceftazidime - resistant . the frequency of infection by resistant strains of bacteria significantly adds to the burden of ssis as more expensive second - line treatments become necessary . published data describing the total economic burden of nosocomial infections and ssis in france are currently lacking . a recent single - centre study found that the mean additional cost per patient experiencing a ssi , taking into account laboratory tests , radiology , surgery , exploratory examinations and antimicrobial agents , was 1814 . the aim of the present study was to estimate the healthcare burden associated with ssis , both nationally and by region , in france and to explore the potential impact of infection control strategies on the overall clinical and economic burden . the frequency of 12 preselected surgical procedure categories ( table 1 ) performed in france between 1 january 2010 and 31 december 2010 was quantified through searching of the programme de mdicalisation des systmes d'information ( pmsi ) using 507 appropriate classification commune des actes medicaux ( ccam ) procedure codes , selected from about 8000 codes used to index hospital procedures in france . the 12 procedure categories were chosen to provide an overview of ssis in france , but do not encompass all surgical procedures performed in 2010 , and therefore the total incidence is underestimated . pmsi is a comprehensive database of hospital visits in france since all hospitals , both public and private , are legally obliged to report activity for collation , otherwise remuneration for the procedure performed is withheld by the local health authority [ 12 , 13 ] . it is recommended as an epidemiological tool by the french guidelines for health economic evaluation . as well as detailing the procedure performed using ccam codes , the database contains details of length of hospital stay , patients details ( such as age ) and an anonymous patient identifier to allow future hospitalizations to be followed . table 1.daily cost of hospital care following healthcare - associated infection by surgical procedure , in public and private hospitalssurgical proceduredaily cost of care in public hospitals ( ) daily cost of care in private hospitals ( ) amputation276171coronary artery bypass268163thoracic or abdominal artery surgery219131thoracic or abdominal vein surgery261kidney or pelvic surgery259162caesarean281160colon and rectal surgery240162bowel surgery304177laparoscopic hysterectomy293189intracranial or cerebrospinal fluid surgery263171prostate surgery166150organ transplant290172 = 2009 euros . daily cost of hospital care following healthcare - associated infection by surgical procedure , in public and private hospitals having identified patients undergoing surgical procedures in france , the incidence of ssis was examined by inspection of patients history subsequent to surgery for the classification statistique internationale des maladies et des problmes de sant connexes ( icd-10 ) codes relating to infection , namely t814 ( infection following a therapeutic or diagnostic procedure , not classified elsewhere ) , l02 ( cutaneous abscess , furuncle , carbuncle ) , and y95 ( nosocomial infection ) . since these codes are not specific to infection of the surgical site , data shown is for hai incidence following surgery . daily costs of care were calculated for both public and private hospitals based on resource use and cost data from the 2009 echelle nationale de cots mthodologie commune ( encc ) . the encc provides costs of care , taking into account costs of a range of factors , including medical professionals , drug costs , and running costs of the medical unit ( e.g. cleaning and laundry ) . it is possible to evaluate daily physician costs for public hospitals , but this is not the case for private hospitals since physicians are reimbursed per ccam procedure performed rather than per day of care administered , and therefore physician costs are captured in public , but not private , hospitals . in this analysis only costs allocated to the days of stay following surgical procedures were included . the mean daily cost of care , in 2009 euros ( ) , following each category of surgical procedure is summarized in table 1 . having collected data on the length of stay in patients with and without hai , a health economic model was constructed to evaluate cost differences between the infection control scenarios . a bespoke model was coded , with sqlite and adobe air used to manage the database and present the results . a 1-year time horizon was used with outcomes evaluated from a hospital perspective . in this model hai incidence could be varied to examine the clinical and economic impact of infection control interventions used . model inputs were length of stay associated with each procedure with and without hai , daily cost of hospital care following each procedure , and hai incidence following each procedure . the model reported outputs of number of hospital stays , number of hospital days of care , and cost of care . as well as examining the burden at current rates of infection , three scenario analyses were performed in which hai incidence was varied . in the first scenario hai incidence this was based on a recent study of the use of triclosan antibacterial sutures , which found that ssi incidence was reduced by 8% ( 7% vs. 15% ) compared to the control arm in which conventional sutures were used . to examine the impact of less focused approaches that could be used to reduce ssis , scenarios with 20% and 30% reduction in incidence were also analysed . these were based on the upper and lower limits of an estimate of the number of preventable nosocomial infections , made by the european centre for disease prevention and control . statistical analysis was conducted to examine differences between clinical outcomes in patients experiencing hai and those not experiencing hai , based on the data collected from pmsi . hypothesis testing of the difference between normally distributed variables was performed using tests , while mann normality tests , to determine whether the data were well modelled by a normal distribution , were performed using kolmogorov the pmsi data showed that of 520 715 selected surgical procedures performed between 1 january 2010 and 31 december 2010 , 30% resulted in infection ( table 2 ) . frequency of hai was correlated with age , with 439% of cases occurring in patients aged 70 years , while only 151% of cases involved patients aged < 50 years the frequency of hai also varied with the type of surgery performed ( table 3 ) . of the selected surgical procedures , the group with the highest incidence of hai was organ transplant , with 131% of surgeries resulting in hai . caesarean section had the lowest incidence of hai , with only 01% of procedures being followed by infection . the clinical burden of hai was greatest following colon and bowel surgery , comprising 491% and 220% , respectively , of the total hai cases . it was found that the highest incidence of infection was in martinique ( 51% ) and the lowest in french guiana ( 07% ) . of the mainland french regions , the highest incidence was found in languedoc - roussillon ( 42% ) and the lowest incidence in alsace ( 22% ) . however , when corrected for case mix it was found that incidence rates were similar across regions . this suggests that differences found between regions were due to the number of high - risk procedures performed , rather than other factors influencing hai incidence . the incidence rates in public and private hospitals showed minimal variation , with 29% and 31% of procedures resulting in hai , respectively . incidence rates by procedure were also found to be comparable for public and private hospitals . 2.incidence of healthcare - associated infection ( hai ) following surgery in the 27 regions of france . table 2.summary of episodes of hospital care included in the analysis ( with and without hai)without haiwith haitotalnumber of procedures505 19515 520520 715median length of stay ( days)7227number of deaths10 326129311 619hai , healthcare - associated infection . table 3.burden of hai by proceduresurgical procedurenumber of procedures performedprocedures resulting in hai ( % ) contribution to the total burden of hai following surgery ( % ) mean length of stay without hai ( days)mean length of stay with hai ( days)mortality rate without hai ( % ) mortality rate with hai ( % ) amputation24 759548617355798coronary artery bypass18 541354114313695thoracic or abdominal artery surgery30 1454179133563143thoracic or abdominal vein surgery35 978030732902104kidney or pelvic surgery20 619304011241369caesarean170 34901157130004colon and rectal surgery93 4308249114294581bowel surgery31 533108220173785124laparoscopic hysterectomy69 27110436190230intracranial or cerebrospinal fluid surgery646565272250131160prostate surgery32 86533699200323organ transplant1742131154262120127total520 7153010014322083hai , healthcare - associated infection.some procedures were classified as more than one type of surgery . incidence of healthcare - associated infection ( hai ) following surgery in the 27 regions of france . summary of episodes of hospital care included in the analysis ( with and without hai ) hai , healthcare - associated infection . the mortality rate in patients experiencing a hai was 83% , compared to 20% in patients who did not ( p < 0001 ) , corresponding to a 415-fold relative increase ( table 3 ) . the largest increase in mortality rate was seen in the thoracic or abdominal vein and thoracic or abdominal artery surgery groups , where mortality rates increased over 50-fold , from 02% to 104% , and 23-fold , from 63% to 143% , respectively . length of hospital stay was found to be non - normally distributed ( kolmogorov smirnov d = 0253 , p < 001 ) . patients experiencing hai had a threefold longer hospital stay than those who did not , with median length of stay increasing from 7 days to 22 days ( p < 0001 ) . when mean length of hospital stay following each procedure was evaluated , it was found that hai was associated with at least a twofold longer stay following all procedures ( table 3 ) . of particular note was the almost tenfold increase following thoracic or abdominal vein surgery , with mean length of stay increasing from 3 days to 29 days following the procedure if hai occurred . the health economic modelling analysis suggested that in 2010 , hais led to an additional cost to public and private hospitals of 43 019 936 and 14 872 779 , respectively ( table 4 ) . this difference was driven not by the rate of infection , since incidence rates were found to be comparable , but by the increased number of procedures performed in public hospitals and the increased daily cost of care . a total of 86 861 more procedures were performed in public hospitals , resulting in 1856 further hais . the daily cost of care was found to be significantly higher in public hospitals for all procedures , ranging from 122 per day increase for care following intracranial surgery ( 293 vs. 171 ) to 88 per day increase following thoracic and abdominal artery surgery ( 219 vs. 131 ) . this was , at least in part , driven by the exclusion of physicians costs in the private sector . table 4.additional length of hospital stay and additional costs associated with healthcare - associated infection following surgery for public and private hospitals in francesurgical procedurepublic hospitalsprivate hospitalsadditional length of hospital stay ( days)additional cost ( ) additional cost per procedure resulting in hai ( ) additional length of hospital stay ( days)additional cost ( ) additional cost per procedure resulting in hai ( ) amputation14 0953 890 220496868141 165 1943078coronary artery bypass13 1663 528 488455684771 381 7512771thoracic or abdominal artery surgery905198 195481829238 2522882thoracic or abdominal vein surgery3297547 3024316kidney or pelvic surgery62 05216 195 5723654415776 735 4742268caesarean40 43910 473 701155416 6322 661 120960colon and rectal surgery48511 363 13142152319375 6782430bowel surgery90102 162 4004800594105 1383540laparoscopic hysterectomy48081 461 63239523096585 1442457intracranial or cerebrospinal fluid surgery34781 019 054849773341 254 1144959prostate surgery63471 669 26128933133469 9501650organ transplant1762510 9805800587100 9643440total164 21043 019 93690 85514 872 779hai , healthcare - associated infection. = 2009 euros . additional length of hospital stay and additional costs associated with healthcare - associated infection following surgery for public and private hospitals in france hai , healthcare - associated infection . the largest cost burden was that of hai following kidney or pelvic surgery , costing public hospitals 16 195 572 per year and private hospitals 6 735 474 per year . an increased number of patients experienced hai in public hospitals , despite similar rates of infection ( 3% in both ) , due to the increased number of procedures performed in public hospitals ( 12 365 in public hospitals vs. 8283 in private hospitals ) . furthermore , daily costs of care following kidney or pelvic surgery were 99 higher in public hospitals than in private hospitals . expenditure following kidney or pelvic surgery equated to 38% and 45% , in public and private hospitals , respectively , of additional expenditure as a result of hai . hais had a particularly large cost burden following caesarean ( 13 134 821 ) , amputation ( 5 055 414 ) and coronary artery surgery ( 4 910 239 ) , comprising 40% of additional expenditure , across public and private hospitals , between these three procedures . an 8% reduction in hai incidence , based on a recent study into the reduction in ssis associated with the use of triclosan antibacterial sutures was predicted to result in a saving of 20 205 hospital days per year , across both public and private hospitals , resulting in annual cost savings of 3 406 520 in public hospitals and 1 178 999 in private hospitals . the bulk of the cost savings came from reduced cost of hai following kidney or pelvic surgery , with public hospitals saving 1 294 299 per year and private hospitals saving 537 516 per year , 40% of total savings ( figs 3 , 4 ) . 3.cost saving by reducing the incidence of healthcare - associated infections ( hais ) following surgery by 8% , 20% and 30% in public hospitals . 4.cost saving by reducing the incidence of healthcare - associated infections ( hais ) following surgery by 8% , 20% and 30% in private hospitals . cost saving by reducing the incidence of healthcare - associated infections ( hais ) following surgery by 8% , 20% and 30% in public hospitals . cost saving by reducing the incidence of healthcare - associated infections ( hais ) following surgery by 8% , 20% and 30% in private hospitals . the analysis in which a 20% reduction in hai incidence was applied , based on the lower limit of an estimate of the number of preventable nosocomial infections by the european centre for disease prevention and control suggested that 3185 hai cases could be avoided annually , resulting in 50 871 hospital days saved . this was made up of 32 758 days in public hospitals and 18 113 days in private hospitals . cost savings of 8 583 202 per year and 2 964 855 per year could be made in public and private hospitals , respectively ( figs 3 , 4 ) . healthcare spending as a result of hais was reduced from 57 892 715 to 46 344 658 . when a 30% reduction in hai incidence was applied , based on the upper limit of a european centre for disease prevention and control estimate of the proportion of nosocomial infections that could be prevented , analysis suggested that 76 373 hospital days per year could be saved as a result of 4781 hai cases being avoided . cost savings of 12 880 036 per year could be made in public hospitals , and 4 454 660 per year in private hospitals ( figs 3 , 4 ) . this saving of 17 334 696 represents a 30% reduction in healthcare spending as a result of hais . the present study has quantified part of the considerable clinical and economic burden of ssis on the french healthcare system , where about 3% of the included surgical procedures resulted in infection . the incidence of hai following surgery increased with patients age and varied depending on the surgical procedure performed . included hais led to a 415-fold increase in post - surgery mortality , a threefold increase in length of hospital stay and an annual cost burden of 57 892 715 . the current analysis underestimates the total burden of ssis on the french health economy for two reasons . first , only a predefined set of surgical procedures were included in the analysis , not all the surgical procedures performed in france in 2010 . furthermore , the icd-10 codes used captured only infections reported during the initial hospital stay . since not all ssi cases were identified in this analysis , the clinical and economic estimates are conservative and the burden to the french health economy is expected to be greater . scenario analysis suggests that reduction in hai incidence by 8% , 20% and 30% will result in savings of 4 588 519 , 11 548 057 and 17 334 696 , respectively . these are substantial cost savings , made predominantly in public hospitals due to the increased number of surgical procedures performed and the increased daily cost of care . the frequency of surgical procedures involving patients at high risk of ssi is increasing , and therefore the use of infection control strategies is likely to become increasingly important in the future . one of the major strengths of the present study is the robust epidemiological data on which the health economic model is based . pmsi provides a detailed account of hospital stays and allows the number of procedures and treatments performed in a given time - frame to be accurately determined . furthermore , the age of the patient and the post - operative clinical consequences can be accurately assessed using the anonymous patient identifier . in this analysis , 507 ccam codes relating to preselected surgical procedures were used to identify treatments of interest , giving a comprehensive list of procedures performed , while the use of icd-10 codes relating to hai allows accurate quantification of hai incidence following surgery during the initial hospital stay . the bespoke health economic model developed for this analysis , based on these data , may be a valuable tool for future economic evaluations of infection control strategies and interventions in the french setting . an assumption of this analysis is that the daily cost of care following surgical procedures is the same in patients experiencing hai and those not experiencing hai . it may be the case that patients with hai would require not only a longer period of care , taken into account through the increased length of hospital stay , but also require more intense and expensive care during hospitalization . this may be through increased volume of diagnostic tests to identify the causative pathogen , intensified antimicrobial treatments ( including more expensive second - line therapy if the causative agent is resistant to first - line treatments ) , and increased nursing staff time . given this conservative assumption , cost savings reducing ssi incidence may be greater than those suggested in this analysis . an important limitation of the present analysis is that it does not capture the costs of achieving 8% , 20% and 30% reductions in hai incidence following surgery . however , the analysis does show the threshold at which an intervention to achieve this reduction will be cost saving . for example , if an infection control strategy resulting in an 8% reduction in hai could be introduced in public hospitals at an annual cost of less than 3 400 000 , healthcare providers would have reduced expenditure . while including the costs of interventions to achieve reductions in hai following surgery was beyond the scope of the present analysis , the potential for significant cost savings as a result of interventions that reduce hai incidence following surgery is worthy of further investigation . the risk of ssi is dependent on the type and virulence of the infecting bacteria , and the immunological status of the host . factors increasing the susceptibility of patients to ssi include advanced age , poor nutritional status , diabetes , smoking , obesity , infection remote to the surgical site and lengthy preoperative hospitalization . due to the increased resource use and cost of hospital care for patients experiencing a ssi , infection control strategies to reduce the incidence are important , particularly in patients at the highest risk . while it is acknowledged that not all ssis can be prevented , evidence suggests that the current level of incidence can be reduced [ 13 ] . the majority of ssis occur at superficial sites around foreign material left in the wound . one potential avenue for improving infection control is in the choice of sutures , which are known to provide a focal point for bacterial colonization , act as a vehicle to transport bacteria into the wound and afford a repository for bacteria in the suture knot . use of triclosan - coated sutures reduces ssis by preventing bacterial colonization of the wound around the suture , due to the presence of an antimicrobial . triclosan is a broad - spectrum antimicrobial agent , possessing mostly antibacterial , but also some antifungal and antiviral properties . use of triclosan - coated sutures has been shown to reduce ssi incidence by 8% ( 7% vs. 15% ) in a comparison with conventional sutures . examination of this benefit in the present scenario analysis suggests that this reduction in ssi incidence may have a substantial impact on both the clinical and economic burden , reducing the number of hospital stays , length of stay and cost of care by as much as 4 588 519 per year . further economic evaluation of the benefit of introduction of infection control interventions such as triclosan antibacterial sutures in the french setting may be warranted based on these observations . the world health organization and the centre for disease control and prevention have issued guidelines detailing pre- , peri- and post - operative infection control actions that can be used to reduce the incidence of ssis [ 17 , 18 ] . preoperatively patients should be treated to resolve infections remote from the surgical site and appropriate disinfection of the skin at the surgical site should take place , with shaving only performed if absolutely necessary [ 17 , 18 ] . if the patient is considered at high risk of ssi , a prophylactic dose of an appropriate antimicrobial should be administered , ideally intravenously , at a time to establish a bactericidal concentration at the operational site [ 17 , 18 ] . surgical team members should follow good practice guidelines for hand washing and preoperative aseptic procedures [ 17 , 18 ] . during the surgical procedure , surgical team members should use surgical masks , caps , gowns , gloves and drapes and replace these items when they become soiled [ 17 , 18 ] . the operating theatre should be cleaned using a hospital - approved disinfectant and good practice used to maintain sterility of surgical instruments [ 17 , 18 ] . air within the operating theatre should be changed at least 15 times per hour , with three of these being with fresh air , and filtering should take place using an appropriate filter [ 17 , 18 ] . in the post - operative period , sterile dressing should be used and sterile techniques should be adhered to during dressing changes [ 17 , 18 ] . patients should undergo post - operative surveillance , including post - discharge if required , with any ssi assessed and reported appropriately [ 17 , 18 ] . ssi rates should then be conveyed to surgical team members [ 17 , 18 ] . the present study used conservative estimates of an overall reduction in incidence of 2030% in ssis . the analyses suggest that substantial reductions in healthcare expenditure could occur if ssi incidence is reduced . with greater decreases in ssi incidence larger cost savings can be expected , as the required number of hospital days of care will be reduced beyond the levels estimated in this study . the present study has quantified the substantial economic burden that ssis following a limited selection of procedures place on the healthcare system in france to be 57892715 per year . however , our analysis was conservative and the total burden is expected to be greater , both clinically and economically . new infection control interventions which reduce ssi incidence have the potential to lessen the clinical and economic burden , providing valuable cost savings to healthcare providers and should be the subject of future health economic evaluations . ludovic lamarsalle is an employee of heva , and barnaby hunt and william valentine are employees of ossian health economics and communications .

summaryover 4 million patients suffer nosocomial infections annually in the european union . this study aimed to estimate the healthcare burden associated with healthcare - associated infections ( hais ) following surgery in france , and explore the potential impact of infection control strategies and interventions on the clinical and economic burden of disease . data on the frequency of hais were gathered from the 2010 programme de mdicalisation des systmes d'information ( pmsi ) , and cost data were taken from the 2009 echelle nationale de cots mthodologie commune ( encc ) . it was estimated that 3% of surgical procedures performed in 2010 in france resulted in infection , resulting in an annual cost of 57 892 715 . patients experiencing a hai had a significantly increased mortality risk ( 415-fold ) and an increased length of hospital stay ( threefold ) . scenario analysis in which hai incidence following surgery was reduced by 8% ( based on a study of the effectiveness of triclosan - coated sutures ) , suggested that , annually , 20 205 hospital days and 4 588 519 could be saved . analyses of 20% and 30% reductions in incidence ( based on an estimate of the number of preventable nosocomial infections ) suggested that annual savings of 11 548 057 and 17 334 696 , respectively , could be made . new infection control interventions which reduce hai incidence during hospitalization for surgery have the potential to provide valuable cost savings to healthcare providers .

INTRODUCTION METHODS RESULTS DISCUSSION DECLARATION OF INTEREST

PMC4617043

chronic disease is considered as one of the major problems and issues of health and social care system in current societies . patients in the last stage of life are patients who are not expected to live more than 6 months based on current medical knowledge . nowadays , paying attention to these patients and their families has gained increasing importance . these patients have different physical , social , and psychological needs , and most of these deaths ( approximately 70% ) occur in hospitals . in hospitals with advanced diagnostic and treatment technologies , . often such patients are deprived of the benefits of professional care , thus impacting their quality of life and death . at present , the main approach to care for these patients in developed countries is hospice care service . generally , hospice means specialized care that provides comfort and support to the patients and their families when long - term treatment has not worked and death is inevitable . this care neither increases the life duration nor decreases it , but they improve the quality of life of end - of - life patients . the goal of hospice care is to support the highest possible quality of life for whatever time remains . hospice care programs cared for more than 885,000 people in the united states in 2002 . cancer remains the most common ( approximately 90% ) primary diagnosis for patients in hospice care . the number of elderly is increasing in the developed world and accelerating in the developing world ; awareness of how we care for this population is gaining increased attention . have shown that there is significant difference between the beliefs and attitudes of american doctors and hungarian ones about end - of - life care . according to successful experience of some developed countries , there is an urgent need for designing and delivering hospice care in developing countries . therefore , the present study is going to investigate beliefs and attitudes of stakeholders of this service ( such as patients and their families , physicians , nurses , hospital administrators , health service managers , and insurance authorities ) toward planning and delivering hospice care for end - of - life patients in developing countries ( case study of iran ) via qualitative methods . the strength of qualitative research is its ability to provide complex textual descriptions of how people experience a given research issue . the participants of the study were employers in tabriz university of medical sciences hospital and faculties , patients who were referred to these hospitals of tabriz , and insurance experts of azerbaijan - e - sharqi province , iran . these people were selected because they had a lot of experience communicating with end - of - life patients and also had broad knowledge and information about hospice care . inclusion criteria for this study included having at least 5 years working experience with end - of - life patients , having adequate and appropriate knowledge and information about hospice and palliative care , and their willingness and ability to participate in this study . purposive sampling , one of the most common sampling strategies , was used for selecting heterogeneous groups ( patients , patients families , service providers , public health officials , and insurance executives ) . individuals who had the largest and richest information and were able to make their data available to the researchers were selected as participants of the study . the point of closure was reached when the acquired information became repetitive and contained no new ideas , in a way that the researcher was reasonably assured that the inclusion of additional participants was unlikely to result in any new ideas . the participants statement was recorded with their consent during the interview by voice recorder set and it was also written by interviewers . after completion of the interview , the researchers listened to each interview several times , and these were transcribed verbatim in microsoft office word software ( version 2007 ) . for analyzing the required data , response validity was used for establishing rigor of the required data . for study rigor at the end of session , the subjects were summed , and were asked to endorse them ( respondent validity ) . also , peer check and immersion strategies ( methods of creating rigor ) were used . to consider ethical issues which are important in qualitative studies , the researchers obtained informed consent from participants of the study . in addition , the objectives of the study were explained to the participants by researchers . the approval of regional committee for research ethics which was located in tabriz university of medical sciences was obtained . demographic variables of participants ( n=65 ) in this study , after analyzing and encoding of the participants statements , five major themes including barriers for designing and delivering hospice care , factors facilitating hospice care , strategies for effective design and delivery of hospice service , participants attitude toward designing and delivering hospice service , and executive members of this service were extracted [ figure 1 ] . five extracted themes from participants prospective towards design and delivery of hospice service in iran barriers for designing and delivering of hospice care service from the perspective of participants were extracted from four major themes and 14 subthemes , which are shown in table 2 . barriers for designing and delivering hospice care from participant perspectives facilitators of designing and delivering hospice care from participant perspective were extracted from five major themes and 12 subthemes , which are shown in table 3 . facilitators of designing and delivering hospice care from participant 's perspective by analyzing the views and attitudes of the participants , two major themes of positive attitude and negative attitude were extracted . we point them briefly : in this study , 49 participants ( 79% ) had positive attitude towards designing and delivering of hospice care . in this regard , participant 4 stated that : i meet cancer patients a lot in our unit , there is no hope in their lives now that you speak about this issue ( design and deliver hospice care ) , i think it 's a great idea and it is useful in our country . or participant 18 in this regard had such an idea : generally the hospice care plan is essential and could be effective and good , if performed with comprehensive and good planning , especially considering the prevalence of chronic disease and aging population . despite the positive attitude of the majority of the participants , some of them did not have positive view or perspective towards hospice care . for example , participant number 32 believed that in my point of view , designing and delivering of this plan will be time consuming and difficult , it 's hard to launch such services . number of participants stated that resistance of some executive authorities is the reason of their negative view . number 16 participant believed that this plan like other projects in iran will meet with failure , due to much resistance and opposition of some people . in this study , participants were asked to present their solution and suggestion for effective and efficient planning and delivering hospice care and eliminating probable barriers . by analyzing and coding the participants suggestion and recommendation , finally 10 outcomes could be extracted which are shown in table 4 with statement of participants . participants recommendations for planning and effective delivering of hospice care and removal of potential barriers at the end of the interview , participants were asked to name the people who should be present in the hospice service team . among 65 participants people who should be present in provider hospice service team from participant 's perspective ( n = 51 ) participants stated that attending physician , nurses , psychologists , clergy , and other specialist are necessary in provider team [ other field specialists - figure 2 ] . in this study , 49 participants ( 79% ) had positive attitude towards designing and delivering of hospice care . in this regard , participant 4 stated that : i meet cancer patients a lot in our unit , there is no hope in their lives now that you speak about this issue ( design and deliver hospice care ) , i think it 's a great idea and it is useful in our country . or participant 18 in this regard had such an idea : generally the hospice care plan is essential and could be effective and good , if performed with comprehensive and good planning , especially considering the prevalence of chronic disease and aging population . despite the positive attitude of the majority of the participants , some of them did not have positive view or perspective towards hospice care . for example , participant number 32 believed that in my point of view , designing and delivering of this plan will be time consuming and difficult , it 's hard to launch such services . number of participants stated that resistance of some executive authorities is the reason of their negative view . number 16 participant believed that this plan like other projects in iran will meet with failure , due to much resistance and opposition of some people . in this study , participants were asked to present their solution and suggestion for effective and efficient planning and delivering hospice care and eliminating probable barriers . by analyzing and coding the participants suggestion and recommendation , finally 10 outcomes could be extracted which are shown in table 4 with statement of participants . participants recommendations for planning and effective delivering of hospice care and removal of potential barriers at the end of the interview , participants were asked to name the people who should be present in the hospice service team . among 65 participants people who should be present in provider hospice service team from participant 's perspective ( n = 51 ) participants stated that attending physician , nurses , psychologists , clergy , and other specialist are necessary in provider team [ other field specialists - figure 2 ] . the results of this study reveal that four major barriers in the planning and delivering of hospice service includes financial barriers , cultural - religious barrier , patients and their family - related issues , and barriers related to health care system . from participants perspective facilitators for designing and delivering hospice care system included facilitators related to families , cultural - religious beliefs , facilitators related to patients , healthcare status , and benefits of hospice service . most of the participants in this study had a positive view and belief toward this service . from participants perspective , 10 different outcomes were presented for designing and delivering efficient and effective hospice care . participants stated that the inclusion of physicians , nurses , psychologists , clergy , and other specialists is necessary in provider team . the participants of this study pointed that financial barriers such as human resources , space and new equipment , lack of insurance support , economic recession , and high costs were the main barriers of delivering hospice care . in the qualitative study of brueckner et al . , in germany , they believed that financial issues and problems is one of the barriers for managing hospice care , too . therefore , conducting a pilot study to determine the cost - effectiveness of the service and financial evaluation studies , as well as appropriate steps to control and reduce the cost of hospice care is essential before planning and delivering this service . the other main barriers mentioned in this study , was cultural and religious beliefs of people . it was noted that the cultures of these countries were different when compared with western countries . as in these countries , there are immigrants who are regarded as minorities . in another study by kapo and macmoran , other related studies showed that cultural differences had a huge impact on the acceptance and use of hospice service . therefore , according to the results of these studies and previous related studies , it can be concluded that for planning and delivering hospice care in each country , cultural difference within that country should be considered . hence , survey of perspectives of population and minorities of the community and applying their comments or suggestions in planning hospice care and designing comprehensive and flexible hospice care can be helpful . other major barriers in terms of participants which can affect planning and delivering hospice care are related to the healthcare system . one of the barriers is lack of motivation and support of service providers , especially physicians . the result of other studies showed that in spite of positive views and attitudes of physicians and other healthcare system providers ; they do n't have great motivation and desire to participate in this service . thus , considering the crucial and effective role of providers , for planning and delivering hospice care in each country at first cooperation and assistance of service providers , especially physicians and nurses is required , and to do so financial and nonfinancial incentives for participating in this study should be remarkable . in countries like iran , where hospice care is not designed and delivered yet , training and education of physicians , nurses , and other people who can be effective in this area by involving them in providing the service can be very helpful . for participants , probable advantages of hospice care over hospital services it was noticed in related studies conducted in other countries trying to compare hospice care and provided services to end - of - life patients , that quality of hospice services were higher and satisfaction rate of hospice service was more than that of hospital services among families . even connor 's study showed that the patients who were cared in hospice centers had survived approximately 29 days more than other similar patients . other advantages of hospice service from participants perspectives was reducing costs , this subject was mentioned in other related studies . it is necessary to point that wherever hospice care was delivered , its benefits were obvious and hospice was compatible with other services in those cases . but the advantages in this study were asserted from participants point of view and could not be said with certainly that it will happen in developing countries and iran , if it delivered . according to the results of this study it can be deduced that the patient 's family can have a key role in providing hospice care . in this context , hauser and kramer believed that families had direct and primary role in delivering hospice care service for patients . , have pointed the role of family members , especially caring skills . hence , for designing and launching hospice service , it is better , that the necessary training should be given to patients family members , and their support and assistance must be used appropriately . in this study , the majority of participants ( 79% ) had positive attitude toward hospice care service . previous studies indicated that majority of participants had positive attitude and view toward hospice service . therefore , positive attitudes and views can be regarded as an opportunity for designing and delivering hospice care and it should be used best . one of the best strategies suggested by participants of this study , was involving patients and their families in planning and delivering hospice service . one of the other strategies which were mentioned in this study was including training material in educational curriculum particularly in the field of nursing and medicine . therefore , revising physicians and nurses educational program to include end - of - life care courses can be effective . also seminars , congress , and training courses can be useful in this regard . the results of previous studies showed the perception of importance and role of it and efforts for educational or communication and cauterizing . this subject is important and sensitive and needs further attention because the participants believed that people in developing countries and iran have a rich culture , but on the other hand they are traditional and adhere to some traditional beliefs . participants of this study believe that presence of physicians , nurses , psychologists , and other specialists in other disciplines and clergy is essential in service provider team . in previous studies , the presence of at least one physician in hospice providers team was necessary . in this study nobody mentioned the necessity of social workers presence in the hospice providers team , but researchers suggest that the presence of social workers were essential . in other previous studies , researches have also pointed out the importance of the people offering these services . two major points should be taken into consideration while assigning the hospice care providers in any country . first , the hospice provider team must be various , according to the type of service , patients , environment , and other variables ; and the members of the team providing this service should have various skills . second , the members of hospice providers team should be selected such that there is maximum cooperation , team work , and high quality service . in order to obtain better one of the weaknesses of this study is that , this article is not a comprehensive review of the topic . rather , it seeks to provide sufficient information to raise awareness of the inequity that exists in current provision of hospice care in developing countries . this is a public health issue that deserves priority among many others and we would not claim it as the most important . however , we would argue that it should not be neglected in the discussion of public health priorities . the result of this study is useful for countries who want to plan and design this service . the need for designing and delivering hospice care service in iran and similar countries is overtly felt considering two main factors : first , increasing load of chronic diseases such as various cancers , cardiovascular diseases , stroke , and increasing population of aging population ; and second , successful experience and results of hospice care in many other countries . for lack of sufficient experiences in delivering such services in iran it seems necessary to conduct basic studies for identifying probable barriers , running appropriate preventive tasks and management , reducing the probable failures of such projects , and taking the benefits from experience of other countries . there are significant challenges against implementing hospice care in iran such as : need for specialized centers and huge investments in the qualification of human resources from technical and applied points of view , to deal with terminality related issues . moreover , a change in curriculum is inevitable , considering teaching introductions of hospice care for healthcare students , especially in medical and nursing studies . this study showed that financial and cultural - religious problems of patients and their families , as well as problems of healthcare system , are main barriers against planning and delivering hospice care service . furthermore , facilitators and suggested solutions proposed in this study would be useful in policy making of hospice care system .

introduction : in view of the recent surge in chronic disease rates and elderly population in the developing countries , there is an urgent felt need for palliative and hospice care services . the present study investigates the views and attitudes of patients and their families , physicians , nurses , healthcare administrators , and insurers regarding designing and delivering hospice care service in a middle income country.materials and methods : in this qualitative study , the required data was collected using semi structured interviews and was analyzed using thematic analysis . totally 65 participants from hospitals and tabriz university of medical sciences were selected purposively to achieve data saturation.results:analyzing the data , five main themes ( barriers , facilitators , strategies , attitudes , and service provider ) were extracted . barriers included financial issues , cultural - religious beliefs , patient and family - related obstacles , and barriers related to healthcare system . facilitators included family - related issues , cultural - religious beliefs , as well as facilitators associated with patients , healthcare status , and benefits of hospice service . most participants ( 79% ) had positive attitude towards hospice care service . participant suggested 10 ways to design and deliver effective and efficient hospice care service . they thought the presence of physicians , nurses , and psychologists and other specialists and clergy were necessary in the hospice care team.conclusion:due to lack of experience in hospice care in developing countries , research for identifying probable barriers and appropriate management for reducing unsuccessfulness in designing and delivering hospice care service seems necessary . input from the facilitators and their suggested solutions can be useful in planning the policy for hospice care system .

INTRODUCTION MATERIALS AND METHODS RESULTS Positive attitude Negative attitude DISCUSSION CONCLUSION

PMC3491278

the first task of public health scholars was to clarify the definition of health system . in the 1990s , the notion of the district health system was seen as pivotal for health sector reform and the modernization of the state , which started in the 1980s . this decentralization strategy consisted of a transfer of responsibilities and decision - making power from central to district authorities within the various functions of health service management ( i.e. , finance , service organization , human resources , and general governance ) to a well defined population in a delineated administrative zone . the main objective was to increase the capacities of district managers to respond to populations needs and bring decision - making services closer to populations . in the world health report 2000 , world health organization ( who ) complemented the definition of the district health system with the concept of health system. who defined a health system as all organizations , people and actions whose primary intent is to promote , restore or maintain health. however , this definition reflected neither the interactions between actors nor the ongoing adaptations within systems in response to the changing environment [ fig . 1 ] . upon examination of the applications generated by this definition , it becomes apparent that the definition was a clear response to both the increasing demand from international donors for better accountability , as well as the need for appropriate health system performance assessment tools and methods . the health system : actors , functions , and objectives ( adapted from who and islam ) defining a health system has become more challenging in a globalized world , due to the multiplicity of actors intervening on different scales and the increasing interactions between global health policies and local health systems . every country has a unique health system characterized by the role of the government in the health system ( e.g. , liberal , socialist ) , the values of the health system ( i.e. , more or less equitable ) , the model of financing ( e.g. , taxes or private insurances ) , and its history . eye care has traditionally been established as an entity separate from the rest of health care , although the vision 2020 strategy has extensively promoted the integration of eye care services . this is because the majority of eye care services do not require the input of any other health services ( e.g. , an anesthetist is not required for adult cataract surgery whereas they might be required for an orthopedic surgery ) . this has led to the existence of eye hospitals and eye clinics which stand alone from the rest of the health care structure . this history has meant that eye care has been late in realizing the importance of health systems as an enabling factor [ fig . 2 ] . the different foundations of the health system vision 2020 : the right to sight aims to eliminate avoidable blindness by the year 2020 . this aim will not be achieved by 2020 by eye care services acting in isolation . eye care staff need to engage with the wider health system , identify ways to interact with their peers , influence decision makers , and advocate for change . increasing magnitude of blindness due to noncommunicable eye diseases such as glaucoma and diabetic retinopathy are dependant prevention following on early detection , and raise the urgency to shift from vertical to more horizontally integrated programs . this change is far more likely to occur , be effective , and sustainable if a health systems strengthening approach is taken . the ministry of health has the responsibility of improving the health status of the populations , ensuring equal access to health services for every socio - economic group of society , ensuring that the resources are distributed so that health services can respond the needs to the population and providing general guidance to the actors of the health sector . the government can use several tools to influence the governance of the health system : elaboration of policies , allocation of budget , elaboration of quality standards and regulations , and introduction of incentives . in the table below [ table 1 ] , examples of governance tools used by governments and their potential impact of the eye health system health services can be organized in various ways , hence the diversity of health systems in the world . eye care services can be delivered through the private sector , the not - for profit sector or the public sector , in facilities or in outreach , by eye care professionals or eye care volunteers , through a vertical or horizontal program.[1319 ] the main issue in eye care is access and demand . access to services and coverage of needs is influenced by the model of service delivery adopted by the country . this encompasses , for example , the degree of decentralization of the health system ( i.e. , at which level of the health system are decision made ? ) , the geographical distance between health facilities and residents ( e.g. , how many facilities are available per population ? ) , the range of services offered at different levels of the health system ( e.g. , what services are offered by community eye care workers ? should we train cataract surgeons?),[2124 ] the level of quality of care offered by facilities . the function of service delivery is under the general responsibility of the ministry of health although service providers , donors and nongovernment organizations ( ngos ) have a key role to play . the following table [ table 2 ] illustrates the types of decisions that can be taken and their potential impact on the health system . financing is a key element of the health system and is an issue of how much money should be invested into eye care but also where to allocate the funds to obtain the best value for money . thus , efficiency and equity are the two objectives of health financing . understanding the financing system of an eye health system consists of identifying the various sources of funding of the health system ( i.e. , taxes , health insurance , user fees , international aid ) and where this money is spent ( i.e. , types of expenses covered ( equipment , maintenance , running costs , consumables , medicine , salaries ) , types of activities covered ( facility - based , eye camps , outreach ) , and type of facility funded ( primary , secondary or tertiary levels ) ) . understanding how money is allocated by government and which sources of funding are used can be of great interest for organizations on health rights to advocate for better equity in the allocation of resources . this is also an excellent way of comparing the volume of public spending in eye care compared with other areas of the health sector ( e.g. , hiv aids , malaria ) . the financing system is governed by all the actors of the eye health system : the users who pay user fees or their insurance premium , the government who collect taxes and redistribute them within the health system , the health providers who collect user fees and receive money from the government and donors and ngos who contribute to the financing of the health system [ table 3 ] . input management concerns the procurement and supply of medicine , the investment and maintenance of equipment and facilities and the recruitment , training , and deployment of human resources . the shortage of drugs has been a key factor for under utilization of health services . this is then essential to ensure that the supply chain of drugs is not interrupted and the quality of medicine is guaranteed ( e.g. , respect of cold chain , monitoring of expiry dates ) . in terms of management of human resources , the areas that need to be considered : the number of eye care professionals and the profile needed in relation to the needs of the population and the country , the volume of the active workforce and its distribution in the country , and the number and profile of eye care professionals leaving the system ( retirement or emigration ) . in the table below , a few examples of strategies are listed to understand how they can have an impact on the health system [ table 4 ] . strengthening health systems requires a wide range of skills not only to be able to table issues in various areas ( e.g. , governance , management , finance ) but also to be able to collaborate with diverse actors that have different agendas and priorities . health system strengthening is a complex intervention that requires a good understanding of how the eye health system functions and how the eye health system is connected with the general health system . in 2012 , innovations were introduced in eye care with the elaboration of the eye health system assessment guidelines to help policy makers evaluate the capacities of the eye health system and the level of integration of eye care into the general health system . however , further efforts and investments are needed to explore the structure of eye health systems and generate evidence on the impact of diverse health system strengthening interventions . in a global effort , academics need to continue to inform policy makers and program managers on evidence available and guide them on how to document their results . in contrast , national eye health authorities and international actors need to experiment and test various interventions and feed academics with data .

understanding health systems have now become the priority focus of researchers and policy makers , who have progressively moved away from a project - centred perspectives . the new tendency is to facilitate a convergence between health system developers and disease - specific programme managers in terms of both thinking and action , and to reconcile both approaches : one focusing on integrated health systems and improving the health status of the population and the other aiming at improving access to health care . eye care interventions particularly in developing countries have generally been vertically implemented ( e.g. trachoma , cataract surgeries ) often with parallel organizational structures or specialised disease specific services . with the emergence of health system strengthening in health strategies and in the service delivery of interventions there is a need to clarify and examine inputs in terms governance , financing and management . this present paper aims to clarify key concepts in health system strengthening and describe the various components of the framework as applied in eye care interventions .

What is a Health System? Examining Eye Care Programs in the Context of Health Systems Governance Service Delivery Financing Input Management Health System Strengthening: A Paradigm Shift

PMC2851510

the study was conducted in the ate - vitarte district ( population 365,473 ) , which is located 12 km from the center of lima . it is a marginal urban area that receives immigrants who come to lima with high rates of tb . the study was reviewed and approved by the ethics committee of the east lima health directorate iv . during july august 2004 , interviews were conducted with a random sample of 150 commuters > 15 years of age who had productive coughs for > 15 days and who came to health services for treatment . a total of 142 persons agreed to participate : 96 were treated at hospitals and 46 were treated at health centers . we obtained demographic and socioeconomic information , as well as information on perceived health , and means of transportation used in commuting . all persons with productive coughs were requested to provide 3 sputum samples ( the first immediately after the interview and the other 2 on 2 consecutive days ) for smear testing . samples were tested by using the ziehl - neelsen method , which is used in all epidemiologic surveillance in peru ( 11 ) . study participants were considered positive for pulmonary tb if > 1 acid - fast bacilli ( afb ) were found ( 11 ) . results were analyzed by using bivariate and multivariate logistic models with spss version 12 software ( spss inc . seventeen ( 11.9% ) of 142 study participants were smear positive ( 29.6% 1 cross , 41.0% 2 crosses , and 29.4% 3 crosses ) . two persons had been discharged from a tb treatment program < 6 months earlier . * more than 3 persons sleeping in the same room * afb , acid - fast bacilli ; or , odds ratio , ci , confidence interval . none of the demographic and socioeconomic indicators analyzed ( table 1 ) were associated with pulmonary tb . the use of a minibus and a commuting time > 1 hour had the highest associations with pulmonary tb . adjusted relationships obtained by logistic regression that controlled for all variables shown in table 2 confirmed that commuting to work by minibus was associated with a positive test result for pulmonary tb ( adjusted odds ratio 4.90 , 95% confidence interval 1.0423.04 ) . we observed a pulmonary tb prevalence rate of 12% in persons with chronic productive coughs who came to health services in the study area . this rate was similar to a prevalence of 11% reported in a similar study conducted in an area of poor socioeconomic status in chiapas , mexico ( 12 ) . the proportion of persons 1544 years of age with pulmonary tb in our study was consistent with data of the world health organization and the peruvian ministry of health , which show that this age group has the highest prevalence of this disease ( 5,13,14 ) . our results also showed that there were no sex - related differences in the frequency of pulmonary tb ( 13 , 14 ) . however , this finding should be interpreted cautiously because of the small sample size , particularly the number of persons who lived in extreme poverty . another factor that could limit our conclusions is accessibility of persons in areas of extreme poverty to public transportation . the fact that the field work phase of our study could not be increased because of shortages of resources and health center personnel time is also a limitation . the relationship of having pulmonary tb with working at home or away from home showed a positive prevalence ratio of 6.06 . among persons working outside the home , commuting by minibus increased the risk of having pulmonary tb by a factor of 4.09 compared with persons who used individual forms of transportation . a commuting time > 1 hour on a minibus also increased the risk for pulmonary tb by a factor of 2.07 ( table 2 ) . minibuses in lima increase the risk for pulmonary tb because they are usually overloaded ( capacity is often doubled ) in the early morning and late evening . overcrowding , exposure to persons with productive coughs while commuting 2 times a day 5 days a week , and closed windows on minibuses , combined with a high prevalence of pulmonary tb in lima , increase the risk of acquiring this disease . because persons with cases of pulmonary tb have more productive coughs in the morning ( when more bacilli are released because of their accumulation at night ) , there is increased risk for transmission of tb to other passengers ( 15 ) , as has already been suggested by other studies in developing and industrialized countries ( 69 ) . the findings that 41% of persons tested were positive with 2 crosses and 29.4% were positive with 3 crosses indicate poor tb prevention and control programs in the study area and higher probabilities of transmission ( 15 ) . despite the limitations of our study , commuting in minibuses was a risk factor for pulmonary tb . consequently , preventive measures need to be taken by health services to encourage persons with productive coughs to avoid this type of public transportation and to come to health services for diagnosis and treatment . health services should also be more accessible to persons with pulmonary tb who , for whatever reason , can not use other forms of transportation .

the association between public transportation for commuting and pulmonary tuberculosis ( tb ) was analyzed in workers in lima , peru . traveling in minibuses was a risk factor for pulmonary tb . preventive measures need to be taken by health services to prevent spread of this disease .

The Study Conclusions

PMC4579740

ultrasonography ( usg ) is a valuable method for imaging peripheral nerves , complementing routinelyperformed diagnostic studies , including the physical examination , electromyography and magnetic resonance imaging . in certain situations , usg becomes the imaging method of choice ; these include evaluating nerves of small diameter ( less than 1 mm ) such as cutaneous nerves , or in cases of diffuse neuropathies , when unlike mri , ultrasonography allows for the assessment of even very long nerve trunks and their branches . as in other types of usg studies , the usg diagnostics of peripheral nerves is noninvasive , well - tolerated by patients , and is relatively inexpensive . however , this diagnostic technique requires substantial experience and a thorough knowledge of the nerves topographic anatomy . it consists of both the static and dynamic evaluation of nerves , the latter during passive or active movements of the extremities ; both components are important in the context of diagnosing musculoskeletal disease with usg . the first reports of the use of usg in the evaluation of peripheral nerves appear in 1992 describing a case of carpal tunnel syndrome . a breakthrough in ultrasound diagnostics of peripheral nerves occurred during the last several years , with the introduction of modern transducers , whose high frequencies allowed for the imaging of fine nerves and their branches . for the imaging of peripheral nerves , a linear probe with a frequency greater than 12 - 14 mhz and a resolution less than 0.3 mm is used ( fig . 1 ) . in the case of obese patients or the evaluation of deeply located nerves , a convex probe may be used , thus ensuring a deeper penetration of the ultrasound waves . although the improved range of the imaging signal corresponds to a poorer image resolution , the image quality is still sufficient for the precise monitoring of nerve injection in regional anesthesia . linear broad - spectrum probes with frequencies of 413 mhz and 618 mhz as well as a convex broad - spectrum probe 18 mhz , all used for the ultrasonographic assessment of peripheral nerves when studying very superficial nerves , distancing add - ons , made of gelous agar , are helpful ( fig . 2 ) . such equipment improves the imaging of set nerves both by improving or eliminating the poor contact between the probe and uneven bony surfaces , as well as by imaging the nerve at the level of the ultrasound wave focus . in particular , such adjuncts are useful in the evaluation of fine nerves of the wrist . the neuron composed of a nerve fiber surrounded by the endoneurium , is too thin to reflect an ultrasound beam , and thus is not visible in usg imaging . only groups of nerve fibers which form nerve bundles surrounded by the perineurium may be pictured with this technique . the perineum contains collagen fibers , fibroblasts , blood and lymphatic vessels , and thus forms a layer sufficiently thick to reflect ultrasound waves . nerve bundles combine to form the trunk of a peripheral nerve , which is surrounded by the epineurium , seen clearly in usg as a hyperechogenic layer . nerves may be assessed in the transverse or longitudinal sections . in the longitudinal view , the peripheral nerve is seen as several parallel hyperechogenic lines representing the perineurium between two more prominent and also hyperechogenic layers of the epineurium . 3 a ) . whereas in the transverse section , the nerve resembles a honeycomb , within which are visible tiny round and hypoechogenic areas representing the nerve bundles with hyperechogenic rims of the epineurium ( fig . a. longitudinal view of the median n erve midway in the forearm ( nerve indicated by arrows ) . b. transverse section of the median nerve at the same level , known as the honeycomb view the transverse image is much more frequently used in clinical practice , as it allows for the nerve to be examined by the so - called elevator technique , nerve bundles in the upper limb may be visualized from the level of the brachial plexus root to that of the proper palmar digital nerves . the only short fragment inaccessible to the usg study is that passing below the clavicle , as this bone obscures the image of the underlying soft tissues ( fig . 4 ) . acoustic shadow of the clavicle with a neurovascular bundle laying behind ( arrow ) the aforementioned elevator technique consists of finding the set nerve at a characteristic anatomic point and tracking it either proximally or distally ( figs . 5 a c ) . in this way it is possible to assess the nerve 's shape , echogenicity , thickness , its relation to the surrounding tissues , the surface area of the nerve and its vasculature . if an abnormality is seen in the transverse view , the nerve should be examined in the longitudinal view , although it may be difficult to obtain a good image , particularly of nerves with a nonlinear course , and thus this view may be limited to short segments . hence peripheral nerves are always evaluated in the transverse view while the longitudinal view is only used in certain fragments . successive images of moving the probe ( in the axis of the limb ) using the elevator technique along the course of the median nerve the ultrasonographic picture of nerves changes from hypo- to hyperechogenic as they are followed more peripherally ; this fact is due to an increasing amount of connective tissue between the nerve bundles . however , the property of anisotropy is seen in cases of nerves with large cross - sections . the shape of a nerve may also be different and vary between individuals : round , oval , triangular , or irregularly shaped , which may change further under compression by the probe or with the movement of a neighboring muscle . moreover , a nerve may change its shape along its course , for example from a triangular to a round cross - section . anatomic variants of nerves should also be remembered , including bifid or even trifid variants of the median nerve ( fig . 6 ) . a bifid median nerve ( arrow ) in the carpal tunnel for localizing fine and deeply - seated nerves , characteristic these are often large vessels accompanying the nerves , which may be seen via doppler imaging . motor and motor - sensory nerves may be evaluated indirectly by analyzing the skeletal muscles which they innervate . in case of chronic denervation , by comparing the image to the contralateral side , muscular atrophy may be evident as a decrease of the muscle 's volume and fatty infiltration , which increases its echogenicity . examples include injury of the suprascapular nerve which is manifested by degenerative changes of the subscapularis muscle ( fig . 7 ) , or trauma to the long thoracic nerve ( which is rarely visualized through usg in healthy persons ) , which may be seen in the anterior dentate muscle by assessing the state of dents of the anterior dentate muscle it is possible to determine the level of the injury to the nerve . unfortunately , an indirect method of diagnosing neuropathies is unreliable in the elderly population , in whom there is a progressive generalized atrophy of muscles , impeding the localization or the reliable assessment of the peripheral nerves . comparative images of the normal infraspinatus muscle ( left ) and the same muscle with signs of neurogenic atrophy ( right ) in a patient with chronic compression of the suprascapular nerve ( infraspinatus muscle asterisk , the dorsal surface of the scapula arrows ) an indisputable benefit of the usg examination is the possibility of confronting the usg image with the patients symptoms , by checking if the place of the visualized pathology is compatible to the location of pain , is it located at the point of entry or radiation ( which occurs with neuromas ) . another advantage of usg study over other imaging techniques is dynamic examination of peripheral nerves enabling diagnostics of a number of pathologies , what will be the subject of the following publications . the median nerve forms on the anterior surface of the axillary artery from branches of the lateral and medial cords of the brachial plexus , at the pectoralis minor 's inferior border ( fig . 8 a ) . in the arm , the nerve runs in the medial bicipital groove of the biceps brachii muscle , initially lateral to , then anterior and distally medial to the brachial artery ( figs . 8 b , c ) . in the cubital fossa , along with the brachial artery , the median nerve crosses deep to the bicipital aponeurosis to enter the forearm between the humeral and ulnar heads of the pronator teres muscle ( figs . 9 a , b ) . at this level , the nerve gives off the anterior interosseous nerve , and then descends in the fascial plane between the fds and fdp ( figs . b. application of the probe perpendicular to the long axis of the forearm , in the median aspect of the cubital fossa . c. the median nerve ( arrow ) below the belly of the biceps femoris muscle ( triangles ) , medial to the brachial artery ( asterisk ) a. application of the probe parallel to the long axis of the proximal forearm . b. the longitudinal cross - section of the median nerve ( arrows ) coursing posterior to the humeral head of the pronator teres muscle ( asterisk ) a. transverse application of the probe at the distal end of the forearm and longitudinal placement at the radial aspect of the forearm . b. transverse cross - section of the median nerve ( arrow ) , with the pronator quadratus muscle ( triangle ) and the radius ( asterisk ) seen in the background . c. longitudinal section of the median nerve ( arrow ) between the fds and fdp muscle bellies it passes the wrist through the carpal tunnel , before dividing into terminal branches ( figs . these are the three common palmar digital branches ( digits 13 ) running deep to the superficial palmar arterial arch along the flexor tendons . at the level of the metacarpophalangeal joint , these divide into seven proper palmar digital nerves , which run toward the fingertips along the radial and ulnar aspects of the proximal and middle phalanges , superficially to the proper palmar digital arteries . b. model showing the nerve coursing below the transverse ligament of the carpal tunnel ( from ossan world of anatomical models ) . c. the median nerve at the level of the carpal tunnel ( arrow ) between the scaphoid ( asterisk ) and pisiform ( triangle ) bones it should be mentioned that branches to the thenar muscles run separately or with the common palmar digital nerves , and along the fpl tendon sheath . the anterior interosseous nerve , directly after branching off the median nerve trunk , runs towards the interosseous membrane , lateral to the anterior interosseous artery . it is covered by the fpl muscle and the belly of the fdp muscle . in the distal part of the forearm , it is covered by the pronator quadratus muscle . to identify this fine nerve the median nerve gives off one more important branch which may be visualized in the ultrasonographic study , this is the palmar branch of the median nerve . its branching point is variable , but with the usg probe it may be sought in the distal third of the forearm . it passes between the fcr and pl tendons , then pierces the fascia usually slightly proximal to the flexor retinaculum . to locate this small branch , it is necessary to track the median nerve 's course in transverse views and search for its branches . the usg study of the median nerve is easy and allows for an assessment of its entire course . in the arm , the nerve courses along the brachial artery , easily identified with the doppler option . in the forearm , it is very well seen between the flat bellies of the fds and fdp muscles . it is best though to begin an examination of the median nerve at the carpal tunnel , where in the transverse view it appears as an oval structure adherent to the flexor retinaculum , and manifests minor anisotropy . the probe should be applied transversely and moved along the limb 's axis , slightly medial to the midline and along the anterior surface of the forearm .

ultrasonography is an established method for imaging peripheral nerves . it serves to supplement the physical examination , electromyography , and magnetic resonance imaging . it enables the identification of post - traumatic changes of nerves , neuropathies secondary to compression syndromes , inflammatory or neoplastic nerve lesions as well as the evaluation of postoperative complications . in certain situations , this technique is the imaging method of choice . it is increasingly used in anesthesiology for regional anesthesia . as in the case of other ultrasound imaging studies , the examination of peripheral nerves is non - invasive , well - tolerated by patients , and relatively inexpensive . this article presents the histological structure of peripheral nerves and their appearance in ultrasonography . it also presents the examination technique , following the example of the median nerve , and includes a series of diagrams and ultrasound images . the interpretation of the shape , echogenicity , thickness and vascularity of nerves is described , as well as their relation to the surrounding tissues . the elevator technique , which consists of locating a set nerve at a characteristic anatomic point , and following it proximally or distally , has been explained . the undisputed benefits of the ultrasound examination have been presented , including its advantages over other diagnostic methods . these advantages include the dynamic component of the ultrasound examination and the possibility of correlating the patient 's symptoms with the ultrasound images . as an example , the proper anatomy and the ultrasonographic appearance of the median nerve were described . this nerve 's course is presented , its divisions , and characteristic reference points , so as to facilitate its location and identification , and enable subsequent use of the aforementioned elevator technique . this article opens a series of publications concerning anatomy , technique of examination and pathologies of peripheral nerves .

Introduction Technique for ultrasonographic imaging of peripheral nerves Ultrasonographic picture of the peripheral nerves (sonohistography) Proper anatomy and ultrasonography of peripheral nerves example of the median nerve

PMC4668141

a flexible flat foot refers to a structural deformity in which the medial longitudinal foot arch has become abnormally depressed under weight - bearing conditions1 , 2 . due to the flat foot , the loading on the foot can not be properly distributed , and more activities of the intrinsic and extrinsic muscles are needed1 . in a pronation deformity , where the valgus position is combined with the pronation of the subtalar joint , the talus , navicular , and first cuneiform bone are placed downward and medially3 , and a biomechanical change occuress2 . furthermore , flat foot causes the hyper - adduction of the knee joint as well as the foot deformity , leading to the general structural deformity of the lower extremity4 . among the lower extremity muscles , the medial gastrocnemius ( mg ) and the long head of the biceps femoris muscle ( bf ) located in the posterior part on the frontal plane , and the tibialis anterior muscle ( ta ) and the rectus femoris muscle ( rf ) as they have the continuity of myofascial meridian , they are mutually involved in posture and balance5 and are activated during walking5 , 6 . however , due to the influence of the flat foot , the lower extremity muscle activity becomes higher or lower than that of the normal foot during walking or when standing on one leg6 , 7 . most studies on the flexible flat foot have used electromyography ( emg ) to investigate the structural deformities2,3,4 of feet and changes in the activities of the lower extremity muscles during walking7,8,9 . recently , studies have used myotonometry to compare muscle tone and stiffness between healthy subjects and neurologically impaired patients10,11,12 . however , no studies have investigated the differences in muscle tone and stiffness of the flexible flat foot . in this study , therefore , differences in the muscle tone and stiffness in both the flexible flat foot and the one - side flexible flat - foot types of both lower extremities , compared to the normal foot type in adults in their twenties , were analyzed to provide basic data for physical therapy and evaluation . this study was conducted with 30 subjects in their twenties who were students of the yeoju institute of technology in yeoju , gyeonggi - do . the subjects consisted of 10 in the normal foot group ( nfg ) , 10 in the group with both flexible flat feet ( bffg ) , and 10 in the one - side flexible flat - foot group ( offg ) . those who had no problem in the vestibular system and the neurological and musculoskeletal system and who had not done any regular exercise for the last six months were selected . we explained the purpose and method of this study to the subjects , and only those who consented to participate were included in this study ( table 1table 1.general characteristics of subjectscharacteristicsnormal foot goup(n = 10)both flexible flat foot(n = 10)one side flexible flat foot(n = 10)gendermale354female756age ( years)21.2 1.121.10 1.421.1 1.5weight ( kg)60.2 10.369.0 10.8foot calibration(pixel / pixel)dominanant : 0.3 0.0non - dominant : 0.3 0.0dominant : 0.41 0.0non - dominant : 0.41 0.0flexible flat foot : 0.3 0.0normal foot : 0.3 0.0dominant legright ( 10)left ( 3 ) , right ( 7)right ( 10)flexible flat footleft ( 0 ) , right ( 0)left ( 10 ) , right ( 10)left ( 6 ) , right ( 4)meansd ) . the study protocol was approved by the local ethics committee of the namseoul university of cheonan ( nsu-150429 - 7 ) . in this study , the global posture system 400 ( gps 400 , redbalance , italy ) was used to determine the flexible flat foot . if the length of the narrowest region of the foot exceeded 0.37 ( pixel / pixel ) of the length of the widest region in the gps system , the foot was classified as a flat foot13 . based on previous studies7 , 14 , the feet were also analyzed using strake s line and marie s line . strake s line refers to the line from the medial line of the fore - foot to the medial line of the rear foot . if the line of the medial sole falls inside marie s line ( the line from the center of the third metatarsal bone to the center of the rear foot ) , it is a flat foot . when both of these two conditions are met , the foot is classified as flexible flat foot and marie s line . strake s line refers to the line from the medial line of the fore - foot to the medial line of the rear foot . if the line of the medial sole falls inside marie s line ( the line from the center of the third metatarsal bone to the center of the rear foot ) , it is a flat foot . when both of these two conditions are met , the foot is classified as flexible flat foot . the myotonpro ( myotonas , estonia ) was used measure the muscle tone and stiffness of the subjects . this device can quickly and safely measure the muscle tone , elasticity , and stiffness of various body regions with high reliability10 , 11 , 15 . every measurement was made in a quiet , isolated room , and the room temperature was 25.5 c . before measurement , the highest regions of the muscle belly of the bf , mg , rf , and the muscle tone and stiffness were measured with the measurement device positioned vertically on the skin marker in a relaxed condition in a prone position for the bf and mg and in a supine position for the rf and ta12 . all the data were encoded and analyzed using the statistical analysis program spss win ( ver . the average and standard deviation of the general characteristics ( age , weight , and height ) were calculated using descriptive statistics for each foot type . the homogeneity was tested with one - way anova , and no significant differences were found ( p > 0.05 ) . for each foot type , the offg was reclassified into a flexible flat - foot side and a normal foot side , and the bffg was reclassified into dominant foot and non - dominant foot . in the case of the offg , the wilcoxon signed - rank test was used to compare the differences between the flexible flat - foot side and the normal foot side , and the differences in the muscle tone and stiffness of the dominant foot and non - dominant foot , compared to the bffg and the nfg . in addition , the kruskal - wallis h test was used to analyze the difference in muscle tone and stiffness between the dominant leg of the bffg and nfg and the flexible flat - foot side of the offg . to investigate the differences in muscle tone and stiffness by foot type , the dominant leg of the nfg and bffg and the flexible flat - foot side of the offg ( one - side flexible flat foot ) were compared . in the measurement results , only the ta and mg stiffness of the nfg and bffg showed a significant difference ( p < 0.05 ) . although it was insignificant , the muscle tone and stiffness was highest in the bffg , followed by the offg and nfg . furthermore , in the nfg , the non - dominant leg showed greater muscle tone and stiffness , on average , than the dominant leg , although the difference was not significant . the comparison in muscle tone and stiffness between the normal foot side and the flexible flat - foot side in the offg showed no significant difference ( table 2table 2.muscle tone and stiffness on each foot typestypemusclesidemuscle tone ( hz)stiffness ( n / m)normal footrectus femorisdominant leg14.1 0.3245.5 9.0non - dominant leg14.0 0.2240.2 7.3tibialis anteriordominant leg19.0 0.8380.6 17.5non - dominant leg19.3 0.6393.5 14.3medial gastrocnemiusdominant leg15.6 0.4253.7 10.7non - dominant leg15.7 0.5254.3 10.9biceps femoris ( long head)dominant leg14.7 0.5236.7 15.8non - dominant leg15.1 0.7244.5 19.1both flexible flat footrectus femorisdominant leg15.3 0.4269.3 10.3non - dominant leg15.2 0.5264.1 13.1tibialis anteriordominant leg20.6 0.8436.7 17.7*non - dominant leg20.5 1.0450.5 26.1medial gastrocnemiusdominant leg16.6 0.3277.0 6.7non - dominant leg16.5 0.3279.4 6.8biceps femoris ( long head)dominant leg15.9 0.4274.8 12.8non - dominant leg16.0 0.3281.7 0.8421.9 27.9medial gastrocnemiusflexible flat foot15.8 0.5263.1 8.5normal foot15.6 0.5254.9 11.0biceps femoris ( long head)flexible flat foot15.3 * significant difference between normal foot and both flexible flat foot in tibialis anterior of dominant leg . significant difference between normal foot and both flexible flat foot in medial gastrocnemius of dominant leg ) . * significant difference between normal foot and both flexible flat foot in tibialis anterior of dominant leg . significant difference between normal foot and both flexible flat foot in medial gastrocnemius of dominant leg the flat foot causes generally negative changes to the body due to the structural deformity4 of the lower extremity joint , changes in muscle activity7,8,9 , biomechanical changes2 , and decreased muscular strength and balance16 . caillet et al.17 claimed that the stiffness of the ankle dorsiflexor was associated with difficulty during walking due to an asymmetric posture and the loss of balance and motor control . therefore , an examination of the stiffness of muscles in the flexible flat foot is a critical part of treatment and evaluation . in this study , a comparison of the muscle tone and stiffness of both lower extremity muscles by foot type indicated a significant difference in the stiffness of the ta and mg muscles of the dominant leg between the nfg and the bffg ( p < 0.05 ) . the muscle tone and stiffness was the highest in the bffg , followed by the offg and nfg , although the differences were insignificant . this means that the muscle tone and stiffness of the flat foot are higher than those of the normal foot . furthermore , a comparison of the muscle tone and stiffness of the normal foot side and the flexible flat - foot side in the offg revealed that the muscle tone and stiffness of the mg were higher on the flexible flat - foot side , whereas the muscle tone and stiffness of the rf , ta , and bf were higher on the normal foot side , although the differences were insignificant . it seems that the differences were insignificant because the muscle tone and stiffness were measured in a relaxation condition . the differences in muscle tone and stiffness could increase during physical activities such as standing or walking . in the case of the nfg , the muscle tone and stiffness of the rf were higher on average in the dominant leg , and those of the ta , mg , and bf were higher in the non - dominant leg , however , the differences were insignificant . the reason for this seems to be that the frequency of raising the dominant leg with the support of the non - dominant leg is higher . among the studies on emg , lee et al.4 compared the muscle activity between the normal foot and the flat foot when standing on one leg and found that the muscle activities of the mg , ta , and vastus medialis ( vl ) of the flat foot were lower , whereas the muscle activity of the rf of the flat foot was higher , however , the differences were insignificant . only the muscular activity of the abductor hallucis muscle of the normal foot was significantly higher than that of the flat foot , suggesting the importance of the intrinsic foot muscles . therefore , more research on the intrinsicfoot muscles is required in the future . in a study that analyzed the muscle activity of the flexible flat foot using the root mean square ( rms ) of the emg , vittore et al.18 mentioned that the ta needs to be strengthened during the rehabilitation of flat foot , because the more severe the flexible flat foot was , the lower the muscle activity of ta in the supine and orthostatic position became . in this study , however , the muscle tone and stiffness of the lower extremity muscles of the flat foot were higher compared to those of the normal foot . the reason for this seems to be that the lower muscle activity increased the muscle tone and stiffness . in this study , only the stiffness of the ta and mg muscles of the nfg and bffg showed significant differences , but on average , the muscle tone and stiffness of both the lower extremity muscles of the flexible flat foot were higher than those of the normal foot . these changes will be a negative factor in walking or other physical functions , and in cases of older age or injury to the lower extremity , the function of the lower extremity will become lower , and more neuromuscular control will be required . for the limitations of this study , the findings can not be generalized to all ages , because the subjects were in their 20s , and differences by gender were not examined . furthermore , this study could not be compared to many previous studies , because there were few previous studies about the muscle tone and stiffness of the flexible flat foot . however , the findings of this study reveal the need for an adjustment of the muscle tone and stiffness of a flexible flat foot during physical therapy , and the results of this study can be used as basic data for various physical therapies such as strengthening exercise , therapeutic massage , and myofascial release .

[ purpose ] the aim of this study was to examine differences in the muscle tone and stiffness of leg muscles according to types of flexible flat foot . [ subjects and methods ] for 30 subjects 10 in a normal foot group ( nfg ) , 10 in group with both flexible flat feet ( bffg ) , and 10 in a group with flexible flat feet on one side ( offg ) , myotonometry was used to measure the muscle tone and stiffness of the tibialis anterior muscle ( ta ) , the rectus femoris muscle ( rf ) , the medial gastrocnemius ( mg ) , and the long head of the biceps femoris muscle ( bf ) of both lower extremities . [ results ] in the measurement results , only the stiffness of ta and mg of the nfg and the bffg showed significant differences . the muscle tone and stiffness were highest in the bffg , followed by the offg and nfg , although the difference was insignificant . in the case of the offg , there was no significant difference in muscle tone and stiffness compared to that in the ngf and the bffg . furthermore , in the nfg , the non - dominant leg showed greater muscle tone and stiffness than the dominant leg , although the difference was insignificant . [ conclusion ] during the relax condition , the flexible flat foot generally showed a greater muscle tone and stiffness of both lower extremities compared to the normal foot . the stiffness was particularly higher in the ta and mg muscles . therefore , the muscle tone and stiffness of the lower extremity muscles must be considered in the treatment of flat foot .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC5192475

the genus streptococcus comprises 116 officially recognized species ( http://www.bacterio.net/ ) partially clustered into six species group ( pyogenic , anginosus , mitis , salivarius , bovis and mutans ) on the basis of systematic 16s rrna sequence study . the exact taxonomic classification of each species within this genus remains challenging , especially in the mitis group , as a result of a high genetic and phenotypic similarity shared within different species , in particular with different species sharing a 16s rrna sequence identity greater than 98.7% . because species belonging to this group gather together highly virulent species involved in pathologies as meningitis , endocarditis , pneumonia and low - pathogenic commensal species , the rapid identification of clinical isolates is mandatory . to reach this goal , several different molecular targets have been tested , including soda , rnpb , tuf and groel . , it has been validated by its use in the classification of different new streptococcal species , , . nowadays , the widespread use of matrix - assisted desorption ionization time of flight mass spectrometry ( maldi - tof ms ) in the clinical and research setting , coupled with the rapid development of next - generation sequencing technology , gives us a new and more complete insight into the taxonomy of the streptococcus genus . in this context , a polyphasic approach to describe new bacterial species , termed taxonogenomics , was proposed in our laboratory in 2004 , combining common phenotypic tests such as api strips , whole genomic sequencing and maldi - tof ms spectra . here we present a phenotypic and genetic comparison of 16s rrna genes , rpob genes as well as whole - genomic - level analysis which led us to propose the validation of streptococcus timonensis strain marseille - p2915 (= dsm 103349 = csur p2915 ) as a new member within the genus streptococcus . in the context of a study aimed at the description of different gut microflora levels by culturomics , a gastric lavage sample from a 60-year - old patient was collected during a gastroscopy performed for medical reasons ( severe anaemia ) . informed and signed consent , approved by the institut fdratif de recherche ifr48 ( faculty of medicine , marseille , france ) , under agreement 09 - 022 , was obtained from the patient . after collection , the sample was immediately placed in an antioxidant transport medium and plated within 2 hours . strain marseille - p2915 's first growth was obtained in april 2016 on 5% sheep 's blood enriched columbia agar medium ( biomrieux , marcy l'etoile , france ) under aerobic conditions at 37c . once isolated on pure culture , proteomic analysis was carried out with maldi - tof ms as previously described , using a microflex spectrometer ( bruker daltonics , bremen , germany ) . strain marseille - p2915 spectra was thus obtained , imported into the maldi biotyper software ( version 3.0 , bruker ) and processed by standard pattern matching ( with default parameter settings ) against the main spectra of 7567 bacteria . the comparison with the biotyper database spectra enabled the rapid matching of the analysed species with those present in the database . the resulting score , if > 2 , enabled the identification at the species level , while a score of < 1.7 did not enable any identification . to obtain the 16s rrna sequence of strain marseille - p2915 , pcr analysis was performed using a geneamp pcr system 2720 thermal cycler ( applied biosystems , bedford , ma , usa ) as previously described . the sequencing step was performed on an abi prism 3130xl genetic analyser capillary sequencer ( applied biosystems ) . chromas pro 1.34 software ( technelysium , tewantin , australia ) was used to fix the sequences , and a blastn ( basic local alignment search tool ) search was performed against the national center for biotechnology information ( ncbi ) database ( default parameters , uncultured / environmental sample sequences excluded ) . the rpob gene sequence of strain marseille - p2915 was obtained using the 1730_f and 3700_r primers as previously described , , , , , , , , , , , . then a blast search was performed against the ncbi database using default parameters . on the basis of the blast research results , 16s rrna reference sequences ( http://www.bacterio.net/streptococcus.html ) of the closest species with standing in nomenclature were downloaded from ncbi . because there is no officially recognized reference for rpob , gene sequences were downloaded directly from the ncbi database after the blastn search . sequences were then aligned using muscle v3.8.31 with default parameters , and phylogenetic inferences were obtained using the maximum - likelihood method with 1000 bootstrap replicates within mega6 software . enriched columbia agar ( biomrieux ) under anaerobic conditions using anaerogen ( oxoid , basingstoke , uk ) , microaerophilic ( campygen , oxoid ) and aerobic conditions . different growth temperatures ( 20 , 28 , 37 , 45 and 55c ) were also tested . the acceptance limit of salinity by strain marseille - p2915 was tested on columbia agar using 10 , 15 and 20% of nacl concentrations . moreover , seven phs were tested : 5.0 ; 5.5 ; 6.0 ; 6.5 ; 7.0 ; 7.5 and 8 . the gram coloration was performed using color gram 2 kit ( biomrieux ) and observed using the dm1000 photonic microscope ( leica microsystems , wetzlar , germany ) with a 100 oil - immersion objective lens . the ability to produce spores the motility test was performed by observing fresh colonies between blades and slats using a dm1000 photonic microscope ( leica microsystems ) with a 100 oil - immersion objective lens . transmission electron microscopic images were obtained using a tecnai g20 ( fei company , limeil - brevannes , france ) at an operating voltage of 200 kev . briefly , cells were fixed with 2.5% glutaraldehyde in 0.1 m cacodylate buffer for at least 1 hour at 4c . a drop of cell suspension was deposited for approximately 5 minutes on glow - discharged formvar carbon film on 400 mesh nickel grids ( fcf400-ni , ems , hatfield , pa , usa ) . the grids were dried on blotting paper , and cells were negatively stained for 10 seconds with 1% ammonium molybdate solution in filtered water at room temperature . a basic biochemical study was performed using the api gallery systems : api zym , api strep and api 50ch ( l medium ) according to the manufacturer 's instructions ( biomrieux ) . oxidase ( becton dickinson , franklin lakes , nj , usa ) and catalase assays ( biomrieux ) were done separately . the antibiogram profile of strain marseille - p2915 was obtained with the disk diffusion method following european committee on antimicrobial susceptibility testing ( eucast ) 2016 recommendations . tested antibiotics included : penicillin g 10 iu , amoxicillin 25 g , ceftriaxone 30 g , erythromycin 15 iu , rifampicin 30 g , sulfamethoxazole 23.75 g , trimethoprim 1.25 g , imipenem 10 g and vancomycin 30 g . cellular fatty acid methyl ester ( fame ) analysis was performed by gas chromatography mass spectrometry ( gc / ms ) . two samples were prepared with approximately 40 mg of bacterial biomass per tube collected from several 5% sheep 's blood enriched columbia agar plates . fames were separated using an elite 5-ms column and monitored by mass spectrometry ( clarus 500-sq 8 s , perkinelmer , waltham , ma , usa ) . a spectral database search was performed using ms search 2.0 operated with the standard reference database 1a ( national institute of standards and technology , gaithersburg , md , usa ) and the fame mass spectral database ( wiley , chichester , uk ) . after a pretreatment by a lysozyme incubation at 37c , dna was extracted on the ez1 biorobot ( qiagen , germantown , md , usa ) with a ez1 dna tissues kit . genomic dna ( gdna ) was quantified by a qubit assay with the high - sensitivity kit ( life technologies , carlsbad , ca , usa ) to 46 ng/l . gdna of strain marseille - p2915 was sequenced with the miseq technology apparatus ( illumina , san diego , ca , usa ) with the mate pair strategy . the gdna was barcoded in order to be mixed with 11 other projects with the nextera mate pair sample prep kit ( illumina ) . the mate pair library was prepared with 1.5 g of gdna using the nextera mate pair illumina guide . the pattern of the fragmentation was validated on an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) with a dna 7500 labchip . the dna fragments ranged in size from 1.5 to 11 kb with an optimal size of 6.920 kb . no size selection was performed , and 678.5 ng of tagmented fragments were circularized . the circularized dna was mechanically sheared to small fragments with optima on a bimodal curve at 675 and 1445 bp on the covaris s2 device in t6 tubes ( covaris , woburn , ma , usa ) . the library profile was visualized on a high sensitivity bioanalyzer labchip ( agilent technologies ) , and the final concentration library was measured at 42.2 after a denaturation step and dilution at 15 pm , the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell . automated cluster generation and sequencing run were performed in a single 39-hour run at a 2 151 bp read length . total information of 7.9 gb was obtained from a 863k / mm cluster density with a cluster passing quality control filters of 94% ( 15 627 000 passing filter paired reads ) . within this run , the index representation for streptococcus timonensis strain marseille - p 2915 was determined to be 11.94% . the 1 865 795 paired reads were trimmed , then assembled in four scaffolds . open reading frames ( orfs ) were predicted using prodigal ( http://prodigal.ornl.gov/ ) with default parameters . the predicted bacterial protein sequences were searched against the genbank and clusters of orthologous groups ( cogs ) databases using blastp . the trnas and rrnas were predicted using the trnascan - se and rnammer tools , respectively . signal peptides and number of transmembrane helices were predicted using signalp and tmhmm , respectively . orfans were identified if their blastp e - value was lower than 1e-03 for an alignment length greater than 80 amino acids . if alignment lengths were smaller than 80 amino acids , an e value of 1e-05 was used . artemis and dna plotter were used for data management and visualization of genomic features , respectively . the mauve alignment tool ( version 2.3.1 ) was used for multiple genomic sequence alignment . the mean level of nucleotide sequence similarity at the genome level between strain marseille - p2915 and other bacteria ( streptococcus oralis strain atcc 35037 ( admv00000000.1 ) ; streptococcus infantis atcc 700779 ( aevd00000000.1 ) ; streptococcus pseudopneumoniae atcc baa960 ( aics00000000.1 ) ; streptococcus sanguinis sk1 ( cp000387.1 ) ; streptococcus mitis atcc 49456 ( aedx00000000.1 ) ; streptococcus parasanguinis atcc 15912 ( cp002843.1 ) ; streptococcus tigurinus strain az_3a ( aoru00000000.1 ) ; streptococcus pneumoniae strain r6 ( ae007317.1 ) ; streptococcus dentisani strain 7747 ( cauk00000000.1 ) ) was estimated by average genomic identity of orthologous gene sequences ( agios ) software . overall , this software combines two other software packages : proteinortho ( to detect orthologous proteins between genomes compared two by two , then to retrieve the corresponding genes ) and the needleman - wunsch global alignment algorithm ( to determines the mean percentage of nucleotide sequence identity among orthologous orfs ) . to evaluate genomic similarity within the strains , we also performed digital dna - dna hybridization ( http://ggdc.dsmz.de/ ) , which exhibits high correlation with dna - dna hybridization . after three failed identification attempts with maldi - tof ms , the reference spectrum of strain marseille - p2915 ( fig . dentisani strain 7747 and 98.8% with streptococcus tigurinus strain az-3a ( fig . 2 ) . because the 16s rrna phylogenetic analysis did not distinguish within different species belonging to the streptococcus mitis group , the rpob gene was sequenced , resulting in a 92.6% sequence identity with the closest streptococcus with an available rpob sequence , streptococcus infantis strain atcc 700779 ( fig . 3 ) . a gel view comparing the spectrum of strain marseille - p2915 with other streptococcaceae species is shown in fig . the streptococcus timonensis strain marseille - p2915 16s rrna accession number from european molecular biology laboratory ( embl)-european bioinformatics institute ( ebi ) is lt576411 . the maldi - tof ms reference spectrum is available online and in the public domain ( http://www.mediterranee-infection.com/article.php?laref=256&titre=urms-database ) . on sheep 's blood enriched columbia agar , after 48 hours of aerobic incubation , cells formed 0.5 to 1 mm punctiform , greyish , -hemolytic colonies with undulated edges . growth was achieved at all tested phs ( 5.0 ; 5.5 ; 6.0 ; 6.5 and 7.0 ; and 7.5 and 8) , whereas no growth was registered in any salt - containing medium ( > 5 g / l of nacl ) . growth was obtained from 20 to 37c under aerobic , anaerobic , and microaerophilic conditions , whereas no growth was obtained at 45 or 55c . 4(a ) ) with a mean diameter of 0.6 m ( range , 0.40.8 m ) ( fig . table 1 summarizes the classification and main features of strain marseille - p2915 . using an api 20 strip gallery , negative reactions were recorded for : voges - proskauer reaction , hippuric acid hydrolysis , esculin hydrolysis , pyrrolidinyl arylamidase , -galactosidase , -glucuronidase , -galactosidase , alkaline phosphatase and arginine hydrolase ; fermentation reactions were negative for d - ribose , l - arabinose , d - mannitol , d - sorbitol , d - lactose , d - trehalose , inuline and d - raffinose . using an api zym strip , positive reactions were observed for esterase ( c4 ) and leucine arylamidase . negative reactions were recorded for alkaline phosphatase , acid phosphatase , -galactosidase , -galactosidase , -glucosidase , n - acetyl--glucosaminidase and naphthol - as - bi - phosphohydrolase , lipase ( c14 ) , esterase lipase ( c8 ) , valine arylamidase , cystine arylamidase , trypsin , -chymotrypsin , -glucuronidase , -glucosidase , -fucosidase and -mannosidase . using an api 50 ch strip , positive reactions were observed for d - glucose , d - fructose , d - maltose and d - saccharose . negative reactions were recorded for d - ribose , l - arabinose , d - xylose , d - mannose , inositol , glycerol , arbutin , d - tagatose , d - arabinose , d - fucose , n - acetylglucosamine , salicin , d - cellobiose , d - trehalose , amidone , erythritol , l - xylose , d - adonitol , methyl- d - xylopyranoside , d - galactose , l - sorbose , l - rhamnose , dulcitol , d - sorbitol , methyl-d - mannopyranoside , methyl-d - glucopyranoside , esculin , d - lactose , d - melibiose , inulin , d - melezitose , d - mannitol , amygdalin , d - raffinose , glycogen , xylitol , gentiobiose , d - turanose , d - lyxose , l - fucose , d - arabitol , l - arabitol , potassium gluconate , potassium 2-ketogluconate and potassium 5-ketogluconate . strain marseille - p2915 resulted susceptible to penicillin , ceftriaxone , imipenem , vancomycin and rifampicin . biochemical characteristics that differentiate strain marseille - p2915 from other related species within the family streptococcaceae are summarized in table 2 . abundant fatty acids were either saturated or unsaturated . moreover , several branched fatty acids were also described at lower abundances . a complete report of fatty acid analysis of strain marseille - p2915 is reported in table 3 . the draft genome of the strain marseille - p2915 is 1 925 331 bp long ( fig . it is composed of four scaffolds ( comprising four contigs ) . among the 2056 predicted genes , 1974 were protein - coding genes and 82 were rnas ( six genes are 5s rrna , six genes are 16s rrna , six genes are 23s rrna and 64 genes are trna genes ) . a total of 1436 genes ( 72.75% ) were assigned a putative function ( by cogs or by nr blast ) . genomic statistics are reported in table 4 , while table 5 lists the distribution of genes into cogs functional categories ( fig . the draft genome sequence of strain marseille - p2915 ( 1925 mb ) is smaller than that of streptococcus pseudopneumoniae ( 2086 mb ) , streptococcus tigurinus ( 2185 mb ) , streptococcus pneumoniae ( 2161 mb ) , streptococcus parasanguinis ( 2154 mb ) and streptococcus sanguinis ( 2303 mb ) , but larger than that of streptococcus dentisani ( 1884 mb ) , streptococcus infantis ( 1857 mb ) , streptococcus mitis ( 1916 mb ) and streptococcus oralis ( 1914 mb ) . the g+c content of s. timonensis ( 38.5% ) is smaller than that of s. dentisani ( 41.1% ) , s. pseudopneumoniae ( 39.8% ) , s. infantis ( 38.9% ) , s. mitis ( 41.1% ) , s. tigurinus ( 40.3% ) , s. pneumoniae ( 39.7% ) , s. parasanguinis ( 41.7% ) , s. sanguinis ( 43.2% ) and s. oralis ( 41.4% ) . the gene content of strain marseille - p2915 ( 1.974 genes ) is smaller than that of s. pseudopneumoniae ( 2.113 ) , s. tigurinus ( 2.114 ) , s. pneumoniae ( 2.125 ) , s. parasanguinis ( 2.022 ) and s. sanguinis ( 2.268 ) , but larger than that of s. dentisani ( 1.797 ) , s. infantis ( 1.876 ) , s. mitis ( 1.793 ) and s. oralis ( 1.847 ) . within species with standing in nomenclature , agios values ranged from 82.23% between s. mitis and s. oralis to 58.20% between s. sanguinis and s. infantis ( table 6 ) . genome - to - genome distance calculator ( ggdc ) values ranged from 59.1 2.8 between s. pneumoniae and s. pseudopneumoniae to 23.1 2.3 between s. sanguinis and s. parasanguinis ( table 7 ) . compared to the closest phylogenetic neighbour according to rpob tree ( streptococcus infantis atcc 700779 t , fig . 3 ) , the probability that both are same species is lower than 5% ( 1.35% , ggdc 2.1 formula 2 , http://ggdc.dsmz.de/ ) . distribution of functional classes of predicted genes according to the cogs categories was realized for the closest species of strain marseille - p2915 ( fig . a strong argumentation to support the recognition of strain marseille - p2915 as a new species within the streptococcus genus is brought by the low value of ggdc ( formula 2 ) obtained with other close species ( highest value 37.4% with streptococcus infantis , probability that both are the same species < 5% ) , particularly when compared to the high value obtained within different streptococcal species with standing in nomenclature ( 59.1% between streptococcus pneumoniae and streptococcus pseudopneumoniae and 48.5% between streptococcus mitis and streptococcus pseudopneumoniae ) ( table 7 ) . other evidence comes from the phenotypic analysis obtained by the api strips , which showed a unique enzymatic profile ( table 2 ) compared to the eight most closely related species . in the family streptococcaceae and in particular within the streptococcus mitis group , it is a common feature to share a high 16s rrna similarity with other streptococcal species ( table 8) . another important point that differentiates strain marseille - p2915 from streptococcus dentisani and streptococcus tigurinus ( the16s rrna closest species with standing in nomenclature ) is the difference in g+c content ( 2.6 and 1.8% , respectively ) . to our knowledge , there is no officially recognized threshold for rpob gene sequence similarity to delineate a new species , but the 92.6% value obtained with streptococcus infantis is well below the 96.7% of the rpob 's gene similarity between streptococcus oralis ( strain kccm 41567 ) and streptococcus cristatus ( strain cip 56.62 ) or the 96.4% gene similarity between streptococcus salivarius ( strain 735 - 09 ) and streptococcus thermophilus ( strain cip 105446 ) ( table 9 ) . on the basis of the phenotypic , phylogenetic and genomic analyses , we propose the validation of streptococcus timonensis sp . nov . within the family streptococcaceae . strain marseille - p2915 is the type strain , and it was isolated from human stomach . s. timonensis is a nonmotile , non - spore - forming , facultative anaerobe and gram - positive coccus . growth is achieved under aerobic , anaerobic and microaerophilic atmospheres in a temperature range of 20 to 37c and at an optimum temperature of 37c . after 48 hours of aerobic incubation on 5% sheep 's blood enriched columbia agar , colonies are pinpoint , greyish and -haemolytic , with undulated edges and with a diameter of 0.5 to 1 mm . streptococcus timonensis strain marseille - p2915 exhibits positive reactions for leucyl - aminopeptidase , esterase c4 and leucine arylamidase . it is able to ferment starch , d - glucose , d - fructose , d - maltose and d - saccharose . strain marseille - p2915 was found to be susceptible to penicillin , ceftriaxone , imipenem , vancomycin and rifampicin . the 16s rrna sequence and whole - genome shotgun sequence are deposited in embl - ebi under accession numbers lt576411 and fmix01000000 , respectively . the type strain is marseille - p2915 (= csur p2915 = dsm 103349 ) , and it was isolated from a human stomach sample in marseille , france .

strain marseille - p2915 t , a gram - positive , facultative anaerobic and nonmotile coccus , was isolated from the gastric lavage of a patient with severe anaemia . the 16s rrna and rpob gene comparison exhibited a sequence identity of 98.7 and 92.6% with streptococcus infantis strain jcm 10157 t , respectively , collocating it within the streptococcus mitis group . on the basis of phenotypic and genomic analysis , we propose the validation of the type strain streptococcus timonensis sp . nov . marseille - p2915 t (= dsm 103349 = csur p2915 ) .

Introduction Materials and Methods Results Discussion Conclusion

PMC4064694

perhaps no amino acid in the bacterial photosynthetic reaction center ( rc ; figure 1a ) has been scrutinized more than the tyrosine situated near the dimeric bacteriochlorophyll ( bchl ) primary electron donor ( p ) and the monomeric bchl initial electron acceptor ( ba ) on the a - side of the rc . this residue is tyr m208 in blastochloris viridis and rhodobacter capsulatus and tyr m210 in rhodobacter sphaeroides . both theoretical work and experiments on mutants have led to consensus that this conserved residue is a significant contributor to extremely rapid and efficient initial a - side charge separation . interestingly , there are no potential hydrogen bond partners available to tyr m208 , which is unique among the roughly 28 ( depending on species ) tyrosines in the rc . consistent with this , a unique chemical shift is observed in the c nmr spectrum for the 4-c - carbon ( the carbon bonded to the oh ) of this unique tyr . replacing the phe residue at the c2-symmetry related site , l181 near bb , with a tyr often has been a design element in eliciting electron transfer ( et ) from p * to the bacteriopheophytin ( bph ) on the b - side ( hb ) . however , p * phb conversion is at best more than an order of magnitude slower than initial charge separation on the a - side . the rc et dynamics have long been modeled in the context of the state free - energy relationships shown in figure 1b . here pba lies between p * and pha in free energy with the analogous b - side states placed somewhat higher and in particular with pbb above p*. these orderings are consistent with a general picture of rapid ( 13 ps ) steps of et from p * to ba and then to ha with details and mechanisms under continuing study . electrostatic calculations by alden et al . suggested that a specific orientation of the hydroxyl group of tyr m208 having the oh pointing toward ba stabilizes pba . in addition , this was found to be the strongly preferred orientation over a second , where the oh is rotated 180 about the c o bond and which , in comparison , destabilizes pba . the calculations predicted that the stabilization of pba induced by the oh of tyr m208 in the preferred orientation is a significant 200 mev . note that a possible corollary of this sensitivity is the expectation that the initial et reactions may be inhomogeneous , depending on the timescale of the oh dipole motion , and in fact nonsingle exponential kinetics are commonly observed for these reactions . sphaeroides x - ray crystal structure ( protein data bank : 2j8c ) . view of the environment of ba as determined in the wild - type rb . note that the numerical values and amino acids of the l and m polypeptides correspond to those of rcs from rb . sphaeroides ) is a ligand to the m - bchl macrocycle of p ( not shown ) . the preferred orientation of the oh dipole is not known experimentally given the current resolution of even the best x - ray structures . therefore , we consider the simulations of alden et al . as providing a working hypothesis for contemplating further protein engineering . an expanded view of the region around tyr m208 and ba is illustrated in figure 2 in a homology model of the rb . therefore , we replaced isoleucine m204 with glutamic acid and glutamine to introduce a potential hydrogen bond acceptor for the oh of tyrosine m208 . as reported previously , to our surprise , the m204q mutation leads to complete loss of ba . ultrafast measurements on m204q rcs gave the expected results of substantial deactivation of p * to the ground state , no a - side et , and a small yield of b - side charge separation , p * phb , that was increased upon changing phe at l181 to a tyr . we report here that substitution of m204 with glutamic acid leads to altogether different results . modeling of glu at m204 using pymol suggests that if a hydrogen bond to the tyr m208 hydroxyl group is formed , the hydroxyl group would be held in an orientation that disfavors p * pha et , noting , though , that the energetic consequences of a hydrogen - bonded tyrosine oh in this orientation may be different from the nonhydrogen - bonded case of the simulations ( in ref ( 8) ) . with the use of uniformly [ 4-c]-labeled tyr , the c nmr spectrum reveals that tyr m208 in m204e no longer has the unique chemical shift associated with tyr m208 not being hydrogen bonded , as in wild - type , consistent with the introduction of a hydrogen bond . ultrafast measurements of m204e rcs reveal that reduction of ha is about a factor of 10 slower than in wild - type and that the room temperature p * decay kinetics are unusually homogeneous . capsulatus rc was changed to a glutamic acid by site - directed mutagenesis , as described previously , using the quikchange mutagenesis kit ( stratagene ) . rc isolation and purification followed published procedures utilizing 0.1% deriphat 160-c for protein solubilization for transient absorption or 0.1% n , n - dimethyldodecylamine n - oxide ( ldao ) for nmr and mass spectrometry . measurements were conducted on rcs in 10 mm tris ( ph 7.8 ) and 0.1% deriphat 160-c , unless noted otherwise . wild - type and m204e rcs were extracted using acetone / meoh ( 7:2 v / v ) and the bchl : bph pigment ratio determined spectroscopically , as according to van der rest et al . the pigment content was also assayed by hplc because of the unusual results reported below . for this , the acetone / meoh extract was dried under a stream of nitrogen and dissolved in acetonitrile / ethyl acetate / meoh / water ( 24:20:47:9 v / v ) . after filtration through a 0.22 m filter , the pigments were analyzed using an agilent infinity 1260 hplc setup equipped with an analytical c18 column ( spherisorb c18 , 4.6 250 mm , 5 m ) and a multiple wavelength detector ( 1260 mwd vl ) . pigments were detected at 770 nm using isochratic elution with acetonitrile / ethyl acetate / meoh / water ( 24:20:47:9 v / v ) at a flow rate of 1 ml / min . capsulatus cells were grown in a bioreactor ( new brunswick scientific , bioflo3000 , 5 l ) under controlled ph and oxygen levels in the dark to maximize rc yield . to incorporate labeled [ 4-c]tyr , a modified rcvpy media was used that substituted peptone and yeast extract with a defined amino acid composition , as according to raap et al . a small amount of antifoam 204 ( sigma - aldrich ) was added before inoculation . the optimal growth conditions were as follows : the culture ph was maintained at 6.8 with a titration solution containing 4 m dl - malic acid with an agitation rate of 500 rpm at a temperature of 34 c . the atmosphere was controlled using a nitrogen / air mixture ( 95:5 ) , and the dissolved oxygen level was monitored using an oxyprobe . the oxyprobe was calibrated using a two - point calibration with 100% air and 100% nitrogen as reference values . the incorporation of labeled [ 4-c]tyr ( cambridge isotopes ) was quantitated by performing lc samples were subjected to in - solution digestion using urea and proteasemax ( promega ) and a combination of trypsin/-chymotrypsin to cut out the peptide of interest that contains tyrosine m208 ( fragment m200-m208 ) . briefly , 50 g protein was precipitated with 4 volumes of 20 c acetone and incubated at 80 c for 20 min . after centrifugation , the white pellet ( blue - green pellet in case of m204e ) was rinsed with 300 l acetone and allowed to air - dry for 5 min . then , 15 l of 8 m urea and 20 l of 0.2% proteasemax were added , and the solution was vortexed for 5 min to solubilize the pellet . the mixture was placed in a shaker at 25 c and 140 rpm for 1 h. after solubilization , the sample was reduced with dithiothreitol , alkylated with iodoacetamide , and digested using trypsin/-chymotrypsin ( 1.8 g in total ) and proteasemax at 37 c for 3 h. the digestion was stopped by addition of 2 l 25% trifluoroacetic acid . to remove particles , peptides were purified using a stage - tip c18 column . in the first step , the column was washed with acetonitrile and in the second step with water/0.1% formic acid . mass spectra were obtained on a thermo ltq - orbitrap velos mass spectrometer equipped with a nanohplc ( c18 , 100 m i.d . the peptides were eluted using a gradient running from water ( containing 0.1% formic acid ) to acetonitrile over the course of 60 min ( 340% for 40 min , 4065% for 10 min , 6595% for 5 min , and 953% for 5 min ) . the mass accuracy of the orbitrap and of the ion trap are below 2 ppm and around 250 ppm , respectively . labeled rcs ( 25 mg ) were reconstituted into lipids and lyophilized to ensure a homogeneous environment and a dry sample following described procedures.l--phosphatidylcholine ( egg pc ) was dissolved in 10 mm tris ph 8 buffer containing 0.58% n - dodecyl - - d - maltoside . twenty - five milligrams rc was added and the total volume was 5 ml ( 1:100 mol % of protein / lipid ) . biobeads - sm2 were washed vigorously with meoh , water , and finally 10 mm tris , ph 8 . the beads were added ( 110 mg , 5-fold w / w excess over detergent ) , and the solution was stirred overnight in the dark at 4 c to remove detergent . the next day , another 110 mg of biobeads - sm2 were added and the solution was stirred for another 3 h. the beads were removed by filtration , trehalose was added as cryoprotectant to a final concentration of 25 mm , and the solution was stirred at 4 c for 15 min . the sample was slowly frozen at 20 c , and then transferred to 80 c and finally frozen in liquid nitrogen . solid - state nmr experiments were performed with an 89 mm wide - bore varian / agilent magnet at 11.7 t ( 125.49 mhz for c ) , varian console , and a home - built four - frequency transmission - line probe with a 13.6 mm long , 6 mm inner - diameter sample coil and a revolution nmr mas vespel stator . samples were spun at 8000 2 hz , using a varian mas control unit and cooled with 30 c nitrogen gas . the proton - carbon matched cross - polarization transfer was performed at 83 khz , and the recycle delay was 2 s. the chemical shift scale was referenced to external adamantane . ultrafast transient absorption ( ta ) measurements were carried out on an apparatus that utilizes 120 fs excitation and white - light probe flashes delivered at a repetition rate of 10 hz . in order to ensure that fresh sample was interrogated on each excitation flash , rcs were flowed from a small reservoir through a 2 mm path length cell used for the pump / probe interrogation . the rcs were kept at 10 c by use of an ice bath to cool the reservoir . the ta data were analyzed using origin ( microcal ) and surface explore ( ultrafast systems ) . the p / p midpoint potential was obtained using a sec - c thin layer quartz glass cell ( basi ef-1358 ) , platinum counter electrode ( basi ef-1356 ) , a ag / agcl double junction reference electrode ( basi mf-2079 , mf-2030 ) and ferricyanide as the oxidizing species . the reference electrode was calibrated using standard solutions . the titration starting conditions were 750 l of 20 m rcs and 4 mm potassium ferrocyanide ( to ensure initial fully reduced rcs ) in 10 mm tris , ph 7.6 , and 0.1% deriphat . small increments ( starting with 0.25 l and gradually increasing to 10 l as the titration proceeded ) of stock 1 m potassium ferricyanide in 10 mm tris , ph 7.6 , and 0.1% deriphat were added directly to the sample in the cell and mixed by gentle pipetting several times over the course of 10 min . the absorbance at 865 nm was then measured ( shimadzu uv-1800 ) and the potential ( voltage ) recorded . the titrations were stopped when addition of ferricyanide no longer altered the measured potential or when the absorption of the ferricyanide began to interfere with reading the rc absorbance ( generally when approaching 50 l total ferricyanide stock added ) . in order to work with the minimal amount of [ 4-c]tyr - labeled media , cell growth of rb . viridis have shown that rc gene expression can be enhanced by growing cells under microaerophilic conditions in the dark . a series of experiments determined the optimum dissolved oxygen level for maximum protein expression . holding the dissolved oxygen level constant around 2% for the last few hours before harvesting cells around od660 5 gave good protein expression ( figure s1 of the supporting information ) , about a 3-fold increase in protein yield ( > 60 mg purified protein per 4 l growth medium ) . to determine the amount of [ 4-c]tyr at position m208 incorporated under our conditions , lc ms / ms was performed on trypsin/-chymotrypsin - digested rc . to our knowledge , mass spectrometry of fragments of bacterial rc proteins has not been previously reported and a more detailed analysis can be found elsewhere . one problem is the hydrophobicity of large stretches the rc , which makes proteolytic digestion difficult in the absence of detergent . in previous work on rcs , detergent was removed by precipitation , and the pellet was treated with 8 m urea . digestion with trypsin or -chymotrypsin yielded only low sequence coverage for h , l , and m subunits ( 54% h , 14% m , and 10% l with trypsin ; 15% h , 15% m , and 18% l with -chymotrypsin ) . in the present study , the pellet was solubilized after detergent removal using proteasemax , which solubilizes during proteolytic digestion , resulting in much higher sequence coverage , in particular for the hydrophobic l and m subunits ( 60% h , 54% m , and 41% l ) . for our purpose , a combination of trypsin/-chymotrypsin was used because this treatment releases a peptide corresponding to residues m200-m208 ( sequence hglsiaaly ) . both labeled and unlabeled wild - type rcs were digested and are compared in figure s2 of the supporting information . the peptide hglsiaaly has a m / z value of 473.76 for the doubly charged ion . comparison with the [ 4-c]tyr labeled wild - type shows that the whole isotope pattern is shifted by m / z = 0.5 , indicating > 99% incorporation of label ( the mass accuracy of orbitrap mass analyzers is < 2 ppm ) . sphaeroides rcs , where a high concentration of [ 4-c]tyr in the media was found to be necessary to compete with tyrosine biosynthesis and ensure quantitative incorporation of label into the rc . in the previous studies , total hydrolysis of the rc was performed to quantify total [ 4-c]tyr incorporation , whereas our approach provides a direct method to quantify site - specific labels using lc ms / ms . nmr spectroscopy is a useful technique to monitor the protonation and hydrogen bond status of the tyrosine hydroxyl group using the chemical shift of [ 4-c]tyr , as shown in numerous studies . figure 3 shows the c cpmas nmr spectrum of [ 4-c]tyr - labeled wild - type and m204e rcs obtained at 230 k. the rb . capsulatus rc contains 31 tyrosines , and this accounts for the major and unresolved [ 4-c]tyr peak between 154 and 158 ppm , typical for protonated and hydrogen - bonded tyr . in wild - type , a small , well - resolved peak this spectrum is similar to that found for [ 4-c]tyr - labeled wild - type rcs from rb . sphaeroides , where this peak could be attributed to tyr m210 ( equivalent to m208 in rb . notably , tyr m208/m210 is the only tyrosine that is not hydrogen bonded and the upfield shift is typical of this effect . the x - ray structure of rb . sphaeroides rcs confirms that there is no hydrogen bond acceptor in close proximity to the tyr m210 hydroxyl group . note that there may also be some contribution to this shift from the ring current of ba . c cpmas solid - state nmr of [ 4-c]tyr labeled rcs at t = 230 k. the unique resolved peak at 153 ppm ( ) in wt ( wild - type ) rcs is assigned to the 4-c carbon ( * ) of tyr m208 . it is shifted back into the poorly resolved broad band corresponding to all the other hydrogen bonded tyrosines in the m204e mutant . the c nmr spectrum of m204e with [ 4-c]tyr ( figure 3 ) clearly shows a significant change compared with wild - type . the unique upfield - shifted peak at 153 ppm is absent in m204e , apparently shifted back into the unresolved peak containing all the other hydrogen - bonded tyrosine residues in the rc . this result suggests that the introduced glutamic acid at m204 creates a hydrogen bond to the hydroxyl group of tyr m208 as designed . as noted above , the m204q mutant assembles without ba , as reflected in the absorption spectrum and determined by analysis of the pigment content . as shown in figure 4 , the 77 k absorption spectrum of m204e rcs has two bands near 800 nm that are assigned to ba and bb . in the wild - type spectrum in figure 4 , the qy bands of ba and bb are not resolved ; however , different detergent or buffer conditions and some mutations also lead to partial resolution of these bands . the rc pigment contents of wild - type and m204e rcs were assayed via the standard spectroscopic method . following extraction of rcs with acetone : meoh ( 7:2 , v / v ) , the solution is filtered before acquiring the uv / vis / nir spectrum in order to minimize the contribution of protein light - scattering . bchl / bph ratios of 1.4 for m204e and 1.9 for wild - type were determined , indicating extraction of only 3 bchls from the m204e rc . the same results were obtained if instead of filtering the extraction solution it was centrifuged for 1 min in a benchtop unit . confirming incomplete extraction of pigment from m204e rcs , the pelleted residue from m204e is blue - green , whereas for wild - type it is pale yellow ( figure s3 of the supporting information ) . attempts to extract a ( presumed ) bchl pigment from the m204e pellet using a variety of solvents were not successful . low - temperature uv / vis / nir spectra obtained at 77 k. the spectra are normalized at 366 nm for comparison . fresh acetone / meoh extract was dried under a stream of nitrogen and dissolved in the solvent mixture used for hplc analysis ( see experimental section ) . figure s4a of the supporting information shows hplc traces recorded at 770 nm . to make an absolute comparison , the analysis was performed on the same concentrations of wild - type , m204e , and a 1:1 mixture of both . the traces are very similar , and there is no difference in retention times between wild - type and mutant . the only difference is the intensity of the band around 7 min , which can be attributed to bchl . the ratio of the integrated peak areas for wild - type , m204e , and the 1:1 mixture is 4:2.7:3.3 , again suggesting incomplete extraction of bchl . the amount of extracted bph is the same for wild - type , mutant , and mix ( at 16 min ) . extraction methods using different solvents or solvent mixtures did not change the observation that one bchl is not extracted from m204e . in an earlier study on a rc mutant of rb . sphaeroides , in which isoleucine l177 ( c2-symmetry partner to m204 ) was substituted by histidine , it was proposed that one bchl might be covalently attached to the protein based on a colored polypeptide band in sds page . for the m204e mutant , no colored polypeptide band is seen in sds page and no chromophore absorption was observed in uv / vis / nir spectra from parts of the gel that were cut out ( figure s4b of the supporting information ) . lc ms / ms analysis of m204e and wild - type was also used to investigate possible covalent attachment of a bchl . the mass spectra of peptide m200-m208 ( hglseaaly ) show no evidence for covalent attachment of bchl ( figure s5 of the supporting information ) . however , the blue - green pellets for m204e ( compared to pale yellow for wild - type ) obtained during the course of processing rcs for both the uv / vis / nir spectroscopic , and the mass spectral pigment assays provide visual evidence that a chromophore is not being solubilized , consistent with the low bchl content obtained from the acetone : methanol extraction method . the time - resolved measurements presented in the next section give no indication of the absence of a pigment from m204e rcs and should be sensitive to such a deficiency . in order to quantify redox potentials of the special pair ( p ) the values obtained are 490 9 mv and 498 3 mv for wt and m204e , respectively . this indicates that the free energies of the charge - separated states ( e.g. , pba ) in m204e are not altered due to a change in the redox potential of the bchl dimer p. figures 5 and 6 show the ta spectra of m204e rcs in the near - infrared and visible regions and kinetic data and fits at select key wavelengths . the data in figures 5a and 6 ( panels a and b ) were obtained with direct excitation of p at 865 nm , and the data in figure 5b were acquired using excitation at 595 nm . the 0.50.6 ps spectra in figures 5 and 6 are that of p * and are identical to p * spectra reported previously . on the red side of the bleaching of the 865 nm band of p , the ta spectra are dominated by stimulated emission from p * with only the far red - edge ( 900920 nm ) of p bleaching observed in the spectra acquired at 100 ps and longer times in figure 5a . decay of the stimulated emission averaged over the 10 nm interval 920930 nm is shown in the inset of figure 5a . this interval spans an 925 nm isosbestic point in the p bleaching spectrum ( see e.g. , figure 5b inset ) . the solid line in figure 5a inset is the fit to the convolution of the instrument response plus one exponential plus a constant ( fit returns zero for the constant ) , giving a p * lifetime of 38 2 ps . there is no indication of a second longer component to the p * stimulated emission decay at this key wavelength interval ( or of a shorter component either ) . to this point , fitting the data to a function with two exponentials does not yield meaningful results , giving , for example , 38 ps for the values of both components global fitting of the data encompassing the entire spectral region shown in figure 5a returns 36 2 ps for the p * lifetime . a transient absorption band at 1017 nm that would indicate formation of a bchl anion ( e.g. , ba ) is not resolved to within a few percent yield . ta spectra and kinetics acquired with 120 fs excitation flashes at ( a ) 865 nm or ( b ) at 595 nm . in both ( a and b ) , the kinetic data ( ) are the absorption changes averaged over the 920930 nm interval of the spectra shown in that panel . the solid lines through the kinetic data are fits to the convolution of the instrument response plus one exponential plus a constant . the time constant ( p * lifetime ) measured in the 920930 nm interval is 38 2 ps ( panel a , inset ) , 38 1 ps ( panel b ) , and 36 2 ps from global analysis of the spectral evolution at all wavelengths shown . ( a ) ta spectra of m204e acquired at the times indicated following 120 fs excitation flashes at 865 nm . ( b ) kinetics averaged between 539 and 545 nm ( ) and fit to the instrument response plus three exponentials plus a constant . the resultant time constants are 35 2 ps , 238 10 ps , and 3 ns ( fixed ) . figure 5b inset shows time - resolved ta spectra acquired using 595 nm excitation flashes which allow probing the entire long - wavelength ( qy ) absorption band of p. the decay kinetics in the same 920930 nm interval are shown in the main panel of figure 5b . the solid line is again a fit to the convolution of the instrument response plus one exponential plus a constant . the p * lifetime returned from the fit is 36 1 ps and again to within a few percent , there is no indication of a second longer component to the decay kinetics at this key isosbestic point . the spectra in figure 5b inset show , on the blue - side of the p bleaching where stimulated emission contributes minimally , that 2025% of p bleaching has decayed at 3 ns . some of this occurs as p * decays , indicating internal conversion of p * to the ground state , and some of the p bleaching decay occurs on the nanosecond timescale and is associated with decay of phb , as will be described below . the ta spectra in the visible region acquired using 865 nm flashes are shown in figure 6a . the spectrum of p * at 0.5 ps is identical to that reported previously for wild - type and numerous mutants . the spectrum at 95 ps ( nearly three 3 multiples of the p * lifetime ) displays features associated with a mix of pha and phb . the expected wavelengths for bleaching of the qx absorption bands are 542/543 nm ( ha ) and 527/528 nm ( hb ) . both such bleaching features are in evidence in the spectrum at 95 ps , with ha bleaching dominating . the peaks of the broad anion bands of ha and hb are known to occur at 665 nm ( ha ) and 640 nm ( hb ) . again , the 95 ps spectrum displays anion absorption indicative of a mix of species but dominated by ha . at 3 ns , the spectrum has largely the characteristics of pqa ( and pqb ) . the data averaged between 539 and 545 nm , encompassing the maximum of the ha bleaching , and fit to the convolution of the instrument response plus three well - separated exponentials plus a constant are shown in figure 6b . this fit returns values of 35 2 ps ( assigned to p * decay ) and 238 10 ps ( assigned to pha decay ) , with a 3 ns component fixed for phb decay . ( previous work with mutant rcs where phb is formed and where there are no mutations in the immediate hb and qb sites has found that the phb lifetime is 2 or 4 ns in the presence or absence of qb , respectively . fixing this value in the present work at either 2 or 4 ns does not significantly alter the fit results ; the 3 ns average value was chosen for convenience and since the qb occupancy is unknown . ) a global fit of the data between 480 and 720 nm to the same function returns values of 36 2 ps and 230 15 ps with , again , the third exponential ( of small amplitude ) fixed at 3 ns . the 230 ps lifetime of pha is identical to that measured for pha pqa et in wild - type rcs in deriphat , and this assignment is made here . analysis of the p bleaching decay kinetics on the blue side of the qy band ( e.g. , 840850 nm ) that includes a 230 ps component does not improve the fits . we can not rule out that the yield of pha pqa et might be slightly less than 100% . in wild - type rcs , pha forms in near 100% yield and bleaching of the qx band of ha reaches essentially the same magnitude as the bleaching of the qx band of p at 600 nm . for the m204e mutant , the maximal bleaching of ha at 543 nm reaches a much smaller maximum amplitude between 90 and 100 ps ( figure 6 ) . this is a consequence of the long 35 ps p * lifetime , the reduced yield of pha , and the 230 ps lifetime of pha . to understand this in detail , kinetic simulations were performed and ranges of values explored for the rate constants for p * internal conversion and et to ha and hb that would simultaneously reproduce the measured p * lifetime in m204e and the measured yields of charge - separated states . the scheme in figure 7 shows a composite model that reproduces the measured p * lifetime of 35 ps and the measured yields of ground state and charge - separated states formed from p*. a number of studies have consistently found that the internal conversion time constant ( 1/kic ) for ( unperturbed ) p * in rb . capsulatus rcs in deriphat - tris buffer at 295 k falls within a range of 180 to 220 ps ( although this value is sensitive to the environment of p , for example , being 100 ps in ldao - tris buffer ) . mutations that perturb p of course would be expected to affect the time constant for p * internal conversion . however , modeling with much smaller or larger time constants for p * internal conversion would result in a respectively larger or smaller yield of ground state recovery from p * than the measured experimental value . photochemical model for m204e rcs . as discussed in the text , the values of the time constants for the wild - type rb . capsulatus rc ( and detergent ) are nominally the same as those shown for the m204e rc , except the first a - side steps in wt are p * pba pha in 3 and 1 ps , respectively , with unity yield . from previous work , and with no mutations introduced around bb , the time constant ( 1/kpb ) for ( presumably superexchange mediated ) p * phb et is expected to be similar to that of p * internal conversion , 200 ps . the measured 1520% yields for both p ground state and p * phb for m204e are consistent with this , and thus the simulations explored a relatively small range ( 150 to 220 ps ) of values for 1/kic and 1/kpb . again , smaller / larger values for 1/kpb and 1/kic would result in higher / lower yields of phb and ground state recovery , inconsistent with the experimentally observed yields . in addition to reproducing the yields of the p * decay products , the values used for kic , kpa , and kpb must reproduce the measured 35 ps lifetime of p * [ i.e. , 35 ps = 1/(kic + kpb + kpa ) ] . from this analysis 1/kpa is determined to be 50 ps and the yield of pha is 65% . in the simulations , the maximal transient buildup of pha is only 4045% and this occurs at 90100 ps . this is in excellent agreement with the experimentally observed time course of ha bleaching , attaining maximal amplitude between 90 and 100 ps ( figure 6b ) . other points of agreement between the experimental data , the simulations , and composite model in figure 7 include the amount of p * that decays to the ground state by internal conversion ( mentioned above ) and the magnitude of p bleaching observed at 34 ns . as a further check of the model in figure 7 , we set the challenge to reproduce the experimentally observed ta spectrum at 100 ps using the known basis spectra of the species involved . the ta spectrum of each individual state p * , pha , and pqa , the latter two both at 100% yield with respect to p * , are known with high fidelity from extensive studies on wild - type rcs . the ta spectrum of state phb is also known , in this case from a mutant in which this state forms in 70% yield as the sole et product of p * decay ( 200 ps internal conversion accounting for the remaining 30% p * decay pathway ) . thus , we have the basis ta spectra needed to reproduce the ta spectrum at 100 ps ( or at any other time ) . figure 8 compares the experimental ( red ) and calculated ( simulated ) ta spectrum at 100 ps , and the agreement is excellent . the calculated spectrum in blue is the following sum of the known ta basis spectra : 9% p * + 48% pha + 15% phb + 15% pqa + 1% pqb + 12% ground state ( no ta change ) . the percentages of these species present at 100 ps were based on the kinetic simulations ( figure s6 of the supporting information ) . beer s law holds here , requiring that the simulation quantitatively reproduce the ta changes referenced to the initial concentration of p * produced upon excitation . in other words , the simulation must replicate not only shapes of spectral features but also the absolute magnitude of the absorption changes and such are achieved in figure 8 . experimental ta spectrum of m204e rcs acquired 100 ps following excitation with 120 excitation flashes at 865 nm ( red ) . the blue ta spectrum at 100 ps is calculated from the basis set of the ta spectra of p * and the charge separated states . in the following , we place the unusually straightforward and homogeneous kinetics observed for the m204e mutant in the context of many attempts to understand the connection between energetics and kinetics in rcs , which in most cases are considerably more complex . glutamic acid at m204 introduces into the environment of the conserved tyr m208 a residue with the potential to hydrogen bond to the tyr hydroxyl group and change its positioning with respect to ba . theoretical work predicts that reorienting the tyr oh can significantly raise the free energy of pba and impede a - side charge separation . first , an nmr signal analogous to one previously assigned in studies of wild - type rb . sphaeroides rcs to a nonhydrogen bonded tyr m210 residue is present in the nmr spectrum of wild - type rb . hydrogen bonded or not , the nmr data clearly indicate that the introduced glutamic acid has significantly affected the environment of tyr m208 . ultrafast measurements reveal that this single amino acid change has substantial effects on charge separation . electron transfer from p * to ha is about a factor of 10 slower than in wild - type and pha no longer forms in 100% yield . specifically , the p * lifetime is 35 ps and p * decays via 50 ps et to ha ( 65% yield ) and via et to hb and internal conversion to the ground state with about equal time constants ( 200 ps ) and yields ( 1718% ) for both , as in figure 7 . in broad view , the m204e mutant has parallels to two rb . sphaeroides mutants that bear a single amino acid change and result in a similar p * lifetime . replacing the conserved tyr m210 in rb . sphaeroides with trp results in p * having an 40 ps lifetime but heterogeneous kinetics . replacing the native gly at m203 with leu in rb . the crystal structure of the m203l mutant reveals steric exclusion of a water molecule , denoted water - a , that in the wild - type crystal structures ( figure 2 ) is positioned such that it could form a hydrogen - bond bridge between his m202 ( in rb . sphaeroides the axial ligand to the mg of one macrocycle of p ) and the ring - v keto group of ba . these three mutants provide glimpses into potential multiple molecular interactions that may stabilize and fine - tune the free energy of pba : the presence of a tyr near ba , optimal orientation of the tyr hydroxyl group with respect to ba , and a hydrogen bond to the ring - v keto group of ba . for all three mutants , pba is suggested to be sufficiently destabilized that it is above p * , thereby lengthening by 10-fold the p * lifetime . electron transfer in the m204e mutant is consistent with prior work on a still relatively small family of rb . capsulatus mutants in which p * decay is partitioned fairly straightforwardly between significant yields ( more than just a few percent ) of internal conversion and et to hb along with a - side et . many , but not all , of these mutant rcs employ a tyr at l181 ( which presumably lowers the free energy of pbb ) and a phe at m208 . however , in m204e , the native tyr m208 and phe l181 are unchanged , as they were in the first two mutants in which et to the b - side was observed , namely the dh and kdh mutants [ where dh = g(m201)d+l(m212)h and kdh = s(l178)k+dh ] . for ease of observing hb bleaching unambiguously , in many of the wrong way mutants the leu at m212 is changed to a his , which results in a bchl , denoted , in place of ha . however , this is not the case in m204e or the yf mutant ( swap of l181 phe to tyr and of m208 tyr to phe ) or in mutants in which there is no pigment in the ha site ( dll mutants ) or no pigment in the ba site ( m204q mutants ) . among these mutants , specifically , deriphat 160-c and ldao impart different photophysical properties to p * and the rates of its decay pathways . not considering mutants aimed specifically at affecting the dimer , the long - wavelength absorption band of p usually occurs near 850 nm for rb . capsulatus rcs in ldao : tris buffer but usually is at 865 nm in deriphat : tris buffer , the latter essentially the same as found when the ( wild - type ) rc resides in its native membrane . for both wild - type and the l(m212)h ( beta ) mutant , the p * lifetime in deriphat : tris buffer is about double that found for these rcs in ldao - tris buffer . similarly , the time constant for internal conversion of p * is about a factor of 2 larger for rcs solubilized in deriphat ( 200 ps ) than in ldao ( 100 ps ) . although this was apparent during early work , it was underscored in the recently revived dll mutant , first made in the youvan lab ca . 1990 , wherein p * decays solely ( 100% ) by internal conversion to the ground state . the recent work established that the time constant for this process at room temperature is 200 ps for rb . capsulatus rcs in deriphat : tris buffer and 100 ps for rcs in ldao : tris buffer . the dll ( ha - less ) mutant has provided basis for understanding a 200 ps component of biexponential p * stimulated emission decay sometimes found in rb . capsulatus mutants in deriphat : tris buffer where the goal has been to manipulate the free energies of the a- and b - side charge - separated states . the amplitude of the 200 ps stimulated emission component has generally ranged between 20 and 40% . meanwhile , a second , and shorter kinetic component is measured for p * stimulated emission decay and measured at wavelengths associated with reduction of electron acceptors ( e.g. , ha/ and hb ) . however , the 200 ps kinetic component is not measured at wavelengths associated with reduction of electron acceptors , rather only at the wavelengths of p * stimulated emission and p bleaching . these results have indicated that attempts to favor et to the b side ( by lowering the free energy of pbb ) and disfavor et to the a side ( by raising the free energy of pba ) can ( and often do ) give rise to a 2040% population of inactive rcs in which charge separation does not occur ( or is substantially impeded ) . for this inactive population , p * has an 200 ps lifetime and decays as in dll by internal conversion to the ground state ( or 100 ps for rcs in the ldao : tris buffer ) . capsulatus rc in deriphat , having a ( relatively ) long p * lifetime and an absence of a population of rcs in which et does not occur . capsulatus dll-1 variant ( formerly denoted dll - fylfm in ref ( 52 ) ) , in which p * decays at room temperature with single exponential kinetics ( to within a few percent ) , as discussed further below . p * populations in which differing rates of et and even differing photochemistry occur are likely rooted in the following inter - related factors . ( i ) in the native rc , the five key electronic states for initial charge separation ( p * , pba , pbb , pha , and phb ) span a modest 0.5 ev in free energy . ( ii ) the mutants designed to influence the primary events aim to shift the free energies of one or more states and likely compress this span by as much as a factor of 2 . this results in very small spacings between any of these states and especially between p * and pba and pbb . sphaeroides ) and p is estimated to be 150 to 200 mev higher in free energy than pha , placing it essentially isoenergetic with ( wild - type ) pba . ( iii ) such small energy spacing makes the rates , yields , and directionality of charge separation very sensitive to different rc or p * conformations or populations that represent a natural landscape of cofactor , protein interactions , static , dynamic , or both . heterogeneous kinetics in wild - type rcs have been ascribed to pigment / protein conformers or populations and links between protein dynamics and et have been much discussed . the presence , position , and orientation of water molecules may contribute , such as one ( water - a ) that may bridge the his ( m202 , equiv of m200 in rb . capsulatus ) ligand on pm and ba via hydrogen bonds , and that has been the subject of much recent study . additionally for rb . capsulatus rcs , p * is 26 mev lower in free energy in deriphat than in ldao , which is 25% of the estimated free - energy difference between p * and pba . this makes even more plausible the differences found between the p * lifetimes for some rb . capsulatus rcs in deriphat versus ldao or for differences between membrane - bound rcs versus rcs in detergent micelles . this mutant was designed to potentially reposition the hydroxyl group of tyr m208 nearly in an orientation postulated to maximally destabilize pba in free energy and thus impede the critical first step of p * pba pha primary charge separation . the calculations of alden et al . indicated that in wild - type rcs , the m208 tyr oh group is confined to an orientation that lowers the free energy of pba by as much as 200 mev ( 40 mev of this is an effect on the p oxidation potential ) . our modeling of possible interactions between a glu at m204 and a tyr at m208 indicates that if a hydrogen bond forms between these residues , the tyr hydroxyl group could be rotated 120 away from the ideal geometry found by alden et al . and only 60 from a geometry that provides the least free energy stabilization of pba . given that pba is thought to be only 50100 mev below p * in wild - type , it is plausible that this state is higher in free energy than p * in m204e ( figure 7 ) . ( note that the energetic consequences of the tyr - oh dipole being hydrogen bonded to a glutamic acid were not modeled in alden et al . ) we can not know with certainty that the hydroxyl group of tyr m208 is hydrogen bonded to the glutamic acid introduced at m204 , but the nmr spectrum is consistent with this and minimally shows that the oh of tyr m208 ( and/or the entire residue ) experiences a different environment . in addition or alternatively , depending on the side chain position and ionization state , a glu at m204 could directly or indirectly affect ba , p , or the functional consequence remains that p * pha et has an 50 ps time constant ( and 65% yield ) , allowing the relatively slow native 200 ps time constants for et to hb and internal conversion of p * to the ground state to compete . additionally , and somewhat unusually , the p * decay kinetics in m204e are quite homogeneous with no discernible contribution of an 200 ps ( or other ) component in the p * stimulated emission decay kinetics and no discernible detection wavelength dependence to the p * decay kinetics . thus , not only is an inactive population absent , the data indicate only a comparatively narrow ( or no ) distribution of p * functional forms . even for dll rcs , where again p * decays solely by internal conversion , a 3-fold wide distribution of p * decay time constants spanning several hundred picoseconds is found at 77 k in both ldao and deriphat . the absence of detection - wavelength dependent kinetics in the m204e mutant may indirectly signal , or at least be consistent with , the introduced glutamic acid negating or constraining some pigment or protein native motions ( conformational changes or rotomers ) as might result , for example , upon formation of a hydrogen bond between glu m204 and the oh group of tyr m208 . some final connections can be made to two other dll variants , ones that led to our making m204e in the first place . in both of these mutants , p * decay was found to be biexponential , but instead of one photochemically active p * population and an inactive p * population with an 200 ps lifetime there were two distinct active p * populations . in one , p * decayed with an 10 ps time constant and gave rise to formation of pba , which state lived for 300 ps and was trapped because ha is absent from all rcs in the dll mutant family . in the second population , p * had an 100 ps lifetime and decayed via a combination of et to hb and internal conversion to the ground state . the dll motif bears a number of changes around the a - side cofactors , including having a phe at m208 . in the dll variants where pba was trapped , m208 was restored to a tyr , and we speculated whether the two populations might reflect orientations of tyr at m208 . the m204e rc was born of the idea of whether it might be possible to pin down tyr m208 and affect the populations . the simple scheme shown in figure 7 , where both pba and pbb are placed higher than p * , suggests that in m204e p * pha et occurs not by a two - step processes wherein pba is a chemical intermediate but rather occurs in a manner posited for p * phb et . all cases of p * phb to date , even aided by a tyr at l181 , are presumed to occur with superexchange assistance of pbb . depending on the mutations involved , the time constant for p * phb et has ranged from 70 to 200 ps ; again , these values are for various mutants in deriphat and 40 ps , the smallest time constant obtained , is for a mutant in ldao . interestingly , the 50 ps time constant for p * pha et in m204e is within this range . on the b - side , the smaller values in a 100200 ps range of time constants have been achieved with a tyr residue at l181 with 200 ps the case for the native phe . electron transfer in the m204e mutant thus conforms well compared to prior observations and analyses . it appears that in this interesting mutant , the free energy of pba is significantly affected ( likely raised above p * ) , and the balance of et in the rc is disrupted even though the native , key tyr at m208 is in place . even though the p * lifetime is a very long 35 ps , pha is still the dominant product of p * decay , though reduced to 65% yield . the native phe at l181 also is in place , and the time constant for p * phb is unchanged from the 200 ps value ( for rb . capsulatus in deriphat ) that has been repeatedly found for mutants with a ( presumably ) native b - side but in which et to hb is observed because of impeded a - side et . similarly , the time constant for p * internal conversion is unchanged from the native capsulatus in deriphat ) . finally , p * decay in m204e appears to occur fairly uniformly in the entire rc or p * population . this suggests a p * population in which pba is elevated well out of the small , congested free - energy window normally occupied by all five key electronic states , whose free energy spacing / ordering is highly sensitive to static / dynamic protein effects that drive the relative contributions of the three p * decay pathways , which give et to the a - side , et to the b - side , or no et at all .

slow , 50 ps , p * p+ha electron transfer is observed in rhodobacter capsulatus reaction centers ( rcs ) bearing the native tyr residue at m208 and the single amino acid change of isoleucine at m204 to glutamic acid . the p * decay kinetics are unusually homogeneous ( single exponential ) at room temperature . comparative solid - state nmr of [ 4-13c]tyr labeled wild - type and m204e rcs show that the chemical shift of tyr m208 is significantly altered in the m204e mutant and in a manner consistent with formation of a hydrogen bond to the tyr m208 hydroxyl group . models based on rc crystal structure coordinates indicate that if such a hydrogen bond is formed between the glu at m204 and the m208 tyr hydroxyl group , the oh would be oriented in a fashion expected ( based on the calculations by alden et al . , j. phys . chem.1996 , 100 , 1676116770 ) to destabilize p+ba in free energy . alteration of the environment of tyr m208 and ba by glu m204 via this putative hydrogen bond has a powerful influence on primary charge separation .

Introduction Experimental Section Results Discussion

PMC3713899

obsessive - compulsive disorder ( ocd ) is one of the most common neuropsychiatric disorders , with a lifetime prevalence of 2%3%.13 ocd is characterized by persistent intrusive thoughts ( obsessions ) , repetitive actions ( compulsions ) , and excessive anxiety . a major characteristic of obsessions and compulsions is that they are excessive and unreasonable , based on the definitions provided in the diagnostic and statistical manual of mental disorders , 4th ed . ( dsm - iv).4 ocd is considered to be among the 20 leading causes of disability in the usa and other countries.5 however , despite widespread recognition of its clinical characteristics , the neurophysiological mechanisms underlying ocd are not yet well understood . recent advances in neuroimaging techniques have now made it possible to assess neurophysiological data aimed at increasing our understanding of the pathophysiology of ocd . although findings from functional , metabolic , and structural imaging studies have suggested dysfunction in the orbitofrontostriatal circuit , recent studies have implicated additional brain regions , including the dorsolateral prefrontal cortex.6 abnormality in the dorsolateral prefrontal cortex , with respect to functional connectivity with the striatum , has also been associated with ocd.7 near - infrared spectroscopy ( nirs ) is a neuroimaging technique well suited to measuring brain function in patients with mental illnesses . the advantages of nirs include no requirement for large devices , improved safety ( not requiring radioactive material or strong magnetism ) , and lower cost ( running costs almost zero ) compared with other techniques used for functional imaging of brain activity ( eg , positron emission tomography , single photon emission computed tomography , and functional magnetic resonance imaging ) . further , nirs has approximately 10-fold higher temporal resolution but lower spatial resolution , and can be measured repeatedly over a prolonged period in a normal posture and state , unlike functional magnetic resonance imaging . therefore , nirs has been used to examine brain function in many psychiatric disorders , including schizophrenia , bipolar disorder , depression , dementia , post - traumatic stress disorder , pervasive developmental disorders , and attention deficit hyperactivity disorder . ota et al8 and okada et al9 recently used nirs to examine ocd as measured during the stroop color - word task and reported a reduced prefrontal hemodynamic response . the verbal fluency task is the most common and well established method of assessing cognitive activation during nirs measurement.10 use of the verbal fluency task allows comparisons between the results of the current study and other studies investigating psychiatric disorders . therefore , the aim of this study was to evaluate hemodynamic responses in the prefrontal cortex in patients with ocd by using nirs during the verbal fluency task and to compare these responses between patients with ocd and healthy controls . twenty patients with ocd and 20 healthy controls matched for age , gender , handedness , and estimated intelligence quotient participated in this study ( table 1 ) . the patients were recruited from the kyoto prefectural university of medicine hospital , kyoto , japan . the medical committee on human studies at the kyoto prefectural university of medicine approved all of the procedures . all participants provided written and informed consent after receiving a complete description of the study . all patients were primarily diagnosed using the structured clinical interview for dsm - iv ( scid ) axis i disorders11 by trained and experienced psychiatrists or clinical psychologists . all controls were screened using the scid nonpatient edition ( scid - np).12 at the time of entry into the study , seven of the 20 patients with ocd had not been taking any psychotropic medication and the other 13 were taking a variety of psychotropic medications , including antidepressants , anxiolytics , and/or antipsychotics , but not mood stabilizers . the exclusion criteria for patients and controls included : significant disease of the neurologic , pulmonary , cardiac , renal , hepatic , or endocrine system , or a metabolic disorder ; prior psychosurgery or traumatic brain injury with any known cognitive consequences , loss of consciousness , or a history of electroconvulsive therapy ; current or past dsm - iv axis i diagnosis of any psychiatric illness other than ocd ; dsm - iv diagnosis of mental retardation or pervasive developmental disorder based on clinical interview and psychosocial history ; and pregnancy . there was no history of any psychiatric illness in the controls , as determined by the scid - np . in addition , none of the first - degree relatives of healthy controls had any history of psychiatric treatment . classification of handedness was based on a modified 25-item version of the edinburgh inventory.13 on the same day as the nirs experiment , all of patients were surveyed for symptoms of ocd using the japanese version of the yale - brown obsessive - compulsive scale ( y - bocs ) symptom checklist,1416 the maudsley obsessive - compulsive inventory ( moci),17 the 17-item hamilton depression rating scale,18 and the hamilton anxiety rating scale.19 the verbal fluency task was administered according to the procedure outlined in a previous report.10 the procedure consists of a 30-second pre - task baseline , a 60-second verbal fluency task ( letter version ) , and a 70-second post - task baseline . during the pre - task and post - task baseline periods , the subjects were instructed to repeat the vowels ( a , i , u , e , and o ) as the japanese counterparts of a , b , and c in english.20 during the verbal fluency task period , subjects were instructed verbally to generate vocally as many words beginning with the japanese syllable mentioned later as possible . the syllables ( first to , a , or na ; second i , ki , or third ta , o , or ha ) were used in this order , which could be counterbalanced across subjects and changed every 20 seconds during the task period , without repetition or resorting to proper words . changes in oxygenated , deoxygenated , and total hemoglobin were measured using a multichannel nirs machine ( foire-3000 functional nirs system , shimadzu , kyoto , japan ) at three wavelengths of near - infrared light ( 780 , 805 , and 830 nm ) . this apparatus measured the relative concentrations of oxygenated and deoxygenated hemoglobin at 42 measurement points in a 9 15 cm area ( figure 1 ) . the distance between probes was 3 cm and channels were defined for each domain measured between the source and detector probes.10 to avoid cross - talk noise , the probe emits light continuously . absorption of near - infrared light was measured with a sampling time of 0.22 seconds . the subjects were instructed to minimize movement , such as head movements , strong biting , and eye blinking , during the nirs measurements , because these movements can produce artifacts or changes in cerebral perfusion unrelated to the task . the nirs shell was placed on the frontal region of the head with the lowest probes positioned along the fp12 line according to the international 1020 system used in electroencephalography.21 first , changes in the oxygenated hemoglobin concentration in each of the 14 channels were analyzed . channel groupings corresponded to approximate positions on the left ( channels 16 , 17 , 25 , 33 , and 34 ) and right ( channels 9 , 10 , 18 , 26 , and 27 ) dorsolateral prefrontal cortex and the prefrontal area near the frontopolar area ( channels 13 , 21 , 22 , and 30 ) , based on an anatomic craniocerebral correlation study ( figure 2a).21 changes in oxygenated hemoglobin have been postulated as the best indicator of blood oxygenation.22,23 therefore , this study focused on the change in oxygenated hemoglobin concentration . nirs data that clearly contained a motion artifact , which is determined by the minute observation about the theme was excluded from the statistical analyses . in addition , a low - pass filter equipped with a 0.1 hz high cutoff was used to exclude any short - term motion artifacts from the data used for analysis . the mean oxygenated hemoglobin concentration during the 60-second verbal fluency task was calculated and the pre - task baseline was calculated using the mean of the last 10 seconds of the 30-second pre - task period.10 the change in oxygenated hemoglobin concentration during the verbal fluency task was calculated by subtracting the oxygenated hemoglobin concentration during the task period from the pre - task baseline oxygenated hemoglobin concentration . a z - score was calculated as follows : d = ( m1 m2)/s , where m1 and m2 represent the mean concentration values during the baseline and trial , respectively , and s represents the standard deviation of the baseline . in order to increase the signal - to - noise ratio , we then compared each of the two groups using student s t - test , and the significance level in the analysis was set to p = 0.05 . since we performed 14 paired t - tests , the correction for multiple comparisons was made using the false discovery rate ( two - tailed ; we set the value of q specifying the maximum false discovery rate to 0.05 , so that there are no more than 5% false positives on average ) . all statistical analyses were performed using statistical package for the social sciences version 17.0 j for windows software ( ibm , armonk , ny , usa ) . twenty patients with ocd and 20 healthy controls matched for age , gender , handedness , and estimated intelligence quotient participated in this study ( table 1 ) . the patients were recruited from the kyoto prefectural university of medicine hospital , kyoto , japan . the medical committee on human studies at the kyoto prefectural university of medicine approved all of the procedures . all participants provided written and informed consent after receiving a complete description of the study . all patients were primarily diagnosed using the structured clinical interview for dsm - iv ( scid ) axis i disorders11 by trained and experienced psychiatrists or clinical psychologists . all controls were screened using the scid nonpatient edition ( scid - np).12 at the time of entry into the study , seven of the 20 patients with ocd had not been taking any psychotropic medication and the other 13 were taking a variety of psychotropic medications , including antidepressants , anxiolytics , and/or antipsychotics , but not mood stabilizers . the exclusion criteria for patients and controls included : significant disease of the neurologic , pulmonary , cardiac , renal , hepatic , or endocrine system , or a metabolic disorder ; prior psychosurgery or traumatic brain injury with any known cognitive consequences , loss of consciousness , or a history of electroconvulsive therapy ; current or past dsm - iv axis i diagnosis of any psychiatric illness other than ocd ; dsm - iv diagnosis of mental retardation or pervasive developmental disorder based on clinical interview and psychosocial history ; and pregnancy . there was no history of any psychiatric illness in the controls , as determined by the scid - np . in addition , none of the first - degree relatives of healthy controls had any history of psychiatric treatment . classification of handedness was based on a modified 25-item version of the edinburgh inventory.13 on the same day as the nirs experiment , all of patients were surveyed for symptoms of ocd using the japanese version of the yale - brown obsessive - compulsive scale ( y - bocs ) symptom checklist,1416 the maudsley obsessive - compulsive inventory ( moci),17 the 17-item hamilton depression rating scale,18 and the hamilton anxiety rating scale.19 the verbal fluency task was administered according to the procedure outlined in a previous report.10 the procedure consists of a 30-second pre - task baseline , a 60-second verbal fluency task ( letter version ) , and a 70-second post - task baseline . during the pre - task and post - task baseline periods , the subjects were instructed to repeat the vowels ( a , i , u , e , and o ) as the japanese counterparts of a , b , and c in english.20 during the verbal fluency task period , subjects were instructed verbally to generate vocally as many words beginning with the japanese syllable mentioned later as possible . the syllables ( first to , a , or na ; second i , ki , or se ; third ta , o , or ha ) were used in this order , which could be counterbalanced across subjects and changed every 20 seconds during the task period , without repetition or resorting to proper words . changes in oxygenated , deoxygenated , and total hemoglobin were measured using a multichannel nirs machine ( foire-3000 functional nirs system , shimadzu , kyoto , japan ) at three wavelengths of near - infrared light ( 780 , 805 , and 830 nm ) . this apparatus measured the relative concentrations of oxygenated and deoxygenated hemoglobin at 42 measurement points in a 9 15 cm area ( figure 1 ) . the distance between probes was 3 cm and channels were defined for each domain measured between the source and detector probes.10 to avoid cross - talk noise , the probe emits light continuously . absorption of near - infrared light was measured with a sampling time of 0.22 seconds . the subjects were instructed to minimize movement , such as head movements , strong biting , and eye blinking , during the nirs measurements , because these movements can produce artifacts or changes in cerebral perfusion unrelated to the task . the nirs shell was placed on the frontal region of the head with the lowest probes positioned along the fp12 line according to the international 1020 system used in electroencephalography.21 first , changes in the oxygenated hemoglobin concentration in each of the 14 channels were analyzed . channel groupings corresponded to approximate positions on the left ( channels 16 , 17 , 25 , 33 , and 34 ) and right ( channels 9 , 10 , 18 , 26 , and 27 ) dorsolateral prefrontal cortex and the prefrontal area near the frontopolar area ( channels 13 , 21 , 22 , and 30 ) , based on an anatomic craniocerebral correlation study ( figure 2a).21 changes in oxygenated hemoglobin have been postulated as the best indicator of blood oxygenation.22,23 therefore , this study focused on the change in oxygenated hemoglobin concentration . nirs data that clearly contained a motion artifact , which is determined by the minute observation about the theme was excluded from the statistical analyses . in addition , a low - pass filter equipped with a 0.1 hz high cutoff was used to exclude any short - term motion artifacts from the data used for analysis . the mean oxygenated hemoglobin concentration during the 60-second verbal fluency task was calculated and the pre - task baseline was calculated using the mean of the last 10 seconds of the 30-second pre - task period.10 the change in oxygenated hemoglobin concentration during the verbal fluency task was calculated by subtracting the oxygenated hemoglobin concentration during the task period from the pre - task baseline oxygenated hemoglobin concentration . a z - score was calculated as follows : d = ( m1 m2)/s , where m1 and m2 represent the mean concentration values during the baseline and trial , respectively , and s represents the standard deviation of the baseline . in order to increase the signal - to - noise ratio , we then compared each of the two groups using student s t - test , and the significance level in the analysis was set to p = 0.05 . since we performed 14 paired t - tests , the correction for multiple comparisons was made using the false discovery rate ( two - tailed ; we set the value of q specifying the maximum false discovery rate to 0.05 , so that there are no more than 5% false positives on average ) . all statistical analyses were performed using statistical package for the social sciences version 17.0 j for windows software ( ibm , armonk , ny , usa ) . age , gender , and estimated intelligence quotient did not differ significantly between patients with ocd and controls ( age , t = 0.34 , df = 38 , p = 0.73 ; gender , 2 = 0.1 , df = 1 , p = 0.75 ; intelligence quotient , t = 0.45 , df = 38 , p = 0.66 ) . for the patients with ocd , the mean y - bocs score was 19.6 8.2 and the mean moci score was 15.5 5.8 , while the mean moci score for control subjects was 4.3 3.5 . there were significant differences in the mean moci score ( t = 7.39 , df = 38 , p < 0.001 ) and verbal fluency scores ( t = 3.892 , df = 38 , p < 0.001 ) between the two groups . there were no significant correlations between verbal fluency scores and age , intelligence quotient , or y - bocs scores in the patients with ocd . the grand - average waveforms of the change in oxygenated hemoglobin in the control group increased during the task period ( figure 1 ) . in contrast , those in the ocd group did not change markedly ( figure 1 ) . in the ocd group , the mean z - score during the task period , compared with the pre - task baseline ( 11.245 ) , was significantly smaller than that of the control group ( 3.173 ) at channel 18 ( t = 2.285 , p = 0.028 , figures 2b and 3 ) , and was not significant after correction for multiple comparisons . the mean z - score in each channel was not significantly correlated with clinical characteristics , ie , gender ( p = 0.485 ) , age ( p = 0.324 ) , intelligence quotient ( p = 0.57 ) , moci score ( p = 0.329 ) , verbal fluency scores ( p = 0.647 ) , y - bocs scores ( p = 0.82 ) , duration of illness ( p = 0.296 ) , and hdrs score ( p = 0.147 ) . there were no significant differences in mean z - score between medication - free and medication groups ( t = 1.562 , df = 18 , p = 0.136 ) , and the mean z - score was not significantly correlated with antidepressant dose ( p = 0.184 ) . in addition , there were no significant correlations between prefrontal hemodynamic responses and verbal fluency scores . age , gender , and estimated intelligence quotient did not differ significantly between patients with ocd and controls ( age , t = 0.34 , df = 38 , p = 0.73 ; gender , 2 = 0.1 , df = 1 , p = 0.75 ; intelligence quotient , t = 0.45 , df = 38 , p = 0.66 ) . for the patients with ocd , the mean y - bocs score was 19.6 8.2 and the mean moci score was 15.5 5.8 , while the mean moci score for control subjects was 4.3 3.5 . there were significant differences in the mean moci score ( t = 7.39 , df = 38 , p < 0.001 ) and verbal fluency scores ( t = 3.892 , df = 38 , p < 0.001 ) between the two groups . there were no significant correlations between verbal fluency scores and age , intelligence quotient , or y - bocs scores in the patients with ocd . the grand - average waveforms of the change in oxygenated hemoglobin in the control group increased during the task period ( figure 1 ) . in contrast , those in the ocd group did not change markedly ( figure 1 ) . in the ocd group , the mean z - score during the task period , compared with the pre - task baseline ( 11.245 ) , was significantly smaller than that of the control group ( 3.173 ) at channel 18 ( t = 2.285 , p = 0.028 , figures 2b and 3 ) , and was not significant after correction for multiple comparisons . the mean z - score in each channel was not significantly correlated with clinical characteristics , ie , gender ( p = 0.485 ) , age ( p = 0.324 ) , intelligence quotient ( p = 0.57 ) , moci score ( p = 0.329 ) , verbal fluency scores ( p = 0.647 ) , y - bocs scores ( p = 0.82 ) , duration of illness ( p = 0.296 ) , and hdrs score ( p = 0.147 ) . there were no significant differences in mean z - score between medication - free and medication groups ( t = 1.562 , df = 18 , p = 0.136 ) , and the mean z - score was not significantly correlated with antidepressant dose ( p = 0.184 ) . in addition , there were no significant correlations between prefrontal hemodynamic responses and verbal fluency scores . in the present study , changes in oxygenated hemoglobin concentration during the verbal fluency task were significantly smaller in patients with ocd than those in healthy controls . these results support the hypothesis that prefrontal dysfunction is associated with adult ocd and suggest the ability of the verbal fluency task during nirs measurement to detect the abnormality . abnormalities in the dorsolateral prefrontal cortex have been reported in studies using other imaging modalities . in functional magnetic resonance imaging studies , nakao et al24,25 showed that patients with ocd had weaker activation than controls in the dorsolateral prefrontal cortex , anterior cingulate cortex , and caudate nucleus . in a positron emission tomography study , martinot et al26 showed that absolute regional cerebral glucose metabolic rates in the lateral prefrontal cortex were significantly lower in patients with ocd than in control subjects . the current results are consistent with these previous reports . in an nirs study , ota et al8 and okada et al9 showed that prefrontal hemodynamic responses were lower in both adult and pediatric patients with ocd using the stroop color - word task , but that the areas with a reduced prefrontal hemodynamic response were different between these two populations . ota et al8 reported a smaller response in the frontopolar region in the pediatric ocd group , while okada et al9 reported a smaller response in the left lateral prefrontal region in adult patients with ocd . in the present study , adult patients with ocd had significantly smaller changes in oxygenated hemoglobin concentrations than controls at channel 18 , localized near the right lateral prefrontal region . the difference between this result and that of okada et al9 may be explained by the difference in the activation task and by differences in the study populations . with respect to laterality , previous neuroimaging studies have reported abnormalities in both hemispheres.2729 further study is needed to resolve these differences . although many findings have been accumulated about cognitive function including verbal memory , learning , and retrieval in ocd , the results are inconsistent in many cases.6,30 the reason for this inconsistency may be that existing neuropsychological examination refects multiple aspects of cognitive function . in our study , there was a significant difference in the mean verbal fluency score between patients with ocd and controls . this result supports not only the previous results for impairment in verbal fluency,30 but also a possibility of this originating in impairment of response inhibition and/or attentional set - shifting . many previous studies have suggested that frontal lobe activity during the verbal fluency task , as measured by nirs , is decreased in many psychiatric disorders.10,20,3134 specifically , reduced responses during the verbal fluency task have been reported repeatedly in the frontal pole in patients with schizophrenia.10,31,33,34 in some cases , the clinical distinction between schizophrenia and ocd is difficult.35,36 therefore , the difference in localization of the response may be useful in the differential diagnosis of these disorders . further direct comparisons between schizophrenia and ocd are needed to explore the diagnostic potential of nirs localization . first , the difference in the right dorsolateral prefrontal cortex between the patients and controls was not significant after correction for multiple comparisons future studies should include a larger sample to confirm the reduced response in the dorsolateral prefrontal cortex , even after correction for multiple comparisons . given that antidepressants have been reported to affect hemodynamic responses,37 a medication effect can not be excluded . our results show that patients with ocd had significantly smaller changes in oxygenated hemoglobin concentration in the prefrontal cortex , especially in the right dorsolateral prefrontal regions . these results support the notion that the dorsolateral prefrontal cortex plays a role in the pathophysiology of ocd .

backgroundnear - infrared spectroscopy has helped our understanding of the neurobiological mechanisms of psychiatric disorders and has advantages including noninvasiveness , lower cost , and ease of use compared with other imaging techniques , like functional magnetic resonance imaging . the verbal fluency task is the most common and well established task used to assess cognitive activation during near - infrared spectroscopy . recent functional neuroimaging studies have shown that the orbitofrontal cortex and other brain regions , including the dorsolateral prefrontal cortex , may play important roles in the pathophysiology of obsessive - compulsive disorder ( ocd ) . this study aimed to evaluate hemodynamic responses in the dorsolateral prefrontal cortex in patients with ocd using near - infrared spectroscopy during the verbal fluency task and to compare these with dorsolateral prefrontal cortex responses in healthy controls.methodstwenty patients with ocd and 20 controls matched for age , gender , handedness , and estimated intelligence quotient participated in this study . the verbal fluency task was used to elicit near - infrared spectroscopic activation and consisted of a 30-second pre - task , followed by three repetitions of a 20-second verbal fluency task ( total 60 seconds ) , followed by a 70-second post - task period . the near - infrared spectroscopy experiment was conducted on the same day as surveys of obsessive - compulsive symptoms , depression , and anxiety . z - scores for changes in the concentration of oxygenated hemoglobin were compared between the ocd patients and controls in 14 channels set over the left and right dorsolateral prefrontal cortex and frontopolar areas.resultsduring the verbal fluency task , significant task - related activation was detected in both the ocd group and the controls . changes in oxygenated hemoglobin concentration in the right dorsolateral prefrontal cortex were significantly smaller in the ocd group than in the controls , but were not statistically significant after correction for multiple comparisons.conclusionpatients with ocd have reduced prefrontal , especially right dorsolateral prefrontal , cortical hemodynamic responses as measured by near - infrared spectroscopy during the verbal fluency task . these results support the hypothesis that the dorsolateral prefrontal cortex plays a role in the pathophysiology of ocd .

Introduction Materials and methods Subjects Verbal fluency task for NIRS activation NIRS measurements Statistical analyses Results Demographic data Correlation between verbal fluency performance and subject characteristics NIRS data from subjects performing the verbal fluency task Discussion Conclusion

PMC3592756

the first course that will be described is a non - majors course called sex , gender , and the brain ( sgb ) . this course combines three hours of class and three hours of lab per week , for fourteen weeks . it is cross - listed between biology and women s studies , and typically fills with a variety of majors . the course begins by broadening the students understanding of sex and gender through the discussion of complex non - human mating systems ( roughgarden , 2004 ; zuk , 2003 ) . then , after an introduction to neurons and the brain , the course addresses topics such as the spectrum of physiological variation along the male - female continuum , focusing on the anatomy and physiology of the male and female brain , and the interaction of environment and social status with the brain ( hines , 2004 ) . throughout the course , students grapple with the primary literature and become conversant with experimental design , basic statistics , and hypothesis testing ( mead , 2009 ) . despite being beginners at science , they bring analytical skills developed in other courses to bear on this new and challenging material . typically , the associated lab includes wet lab observations and experiments based on the brain , physiology , and gender ( see mead for some examples ) . in the spring 2010 version of the course , the last six weeks of lab was replaced with a sl component involving brain outreach to second graders . this sl unit consisted of a cluster of learning activities ( crafts and games ) relating to the brain and to sensory physiology . we spent two weeks preparing and practicing the activities , and then divided into groups of four and visited six second grade classrooms in newark , ohio each week for a total of twenty - four classroom interactions ( see mead for more details ) . the learning goals for the college students during the sl outreach were 1 ) to develop their knowledge about the brain , and 2 ) to theorize and to test hypotheses about gender - based responses to different types of learning activities . for instance , we read studies suggesting that boys and girls prefer different types of toys , with boys gravitating to balls , blocks , bikes , trucks , cars , weapons , and male figurines , and girls gravitating towards board games , puzzles , crayons , and dolls ( berenbaum and hines , 1992 , hines , 2004 , berenbaum et al . , 2008 ) . theory suggested that boys would spend more time on rough and tumble games ( fabes et al . , 2003 ; hines , 2004 ) , although some deviation from these patterns was acknowledged ( sandberg et al . , 1993 ) , with many factors influencing the gender - specificity of play ( hines , 2004 ; mchale et al . , 2004 ; mchale et al . , 2005 ) given these studies , we hypothesized that girls would be more engaged than boys with the brain puzzle , that the sensory activities ( mystery socks , jelly beans , mystery noises ) would be gender neutral , and that the running around and competitive activities ( message transmission , neuron chain tag ) would be more engaging for boys than for girls . we collected data for our hypothesis testing by monitoring student engagement multiple times over each of the activities and by comparing the engagement of boys and girls . students were considered engaged when they watched the denison students leading the activity , followed directions , interacted with peers when instructed ( as in the tag and message games ) , and worked independently as assigned ( as in the brain puzzle , see fig . students were considered unengaged when they were not looking at the students leading the activities , were talking out of turn , had their head down on their desk , or were otherwise not following directions . briefly , we found that girls were indeed more engaged in the brain puzzle , but were also more engaged in some of the sensory physiology activities ( which we had expected would be gender neutral ) , and the competitive and running around games were gender neutral , rather than biased towards the boys . more details on our methodology and our results are given in mead ( 2010 ) . most denison students ( 78% ) thought that the experience of teaching about the brain enhanced their understanding of the content material , because they had to simplify it , explain it , and answer questions . furthermore , students successfully made the connection between articles read in class and the behaviors of the second graders . the students who did not feel that service learning helped them directly with course content acknowledged that they learned more about learning styles , but typically did nt make the connection to the underlying theory . 87% of students said that they had developed a greater sense of themselves as agents of change , 91% of students said that the experience gave them more tools for solving community problems , and all agreed that it was important to engage in the community . the other neuroscience course with a service - learning module is , by contrast , a course required for neuroscience concentrators called introduction to neuroscience ( neur 200 ) . the course meets for three hours per week of lecture / discussion , with an additional three - hour per week laboratory . historically , the course enrolls students at the sophomore and junior levels who plan to pursue the neuroscience concentration , complementing a major in either biology , psychology , or ( more infrequently ) , biochemistry . students are required to have completed courses in introductory psychology and introduction to the science of biology prior to enrolling in neuroscience 200 . in the spring of 2011 , a service - learning requirement ( termed a cip , for community involvement project ) was piloted in the course . two of the weekly labs were dedicated to the cip project , but the rest of the cip project took place outside of class time , either in group meetings with the course instructor , or on site visits by the students . early in the semester , students were provided with an overview of the semester long projects , and were given the opportunity to designate their top choices for the available projects . working with the alford center for service learning on campus , four possible off - campus sites were deemed to both meet neur 200 learning goals while having needs that could be met by the students : students worked in small groups ( typically , four students per group ) on their cip projects , which were as follows : four students worked with the yes clubhouse , an afterschool program in newark , to develop a presentation to teens about alcohol and recreational ritalin s effects on the brain . they used a jeopardy quiz - show format , with candy prizes , in addition to spending time with the teens and creating brochures that provided information about how ritalin works in the brain , the general physiological effects of stimulants like ritalin , and the potential effects of ritalin abuse . they found that the yes clubhouse teens had very little factually correct information about ritalin and about recreational drugs in general . a second group of students worked to collect the most recent information about sports concussions , and created a presentation for local high school coaches about short - term and long - term brain responses following concussion . these focused on the importance of sleep , now thought to aid in recovery , and on the value of baseline monitoring . after a number of scheduling problems , the coaches were receptive , and felt validated regarding their current use of the impact test , a computerized cognitive assessment used by athletic trainers to collect baseline data of the athletes mental performance prior to any injury . a third group of students worked with the chapel grove retirement facility to create a resource guide about keeping the aging brain healthy . the students worked with the staff to develop materials in support of a four - pronged approach . the four main tenets were : 1 ) a healthy and nutritious lifestyle including anti - oxidants , omega-3 fatty acids and a balanced diet ( singh et al . , 2011 ) , 2 ) the role of physical exercise in supporting favorable changes in neuronal structure including dentritic spines , the dentate gyrus , the hippocampus and the entorhinal cortex ( stranahan et al . , 2007 ) , 3 ) cognitive exercise , which is thought to reduce the risk of alzheimer s disease ( friedland et al . , 2001 ) , and 4 ) psychological well - being , including social support , religious involvement and personal meaning ( fry , 2000 ) . in addition to the presentation and resource guide , the students created multiple copies of an interactive book containing mental exercises that residents could practice ( see fig . the final group of students worked with the newark health care center to create a presentation for family and medical caregivers of alzheimer s patients about the potentially dangerous stress - related effects of care giving , role of support , etc . the information shared included the immunoprotective role of social support ( bodnar and kiecolt - glaser , 1994 ) , the importance of becoming informed about alzheimer s care and institutional support and the resultant reduction of stress , the role of exercise in combating oxidative stress and thus psychological distress , and the positive effects of artistic and sporting outlets ( miyazaki et al . , 2001 ) . students worked on their projects in phases throughout the semester , guided by pre - determined deadlines established at the beginning of the semester . during the month of february , they met with the course instructor as often as needed for feedback and guidance , prior to the site visit . during the month of march , students completed their on - site visits ( one on - site visit per group ) , and began the creation of an informative brochure based on their projects to be distributed at a local health fair . students in neuroscience 200 participated in denison s big red s big day , a community outreach event ( fig . 3 ) , and attended the licking county health fair in early april , where they interacted with health fair visitors from the community , talked with community members about their topics , and distributed their created brochures . finally , students presented their projects as a power point to peers and to members of the neuroscience faculty on the last laboratory meeting day of the semester ( early may ) . students were asked to provide responses to three questions generated by the office of service learning at denison : 1 ) did your service to the community in this course enhance your learning of the class s subject matter ? 2 ) as a consequence of your community service in this class , did you increase your awareness of social , economic , or political issues that face our community ? if so , what are those issues ? can you give a summary sense of what you learned about this issue ? 3 ) what possibilities are there for the beneficial application from this course to address problems in the licking county community ? i was proud of the mere idea that i would be completing a service project as part of my college neuroscience course . the cip projects were an excellent opportunity to apply neuroscience outside of the classroom . it also made me prioritize and remember that these are the people that my future research in neuroscience could help . this neuroscience project taught me that is not just my job to be a critical consumer of information , but it is also my responsibility to distribute this information to the community . the cip was one of the most rewarding semester long projects i have been a part of at denison . i met people in our class that i normally would not have gotten a chance to work with ..i felt proud that we contributed to the everyday lives of the chapel grove residents . at the beginning of the semester , i will admit that i was slightly skeptical as to the educational value of a cip in a neuroscience class . however , i now agree that community service can be an invaluable tool to promote active learning in any academic department . having the opportunity to participate in the cip has been an uplifting , rewarding , and educational experience . the sl projects counted for 1520% of the final grades in both courses . for the sex , gender , and the brain course , the 15% was broken down into 3% for their presentation of proposed activities , 4% for four weeks of journal entries about the process , 5% for leadership and participation during the outreach events , and 3% for answers to essay questions relating neurophysiological theories to their observations and analyzing the data on the final . the neur 200 students were assessed on their attendance and participation in group meetings with the course instructor , their work with other group members on the two laboratory days dedicated to the project design and to their oral presentation at the end of the semester , and on peer evaluations from other group members . peers evaluated fellow group members on overall attendance and participation at all group meetings , on their contribution to the project ( research , brochure creation , and on their overall team effort ) . ideally , the sl projects should also be assessed in terms of how they facilitated progress toward learning goals and in terms of outcomes for the community partners . as described above , most students reported that the sl experience advanced their learning goals . in future iterations assessing the effects of our sl projects on the community requires more thought . some of the populations that we worked with were protected populations , with the result that direct surveys of increases in knowledge might take more planning before they are approved by our human subjects review board . for example , working out questions that would both accurately assess increases in brain knowledge or interest in second graders and be answerable in five minutes will be challenging . however , the creation of pre- and post surveys at the licking county health fair is more tractable , and should be done in the next iteration of cips in neur 200 . furthermore , even if we ca nt parse out the effects of each community visit , we might be able to consider the role of multiple interventions as the campus - community relationships continue . as multiple denison groups visit the yes clubhouse or the elementary and middle schools , we could ask if rates of recreational drug use decrease and graduation rates increase . while the service learning experience resonated with both male and female students , the link to community service may have struck a deeper chord in female students , especially in the non - majors course . despite denison s campus being fairly evenly male and female , 76% of the students in the sgb class were female , possibly suggesting that more females than males were attracted to this type of class . the preponderance of female students in these service - learning classes is supported by an unpublished survey documenting volunteering in 2009 at denison ( fisher , unpublished ) . nearly twice as many female students as male students participated in service learning classes in 2009 ( 206 vs. 113 ) , and the women who participated gave more than 1.5 times more hours to their service learning projects than the men gave to theirs ( 19.8 vs 12.6 hours ) . when activities outside of the classroom are included , 78% of females versus 69% of males participated in some philanthropic activity in 2009 , and female students spent nearly 2.5 times more hours volunteering than males did ( 28.2 hours vs. 11.4 hours ) . looking beyond our campus , this type of gender difference in altruistic behaviors is supported in some areas ( andreoni and vesterlund , 2001 ; themudo , 2009 ; trudeau and devlin , 1996 ) , but not universally ( wilson , 2000 ) . . this could be partly because many of our female students already have experience with service learning or other forms of volunteering , so that the service learning portion of the laboratory experience seems comfortable and familiar , even if the topic is new and challenging . furthermore , the service learning model of introductory biology allows students the opportunity to engage in problem - solving outside of the classroom . according to yang ( 2010 ) , college women who are not science majors are more likely to want to engage in science problem - solving outside of the classroom rather than within the classroom . also , college women who are not science majors are apt to see science as only moderately or hardly relevant to life , rather than highly relevant to life ( yang , 2010 ) . service learning experiences are likely to give the scientific content a more meaningful and relevant context for these students ( bhattacharyya , 2009 ; george and brenner , 2010 ; reynolds and ahearn - dodson 2010 ) . while service learning can play an important role in many types of neuroscience courses , different goals and pressures surface in courses for non - majors versus courses for neuroscience concentrators or majors . required courses for majors or concentrators tend to contain more content pressure than courses for neuroscience majors . this can be dealt with via judicious choice of community partner , or by planning the service learning activities outside of class or lab time . this latter solution can make group meetings difficult , and can lead to over - scheduling , but may be a good way to initiate small - scale sl projects into a neuroscience program . all students are likely to experience the service learning component as an important preview of real - world neuroscience . for majors , this experience may lead to the identification of future topics for graduate school . for non - majors , the preview is more in the nature of a case study for how science is done , and how neuroscience in general can relate to social issues . in both groups , service learning can help the students form connections among their classes , whether within the science division or among all divisions . working out the logistics may be difficult and time - consuming . however , sl gives introductory students an opportunity to see neuroscience in action , and links class material to the real world . if serving a diverse student population is part of the mission of your institution , sl can be a potent tool for motivating and retaining students . sl benefits the community by providing access to important information . linking universities to their communities is especially critical for both partners in a poor economy . in addition , sl gives students opportunity for self - reflection . as a consequence of their sl experiences , nearly all of the students realized the importance of education , were more aware of their position of privilege , and realized the importance of working in the community . students also remarked upon the benefits of peer interaction , both within their sl team or group , and with their community partners . while there are many benefits to engaging in sl activities , there are also some distinct challenges . for most faculty members , it is easier to support meaningful sl activities in small classes of 25 students or fewer . while it is possible to engage in sl activities independently , it is much easier , less time - consuming , and more likely to lead to long - term , sustainable benefits for all parties when institutional support is present . ideally , this support consists of a service learning center , administrative awareness that sl is a valuable activity , and a budget . some sl models require additional time outside of class ; this poses a potential burden on already busy students . if sl occurs within the class - time framework , this may take time away from other potential neuroscience class activities . lastly , it is important for all participants to remember that academics and community members often operate on different time - frames , needs , and budgets . given these opportunities for benefits and the possibilities of challenges , we have developed several keys to success . first and most important , make sure that you are addressing a real community need , and not imposing on community members or institutions that may already be stretched to the limit in terms of staffing and mission ( bowers - sipe , 2001 ; butin , 2006 ) . this is where any campus or community service learning center can be invaluable in terms of connections , experience , and community assessment . it is also essential to start early : even if a campus service learning center is available , it is best to start planning early in the semester prior to your anticipated sl launch , both to make sure that you and your community partner are on the same page and are prepared , and to be able to provide accurate course descriptions ahead of registration deadlines . without a service learning center , it may take 23 years to develop relationships with community partners before engaging in any type of meaningful exchange . however , given the broader impact and outreach components required by many funding sources , there may be campus colleagues already engaged in this type of effort . finding ways to engage with partnerships already in existence both speeds up the planning process and can increase the robustness and longevity of the relationship . it is also practical to start small , with a limited number of projects . in our current educational climate , it is a good idea to plan for assessment of learning goals , and to consider assessment of service learning goals from the point of view of your community partner . if possible , plan for collection and analysis of data ( publish in june ! ) . once you have identified your project , it is important to get your colleagues and your students onboard . if your community partner organization can spare the personnel , ask if someone can speak to your students about what they will be doing , and why their assistance is critical . if no one is available , perhaps someone from the service learning center can speak more generally about the value of this endeavor . often even really gifted students need extensive guidance about their role and their interaction with community . especially students who have not participated in the workforce will need to know about professional behavior , including the importance of promptness , dress code , respect for all of the people with whom they interact , and appropriate thank - yous . most students will need guidance about working with protected populations , such as minors or clients at health care centers , as well as site - specific regulations about photographs , survey instruments , activities , etc . despite these challenges , the benefits of sl cause these authors to remain convinced that sl courses add valuable experiences for both our non - major students and our neuroscience concentrators . in fact , we think that the interdisciplinary nature of neuroscience may lend itself particularly well to enriching sl opportunities . we both plan to continue to offer sl experiences , and hope to refine our methodology as we learn more about this activity ourselves ! anyone interested in a copy of the brochures created by the neuroscience 200 students should contact susan kennedy . additional resources can be found below : general information http://www.compact.org/wp-content/uploads/resources/downloads/aag.pdf ( this is an excellent , annotated study of what is known about service learning and its effect on students and on the community)http://www.learnandserve.org/ ( a link to the national clearinghouse for service learning)http://www.service - learningpartnership.org / site / pageserver ( this site provides a network of over 10,000 individuals involved in service learning , including educators , parents and policy makers.)http://www.aee.org/ ( another site connecting educators to timely research findings and resources in service learning ) http://www.compact.org/wp-content/uploads/resources/downloads/aag.pdf ( this is an excellent , annotated study of what is known about service learning and its effect on students and on the community ) http://www.learnandserve.org/ ( a link to the national clearinghouse for service learning ) http://www.service-learningpartnership.org/site/pageserver ( this site provides a network of over 10,000 individuals involved in service learning , including educators , parents and policy makers . ) http://www.aee.org/ ( another site connecting educators to timely research findings and resources in service learning ) syllabi in sl http://www.compact.org/syllabi/ ( access to hundreds of sample syllabi across disciplines for those wishing to incorporate service learning into their classes)http://www.servicelearning / org / instant_info / fact_sheets / the_facts / discipline/ ( access to sample syllabi in service learning courses , including syllabi from courses that include interdisciplinary perspectives ) http://www.compact.org/syllabi/ ( access to hundreds of sample syllabi across disciplines for those wishing to incorporate service learning into their classes ) http://www.servicelearning/org/instant_info/fact_sheets/the_facts/discipline/ ( access to sample syllabi in service learning courses , including syllabi from courses that include interdisciplinary perspectives ) working with a community http://www.cbprcurriculum.info/ ( offers a model for working with members of the community in order to facilitate service learning)http://ctb.ku / org/ ( a detailed resource offering information on the skills necessary to effectively build community and create change ) http://www.cbprcurriculum.info/ ( offers a model for working with members of the community in order to facilitate service learning ) http://ctb.ku/org/ ( a detailed resource offering information on the skills necessary to effectively build community and create change )

incorporating service learning ( sl ) components can be a very powerful way to engage students , add relevance , and develop community - building skills . sl experiences can play important roles in neuroscience classes , although the roles can be different depending on the needs of the classes . in this paper , we will present two models of incorporating service learning into neuroscience courses . the first model gives an example of using sl in a non - majors course , and the second model gives an example of using sl in a neuroscience class for neuroscience concentrators . after describing the two sets of experiences , we summarize the positive aspects and the challenges involved in creating sl components in neuroscience courses , develop some keys to success , and then provide a list of additional resources .

COURSE DESCRIPTIONS GRADING AND ASSESSMENT DISCUSSION

PMC3964730

assessing the potential risk that a substance poses to human health or the environment involves a combined quantitative analysis of both exposure and hazard , including the estimation of associated uncertainties . for decades , the evaluation of toxicological hazards in the context of regulatory risk assessment has relied heavily on animal experimentation , often described in internationally recognized test guidelines ( e.g. , oecd ) . animal experiments are typically designed either to indicate the dose at which substance - induced pathological effects , other than cancer , are first observed after a defined treatment duration ( e.g. , lowest observed adverse effect level , loael ) or to infer the dose below which no effect is expected to occur ( e.g. , no observed adverse effect level , noael ) . then , through a process of extrapolation using empirically derived uncertainty factors , the assessor can arrive at various hazard reference values ( e.g. , a derived no effect level , dnel ) useful for an ecological or human health risk assessment . throughout this process , information about the mechanistic basis of the observed adverse effects is often considered nice to have rather than necessary to have . despite its shortcomings , over the years this traditional approach to toxicity assessment has been generally considered as offering an acceptable level of protection to our health and the environment . however , we are arguably positioned to shift toward a new paradigm for toxicity assessment because of the limitations of this process in addressing increasing demands imposed by modern society together with the opportunities offered by considerable developments in biomedical research tools to do better . the possibilities of improved safety from applying systems toxicology ( figure 1 ) approaches provide a scientific impetus to bring about change . systems toxicology is aimed at decoding the toxicological blueprint of active substances that interact with living systems . it integrates classic toxicology approaches with network models and quantitative measurements of molecular and functional changes occurring across multiple levels of biological organization . the multidisciplinary systems toxicology approach combines principles of chemistry , computer science , engineering , mathematics , and physics with high - content experimental data obtained at the molecular , cellular , organ , organism , and population levels to characterize and evaluate interactions between potential hazards and the components of a biological system . it is aimed at developing a detailed mechanistic as well as quantitative and dynamic understanding of toxicological processes , permitting prediction and accurate simulation of complex ( emergent ) adverse outcomes . thereby , the approach provides a basis for translation between model systems ( in vivo and in vitro ) and study systems ( e.g. , human , ecosystem ) . systems toxicology , therefore , has an ultimate potential for extrapolating from early and highly sensitive quantifiable molecular and cellular events to medium- and long - term outcomes at the organism level , and its application could be part of a new paradigm for risk assessment . there are numerous regulatory drivers promoting the development and application of innovative methodology for risk assessment , many of which have particular emphasis on alternative in vitro and computational methods that avoid or limit the use of animals . since 1986 , the european union ( eu ) has had legislation in place covering the use of animals for scientific purposes , in particular directive 2010/63/eu , which came into full effect in january 2013 , restricting the use of animals for assessing the toxicological properties of substances for either regulatory or research purposes to cases where it can be clearly demonstrated that no alternative method is available . the eu cosmetics regulation bans the marketing of cosmetics products in the eu that have had any ingredient tested on an animal since 11 march 2013 , irrespective of the fact that full replacement methods are not yet available for complex health effects such as chronic systemic toxicity , reproductive toxicity , and cancer . legislation dealing with risk management in other sectors , such as the registration , evaluation , authorization and restriction of chemicals ( reach ) , the plant protection products regulation , and the biocides regulation , still allows the use of animal testing to satisfy information requirements ; however , the use of alternative methods is strongly encouraged and , in some cases , obligatory if available and accepted by regulatory authorities . thus , never before has there been such demand for animal - free solutions to toxicological testing and assessments that are fit for the purpose of regulatory decision making . considering the diversity of chemical exposures under real - world conditions , there is currently no mechanism within the framework of eu legislation to facilitate the systematic , comprehensive , and integrated assessment of the combined effects of chemicals resulting from different routes and times of exposure . although risk assessment for mixtures is typically driven by exposure considerations , a key question is whether certain combinations of chemicals have independent , additive , or even synergistic toxic effects . however , mechanism - based mixture toxicology is severely hindered because the mechanism(s ) or mode(s ) of action of most chemicals is unknown or poorly described , there are no established criteria for characterizing mechanisms or modes of action , and there is no agreed terminology or formal description thereof . in addition , although assessing the combined action of co - acting chemicals may be practically possible when they have the same basic mechanism of action ( e.g. , act on the same molecular target ) , the problem becomes much more difficult when considering chemicals that impact the same biological system but act through different mechanisms . these issues have emerged prominently , for example , in the contemporary dialogue regarding endocrine - disrupting chemicals ( edcs ) because there is much debate regarding what constitutes the endocrine system and criteria to identify edcs . tackling issues such as non - monotonic dose response and the existence of thresholds is severely hampered by an over - reliance on empirical evidence derived primarily from animal experiments , a lack of emphasis on understanding the fundamentals of endocrine system dynamics , and the limitations of analytical and computational tools suitable to characterize and model the problem at hand . in the context of chemical risk assessment , the application of the new tools of experimental and computational systems toxicology is anticipated to occur first for the large numbers of environmental chemicals that have little or no toxicological data in the public domain . in this manner , there can be a prioritization of chemicals to evaluate for endocrine - disrupting activity ( see http://www.epa.gov/endo/pubs/edsp21_work_plan_summary%20_overview_final.pdf ) . toxic substances control act ( tsca ) ( see http://www.epa.gov/agriculture/lsca.html ) once the methodologies are capable of deciphering modes of action beyond those related to the endocrine system . for drug development , motivators for the application of systems tools include identification and characterization of disease - related therapeutic targets and uncovering chemical liabilities early in the drug - discovery pipeline , thus avoiding costly failures once a candidate molecule reaches clinical trials . drug safety objectives are being addressed , for example , by a large public private partnership , the etox project in the european union ( see http://www.etoxproject.eu ) , under the innovative medicines initiative ( see http://www.imi.europa.eu/ ) . the aim is to develop a drug safety database for the pharmaceutical industry and novel tools to predict better the toxicological profiles of molecules in early stages of the drug - development pipeline . another application on the horizon is the assessment of alternative chemicals designed and produced within the framework of green or sustainable chemistry . the aim in this case is to minimize the use and generation of hazardous substances . without an understanding of the toxicological liability of proposed alternatives , the push for greener chemicals is limited to using chemical engineering tools to reduce the use of known hazardous precursors , maximize atom economy in synthetic processes , and increase the energy efficiency of reactions . national academy of science has recently undertaken a project to provide a framework for evaluating safer substitutes ( see studies in progress at http://www8.nationalacademies.org/cp/projectview.aspx?key=49569 ) , including , in particular , systems toxicology . together with regulatory drivers , an overarching expectation is that systems toxicology approaches , applied in a systematic and rigorous manner , will provide more sound information on which to judge how chemicals cause biological perturbations , moving knowledge beyond knowing only what phenotypes are altered . the resulting understanding of biological responses to chemical exposure and biomolecular interactions is expected to reduce uncertainties in species extrapolations , in high - to - low dose extrapolation , and to shed light on life stage and other susceptibility factors such as genetics and pre - existing diseases . when paired with advances in hazard detection and biomonitoring strategies , systems toxicology holds the long - term promise , at least in part , to address these challenges . it will enable the gradual shift from toxicological assessment using solely apical end points toward understanding the biological pathways perturbed by active substances . systems toxicology is expected , therefore , to create knowledge regarding both the dynamic interactions between biomolecular components of a complex biological system and how perturbing these interactions with active substances alters homeostasis and leads to adverse reactions and disease . this knowledge , represented as adverse outcome pathways ( aops ) , is then expected to enable our system - level understanding of exposure to active substances and to lead to more effective risk assessment . systems toxicology is rooted in the premise that morphological and functional changes in cellular , tissue , organ , and organism levels are caused by , and cause , changes at the genomic , proteomic , and metabolomic level . a molecular system can be represented initially by high - level static biological network ( bn ) models that employ basic graph representations to map the molecular entities ( nodes ) and their interactions ( edges ) . although these basic bn models serve to map the underlying molecular wiring of a biological system , they are too simplistic in nature to describe a toxicological pathway and its dynamic nature . the description of a toxicological pathway requires the development of more sophisticated computable bns by incorporating mechanistic information through the encoding of causal relationships between the nodes . such models enable the computational analysis and contextualization of experimental data , for instance , for the quantification of network perturbation , which is further described in this perspective . ultimately , the description of dynamic toxicological processes requires the development of even more sophisticated executable bn models by incorporating the mathematical description of the dynamic behavior of these causal relationships . such bns are capable of simulating changes in state of the molecular network caused by external perturbation or modification of boundary conditions . to be useful in current risk - assessment frameworks , however , mathematical models need to be multiscale or able to traverse the different levels of biological organization ultimately to relate the dynamics at the bn level to apical adverse outcomes on which regulations are based . the most direct causal path , traversing multiple levels of organization and linking exposure , binding of the toxicants to biomolecules , intermediate key events , and apical adverse outcome , is what characterizes aops ( figure 2 ) . steps that define the systems toxicology paradigm , from biological network models to dynamic adverse outcome pathway ( aop ) models . the development of dynamic aop models enabling the simulation of the population - level effects of an exposure is the ultimate goal of systems toxicology . the first level consists of the development of causal computable biological network models that link the system s interaction of a toxicant with the organ - level responses . such models can be used to quantify the biological impact of an exposure in the context of quantifiable end points such as histology or physiological measurements . in a second step , as more mechanistic knowledge derived from quantitative measurements accumulates , dynamic models linking the exposure with the organ - level responses can be developed . ultimately , the third level of maturity is reached when the link between the exposure and the population outcome can be represented by mathematical models that enable the simulation of population - level effects of an exposure . aops are tools for describing the entire toxicological process from the molecular level , triggered by an active substance , to an adverse outcome of regulatory concern , typically observed at a tissue , organ , organism , and potentially population level . although not traditionally associated with systems toxicology , an aop is , in fact , a model that discretizes a process into key events ( ke ) ( nodes ) and the causal relationship between them ( edges ) . an aop is developed first on the basis of a qualitative model that captures kes and their high - level relationships . gradually , as detailed mechanistic knowledge accumulates , they will capture the causal relationships between the kes and the underlying molecular events . for example , such aops can be used to map quantitative information for visualization purposes or to compare and identify common features and intersections between aops . as aops are developed to include more detailed causal and quantitative information , they are expected to become computable and eventually executable , making them quantitative , sensitive , and predictive of outcome for risk assessment . the implementation and practice of systems toxicology requires several experimental approaches to generate data relevant to the question at hand . first , the exposure needs to be characterized both in terms of composition , dose , and duration and in terms of biomarkers of exposure at the subject level . the next step is to examine the effect of these exposures in biological model systems such as cellular and organotypic cultures relevant to the exposure and the observed toxicological effects . the results obtained from these in vitro experiments can then further guide the selection of relevant models of human diseases and environmental health . all of these studies will , however , yield quantitative phenotypic readouts in the context of quantitative system - wide molecular measurements . the third step is to integrate the data from these large - scale experiments , to analyze them to decipher the biological mechanisms by which adverse effects arise from exposure , and to build a functional model thereof . a new framework for risk assessment should therefore integrate this mechanistic knowledge . this perspective , derived from the presentations and discussions held at a recent symposium on systems toxicology , offers an integrated view on this trajectory from experimentation to computational analysis and risk assessment . xenobiotic exposures are acknowledged to play an overwhelmingly important role in common chronic diseases such as cancer , diabetes , cardiovascular , and neurodegenerative diseases , which constitute the major health burden in economically developed countries . it has been estimated that 90% of cancer deaths and half of heart disease mortality can not be explained by genetic factors , suggesting an environmental origin . sequencing the human genome has permitted identification of individual susceptibility to disease through genome - wide association studies , but so far these have explained comparatively little of the variability in chronic disease prevalence . in contrast , there has been a relative paucity of comparable tools for exposure assessment and exposure estimates ( e.g. , in epidemiology studies ) have relied primarily on questionnaires , geographic information , and a few targeted measurements . in environmental toxicology , exposure assessment is commonly based on quantifying chemical concentrations in the exposure medium ( e.g. , water or soil ) , although there is increasing recognition that quantification of intraorganism concentrations provides a better means to link exposure to toxicological effect . remarkable advances in analytical sensitivity and capacity , including omics technologies , have now made environment - wide association study ( ewas ) feasible , permitting detection of associations between biomarkers and human health outcomes on a much larger scale than previously possible . wild , therefore , proposed the concept of the exposome , representing all environmental exposures ( including diet , lifestyle , and infections ) from conception onward , as a complement to the genome in studies of disease etiology . in this concept , exposures are therefore not only restricted to chemicals ( toxicants ) entering the body from air , water , or food but also include endogenous chemicals produced by inflammation , oxidative stress , lipid peroxidation , infections , gut flora , and other natural processes . this internal chemical environment continually fluctuates during life because of changes in external and internal sources , aging , infections , life - style , stress , psychosocial factors , and pre - existing diseases . a challenge lies in choosing on which of the thousands of measurable biologically active chemicals to focus . paraphrasing abraham lincoln , stephen rappaport has suggested , we can not measure everything all the time , but we can measure most things some of the time , and we can measure some things most of the time . thus , the primary purpose of biomarker - based exposure monitoring should be to identify risk factors for use in epidemiological studies rather than to provide exhaustive personalized exposomes that can not be interpreted at an individual level . even a partial exposome characterization can lead to major public health benefits , for example , one or more cross - sectional exposure measurements at different time points in a prospective cohort study . because the serum exposome is enormously complex , an ewas focuses on measuring network features , such as n - linked glycoproteins or reactive electrophiles stabilized as adducts of human serum albumin . in this way the issue of timing of exposure assessment may have to be tailored to specific hypotheses by examining exposure in cohorts of different ages . the term biomarker can relate to any interaction between an external environmental agent and a biological system such as the human body . in the context of risk assessment , biomarkers of interest have been defined in terms of whether they relate quantitatively to a particular exposure , its effect in a biological system , or to the way in which the system reacts . these biomarkers are distinguished as follows:biomarker of exposure : an exogenous substance or its metabolite or the product of an interaction between a xenobiotic agent and some target molecule or cell that is measured in a compartment within an organism;biomarker of effect : a measurable biochemical , physiological , behavioral or other alteration within an organism that , depending upon the magnitude , can be recognized as associated with an established or possible health impairment or disease;biomarker of susceptibility : an indicator of an inherent or acquired ability of an organism to respond to the challenge of exposure to a specific xenobiotic substance . biomarker of exposure : an exogenous substance or its metabolite or the product of an interaction between a xenobiotic agent and some target molecule or cell that is measured in a compartment within an organism ; biomarker of effect : a measurable biochemical , physiological , behavioral or other alteration within an organism that , depending upon the magnitude , can be recognized as associated with an established or possible health impairment or disease ; biomarker of susceptibility : an indicator of an inherent or acquired ability of an organism to respond to the challenge of exposure to a specific xenobiotic substance . however , the boundaries between exposure and effect are fluid and it is therefore helpful to describe the measurement of xenobiotics and their metabolites in biological systems as biological monitoring and to restrict the term biomarker to consequences of functional or structural change ( e.g. , protein and dna adducts , chromosome aberrations , urinary proteins , and enzymes ) that represent an integration of exposure and various biological processes . for example , chemical - induced dna adducts vary at the individual level because of carcinogen metabolism , dna repair , and cell turnover , at least partially as a result of interindividual genetic variation . therefore , such biomarkers may not strictly correlate with exposure but are more likely to be associated with disease outcome , for example , cancer risk , because they reflect the consequences of exposure on a pathway relevant to carcinogenesis . in assessing biomarkers , particularly omics - based biomarkers , it is therefore important to separate those generated by environmental exposures ultimately leading to disease ( causal pathway ) from those that are manifestations of disease ( reactive pathway ) . this process of biomarker identification and validation can be separated into two main phases . in the discovery phase , ewas is used in an untargeted , data - driven way with a focus on small molecules and proteins . the main objective is to identify discriminating features that are consistent across studies , ideally those with a prospective cohort design . the exact chemical nature of the discriminating feature has to be identified , a process that may involve searching for matches in existing databases as well as additional experiments using a battery of different analytical techniques . in the second phase , candidate biomarkers are investigated in targeted , knowledge - driven follow - up studies to establish causality , prevention , diagnosis , prognosis , and treatment of diseases . an example of such a stepwise approach to the identification of biomarkers is the screening of several thousand small nonpolar molecules in serum to discover a molecular biomarker of susceptibility to colorectal cancer linked to low serum gta-446 anti - inflammatory fatty acid levels . unraveling complex environmental and genetic disease etiologies demands that both environmental exposures and genetic variation are reliably measured . characterizing the exposome represents a technological challenge at least as large as that for the human genome project , which began when dna sequencing was in its infancy . the data generated by application of omics technologies , potentially in a longitudinal fashion , poses enormous bioinformatics and biostatistics challenges , requiring substantive interdisciplinary collaboration . however , exposomics methods and goals are not as standardized as they were for dna sequencing , and , consequently , the exposome does not translate as easily to scale - up as did sequencing the human genome . nevertheless , systems biology and systems toxicology , when linked to epidemiology , offer the prospect of examining gene environment interactions and identifying biomarkers for causative pathways of diseases , monitoring their progression , and guiding treatments . a fundamental building block of systems toxicology is that exposures lead to changes at the molecular level , some of which may induce morphological or functional changes at the cellular and organism level that contribute to toxic outcomes . over the past decade , there has been unprecedented development in technologies that enable the collection of very large - scale data sets composed of quantitative information regarding molecular responses on the basis of changes in all major classes of biomolecules in living organisms . furthermore , advanced imaging methods enable the quantitative analysis of cell - level changes . hereafter , we will highlight a few of the recent developments in transcriptomic , proteomic , metabolomic , and lipidomic measurement technologies as well as high - content screening as the main emerging molecular methods that are critical enablers of systems toxicology . a broader range of technologies , including epigenetics , and their applications to toxicology have been described recently . transcriptomics or gene - expression profiling is perhaps the most widely used measurement technology in systems toxicology and is used to study the changes in expression of all mrnas in a cell population , organ , or organism . transcriptomic analysis is also the best established approach for identifying perturbed biological networks ( see below ) and thereby gaining mechanistic insight into the system s response to an exposure . the current technological basis of transcriptomics measurements for systems toxicology studies has involved oligonucleotide microarrays and , more recently , next - generation sequencing ( ngs ) . oligonucleotide microarrays represented the major technological advance in transcriptomics of the past decade and were introduced and generalized with the invention of high - density array printing by affymetrix . today , it is possible to design an array of oligomer probes that covers the whole transcriptome of any organism for which the genome sequence is known and the possible open reading frames and gene models have been identified using well - established bioinformatics analysis pipelines . these microarrays enable high - throughput parallel analysis of many samples derived from model systems as well as clinical or environmental samples . with the concomitant progress in full cdna sequencing and rna sequencing by next - generation sequencing technologies , current microarrays can cover each gene or its exons ; such exon arrays are now available for human , mouse , and rat . the use of exon arrays has improved the analysis of alternatively spliced rna transcripts as well as the accuracy of the overall gene - expression measurements . ngs technologies are emerging as the measurement methods that may supersede microarray technologies in the near future on the basis of greater accuracy , providing exact transcript counts and results that closely approach quantitative pcr . furthermore , ngs methods are more flexible as they enable gene - expression studies in organisms for which microarrays are not available , such as model systems used in environmental toxicology . finally , they are likely to offer a higher throughput than microarrays as new developments will likely allow for the analysis of thousands of transcriptome samples in a single sequencing run . the proteome represents the full complement of the proteins in a cell , organ , or organism , and proteomics is a systematic approach to characterizing all or an enriched subset of proteins therein . measuring changes in levels and modifications of proteins is applied to diagnostics , drug discovery , and investigating toxic events . proteomic data are of particular value in systems toxicology because the proteome is an important mediator of altered biological responses as a consequence of exposure to active substances . increases or decreases in protein levels may be a direct consequence of corresponding mrna - expression changes , but increases or decreases in protein function may also be influenced by post - translational modifications . for example , protein phosphorylation , which can be further addressed by high - throughput phosphoproteomics , enables the characterization of molecular events proximal to disease - related signaling mechanisms . mass spectrometry ( ms ) is widely considered to be the central technology for modern proteomics , mainly because of its unsurpassed sensitivity and throughput . thanks to the high accuracy of ms , peptides in the subfemtomolar range can be detected in biological samples with a mass accuracy of less than 10 ppm . this level of accuracy is necessary to compare proteins between samples derived from exposed and control systems . isotope tagging for relative and absolute quantification ( itraq ) is used in comparative proteomics for systems toxicology because it enables the relative quantification of protein species between samples in a nontargeted manner . this method can be further complemented with a targeted method of even higher accuracy : selected reaction monitoring ( srm ) . it leverages a unique property of ms to quantify peptides , produced by a controlled enzymatic digestion of the proteome , as a way to quantify their corresponding proteins . because it is a targeted approach , any proteomics analysis by srm requires the a priori selection of the proteins to quantify . this is generally achieved using the results of previous experiments using nontargeted approaches such as itraq combined with a review of the scientific literature to leverage prior knowledge . the list of selected proteins is then processed with bioinformatics tools to identify at least two proteolytic peptides that optimally represent the protein and distinguish it from all others . this step is followed by the selection of srm transitions for each of those peptides and several optimization and validation steps to ensure unique identification and accurate quantification . this method enables a multiplexed approach by which hundreds of proteins can be quantified in a single ms run . targeted approaches for protein quantification have been developed to produce accurate and high - sensitivity data for defined subproteomes . an emerging example is the development of surface - capture probes to interrogate the surface - reactive subproteome , a critical response gateway for active substances , as recently demonstrated for hepatocytes . antibody - based approaches are also important in targeted proteomics . for instance , reverse protein arrays ( rpas ) have evolved from classical enzyme - linked immunosorbent assays ( elisa ) and enable high - throughput analysis of up to 192 different lysates , including post - translationally modified proteins , on a zeptochip with over 200 validated antibodies . other antibody - based targeted methods , such as luminex , provide alternative measurement platforms for multiplexed , high - sensitivity and -specificity targeted proteomic data generation . these data can lead to the refinement of mechanistic details of toxicology pathways that were mainly based on transcriptomic data . metabolomics involves a comprehensive and quantitative analysis of all metabolites or low molecular weight organic or inorganic chemicals that are products or substrates of enzyme - mediated processes . in the context of systems toxicology , metabolomics is unique because it can be used to define amounts of internalized xenobiotic chemicals and their biotransformation products as well as the perturbed endogenous metabolome , which represents the ultimate change in the levels of chemical species resulting from molecular perturbations at the genomic and proteomic levels . in the first case , an understanding of the kinetic behavior of xenobiotic toxicants and their metabolites ( as well as related biomolecular adducts ) is necessary to identify candidate biomarkers of exposure for human and environmental monitoring . the second is a more conventional systems biology perspective of metabolomics , which involves both identification of metabolites and quantification of changes in their abundance and rates of production caused by an exposure . from a technical perspective , metabolomics most commonly involves nmr spectroscopy and/or ms analysis techniques in untargeted profiling or targeted analysis strategies . profiling of a metabolome may entail global detection and relative quantification of a large number of metabolites without a priori knowledge . however , targeted experiments involve the absolute quantification of a small number of metabolites ( around 20 or less ) to test a defined hypothesis . in systems toxicology , metabolomics may be accompanied by concomitant transcriptomic and proteomic measurements to provide the full context of the exposure . integrated analysis of these diverse data types is necessary to enable a full understanding of the mechanistic events driving metabolomic changes . finally , emerging application areas include the characterization of effects of mixtures on chemical responses in microfluidic organ model systems . the lipidome is the complete lipid profile of a biological system and is an example of a specialized subset of the metabolome . by extension , lipidomics is a systematic approach to characterize and quantify lipids in biological samples using , as with metabolomics , analytical methods based on nmr and ms . it has been recognized recently that alterations of lipid homeostasis contribute to several pathophysiological conditions . lipids are not only an important energy store but are also essential constituents of all cellular membranes and exert a number of signaling functions . indeed , profound changes in the cellular and tissue lipid composition occur in response to exposure to active substances and environmental factors . lipidomics is thus a powerful method to monitor the overall lipid composition of biological matrixes and has been shown to have great potential to identify and detect candidate biomarker signatures indicative of toxicity . there have been rapid recent advances in ms - based techniques capable of identifying and quantifying hundreds of molecular lipid species in a high - throughput manner . the lipid extracted from various biological matrices can then be analyzed by multiple ms platforms , either by detecting the lipids by shotgun lipidomics or after separation by liquid chromatography to detect and quantify lipids of lower abundance . performing concomitant transcriptomics analysis in samples subjected to lipidomic profiling enables the integrated analysis of these complementary data sets to derive mechanistic information regarding the toxicological meaning of lipid alterations . molecular changes measured in systems toxicology studies should , as often as possible , be correlated with cellular or tissue - level changes measured under the same conditions . although histopathology is the main approach to gather such data , phenotypic assessment of cells in culture can be performed using high - content screening ( hcs ) methods . this technique is based on computer - aided visual detection of a panel of functional biomarker measurements providing precise temporal , spatial , and contextual information that define the biological status of the cells or organs and structure of small organisms ( e.g. , early life stages in zebrafish ) . the measurement of the different biomarkers can be achieved on either a fixed specimen labeled with fluorescent reagents or directly on a living specimen during the time of the exposure . a broad panel of biomarkers can be analyzed in a multiplexed manner to provide quantitative measurements of the level of abundance and localization of proteins ( e.g. , phosphorylated protein ) and/or changes in the morphology of the cell . each biomarker can be adapted to the cell type relevant to a particular exposure modality to quantify key cellular events such as ( 1 ) apoptosis and autophagy , ( 2 ) cell proliferation , ( 3 ) cell viability , ( 4 ) dna damage , ( 5 ) mitochondrial health , ( 6 ) mitotic index , ( 7 ) cytotoxicity and oxidative stress , ( 8) nascent protein synthesis , and ( 9 ) phospholipidosis and steatosis . hcs is enabled by computer - aided automated digital microscopy ( e.g. , arrayscan ) or flow cytometry for data analysis and storage . advanced experimental technologies for systems toxicology produce unprecedented amounts of data , the management of which is a major challenge for scientists . it is expected that this trend will accelerate because of ongoing technology developments and concomitant cost decreases . therefore , we are witnessing many efforts globally to develop methods and tools to manage , integrate , and process this data . as a consequence , a wide range of open - source software solutions ( e.g. , emma and mimas ) are continuously being developed and made available to store and manage data along with the information to describe accurately the experimental setup and the conditions that led to the data . this latter point is of crucial importance to data integration , experimental reproducibility , and proper data analysis and interpretation . national academy of sciences has recently specified experimental transparency as one of the major requirements for translational omics . minimum information about microarray experiment ( miame ) -based exchange format mage - tab provides a solution for microarray data , which has also become the standard for data sets deposited in public databases such as arrayexpress ( http://www.ebi.ac.uk/arrayexpress/ ) or gene expression omnibus ( geo ) ( http://www.ncbi.nlm.nih.gov/geo/ ) . a similar standard exists for proteomics experiments , namely , the minimum information about a proteomics experiment ( miape ) . the analysis of complex data sets is greatly facilitated by computational pipelines or workflows that automate a sequence of computational tasks , often performed by independent software components . in this context , computational biology workflow management systems ( e.g. , galaxy and taverna ) have been developed to implement reproducible data analysis processes . caarray and cagrid from the national cancer institute biomedical informatics grid ( nci cabig ) are prominent examples of a systems biology data - integration environment that includes knowledge management as well as workflow systems . for instance , this environment allows integration with commercial data analysis and biological pathway analysis systems . as outlined above , the core objective of systems toxicology is to elucidate the mechanisms that causally link a well - characterized exposure to active substances with quantifiable adverse events and disease . it requires collection of experimental data reflecting molecular changes in the context of quantifiable cellular , tissue - level , or physiological changes that are linked to disease phenotypes or adverse events at the organism level . thus , systems toxicology heavily relies on computational approaches to manage , analyze , and interpret the data generated by the large - scale experimental approaches described earlier . the ultimate goal is to develop predictive in silico models that can be used in risk assessment . to reach this ambitious goal , computational systems toxicology has four major areas of focus:analyzing the massive amounts of systems - wide data generated by high - throughput methods and their integration with the structural description of the substances involved in the exposure.representing the relevant mechanisms leading to adverse outcome as biological network models that adequately describe both the normal state and the causal effect of their perturbations upon exposure to active substances ( figure 3).quantifying the dose - dependent and time - resolved perturbations of these biological networks upon exposure and assessing their overall biological impact.building and validating adequate computational models with predictive power that can be applied to risk assessment ( figure 3 ) . analyzing the massive amounts of systems - wide data generated by high - throughput methods and their integration with the structural description of the substances involved in the exposure . representing the relevant mechanisms leading to adverse outcome as biological network models that adequately describe both the normal state and the causal effect of their perturbations upon exposure to active substances ( figure 3 ) . quantifying the dose - dependent and time - resolved perturbations of these biological networks upon exposure and assessing their overall biological impact . building and validating adequate computational models with predictive power that can be applied to risk assessment ( figure 3 ) . biological network model - development process . initial static bn models can be constructed using biological facts derived from the literature . these initial models can serve as the basis to guide the development of computable bn models . these models rely on biological facts ( key events ) derived from both the literature and new experimental data and are expressed in a computable format such as the biological expression language ( bel ) . computational methods such as reverse causal reasoning and reverse engineering are used to support the model - building process . such networks can then serve as the foundation to build executable bn models in which edges are expressed with equations such as ordinary differential equations . the main applications of these broad classes of bn models are shown in light blue boxes . hereafter , we highlight just a few applications of computational systems toxicology covering computational toxicology , biological network model building , biological impact quantification , and predictive modeling to exemplify some recent developments in these four areas . computational toxicology , recently reviewed by kavlock and colleagues , is a growing area of research that integrates molecular and cell biology with chemistry and computational approaches . the objective is to increase the predictive power of toxicology by more efficiently and effectively ranking chemicals based on risk . with the advent of high - throughput experimental setups , relevant cell - based assay systems can be exposed to a large number of substances in a dose - dependent manner , and many phenotypic end points can be measured quantitatively by high - content ( or visual ) screening methods . furthermore , cell - free enzymatic and ligand - binding assays , including g - protein - coupled and nuclear receptors , kinases , phosphatases , cytochrome p450s , histone deacetylases , ion channels , and transporters , can be used in a high - throughput screening mode to profile the mechanism(s ) of action of chemicals . these approaches enable the screening of numerous substances for their impact on toxicity pathways . this will eventually lead to the creation of a comprehensive landscape of adverse cellular effects induced by biologically active substances . the integration of the biological end points with the chemical structures of the tested substances enables computational mapping ( interpolating ) of untested chemical analogues into the toxicity data , thereby providing a prediction of their relative toxicological hazard potential . toxcast aims to understand the potential health risks of a large number of diverse chemicals including failed pharmaceuticals , commodity chemicals , and pesticides by employing a large array of high - throughput screening methodologies . the first phase , or proof of concept , involved the use of almost 300 chemicals that had been well - characterized for their toxicological potential in animal models that were studied in more than 600 high - throughput screening assays . the results of this phase included a large number of expected perturbed biological pathways and the development of several predictive models based on a multifactorial data analysis . the second phase of the toxcast initiative ( http://www.epa.gov/ncct/toxcast/ ) is currently screening over 1000 such compounds against many of the same targets employed in phase i , and the data for this component has been released ( see toxcast website ) . a key element of the toxcast program involves the interpretation of the in vivo relevance of in vitro activity concentrations through a process called reverse toxicokinetics . combining this information with high - throughput exposure assessments , as introduced by wambaugh and colleagues , provides for a risk - based process of prioritization of chemicals for more intensive study of their potential to impact human health . by necessity , all descriptions of complex systems need to be simpler than the system itself . indeed , bn models are represented as diagrams of nodes ( entities ) and edges ( relationships ) . initial models can be constructed using existing knowledge drawn from both pathway databases and literature - derived information ( figure 3 ) . although publicly accessible databases of manually curated biological networks and pathways , such as the kyoto encyclopedia of genes and genomes ( kegg ) and wikipathways , are available , they generally provide a high - level static representation of our understanding of biological processes and offer only a limited representations of the adverse event mechanisms in the context of a particular exposure . therefore , they need to be further supplemented with new nodes and edges derived from experiments adequately designed to link observed molecular changes with phenotypic changes in the context of a given exposure . to extract the key molecular entities and their relationships from these data sets , sophisticated reverse - engineering algorithms , such as gns healthcare s reverse - engineer and forward simulation predictive framework or those reviewed by lecca and priami can be applied . second , these high - level pathway maps have limited computational value for the analysis of systems toxicology data sets . even though recent network informatics tools allow us to paint pathway maps according to the differential abundance of molecular entities ( such as gene - expression data ) , they generally fall short of providing a computational infrastructure for causal reasoning and hence a more complete interpretation of high - throughput data sets . the transition from static to computable bn models is thus a necessary step to analyze experimental data fully and to build our collective knowledge of the toxicological effects of exposure to biologically active substances . this transition requires a formal language to describe the causal nature of the interactions between nodes to complement the gene ontology that already provides a coherent framework for the description of the nodes themselves . the recent development of the biological expression language ( bel ) , for instance , enables this transition because it allows the semantic representation of life - science relationships in a computable format and thus the development of computable bn models composed of cause - and - effect relationships . bel was designed to capture biological cause - and - effect relationships with associated experimental context from disparate sources . bel thereby facilitates the encoding of directional relationships within computable biological network models . the bel framework is an open - source technology for managing , publishing , and using structured life - science knowledge ( http://www.openbel.org/ ) . this is unlike the biological pathway exchange ( biopax ) , which focuses on the integration and exchange of biological pathway data across a large array of existing pathway resources . building causal computable bn models is a process that has been previously described in several publications and is briefly summarized below . these models consist of qualitative causal relationships to represent biological processes and are coded in bel . the model - building process starts with the definition of its contents and boundaries by including all necessary mechanistic building blocks relevant to the bn under consideration . this step is guided by literature investigations of the signaling pathways relevant to the bn model under construction . these network boundaries are then used to screen the literature and to derive a collection of cause - and - effect relationship statements describing the relevant and known biology linked to the bn model in a given tissue context . from this the model consists of nodes , representing biological entities , such as mrna expression or protein abundance , and edges , representing causal relationships between the nodes . during this model - building process , reverse causal reasoning ( rcr ) analysis of relevant gene - expression data sets is used to guide the selection of nodes for model building . rcr interrogates the experimental data against a knowledgebase of possible causal relationships for all of the context - specific nodes and edges in the literature model to identify upstream controllers of the mrna state changes ( gene - expression changes ) observed in the data set . the potential upstream controllers identified by rcr are statistically significant potential explanations for the observed mrna state changes . these potential upstream controllers are then included as new nodes in the bn model if they have literature support for a mechanistic role in the biological process and context of interest . this allows uncovering relevant nodes and edges that were not identified during the construction of the literature - based network . rcr - based augmentation of the network model can be performed using proprietary or publicly available gene - expression data sets ( e.g. , geo or arrayexpress ) . as a final step in the construction of the network model , the nodes and edges are manually reviewed and refined to include relevant context and pathways ( e.g. , by additional literature review ) , producing the final network model . the review process should be conducted with each model periodically to ensure that newly available data sets and new literature are taken into account and thereby ensure that each model is kept up to date . furthermore , bn models need to be verified to prove that they capture context - specific nodes and edges that are supported by experimental observations . this verification can be achieved on three levels . first , existing data sets not considered during model building can be used to verify specific areas of the model . this approach is , however , constrained by the availability of relevant publicly accessible data sets that fulfill the context - specific boundary criteria . second , the structure of the model can lead to the design and conduct of new experiments designed for verification purposes , as exemplified by recent work on the computable cellular proliferation network model for normal lung cells . finally , research communities can be engaged to curate network models as implemented in wikipathways or through collaborative competition leveraging the wisdom of the crowd . by systematically investigating and describing the major mechanisms involved in toxicology , the scientific community will gradually build a compendium of computable bn models covering the crucial mechanisms involved in toxicity . three layers of information and knowledge need to be addressed to tackle this daunting task . first , the molecular entities ( i.e. , the nodes in the bn models ) will need to be further characterized , and their often diverse biological functions need to be captured in curated knowledge bases such as uniprot . second , bn model construction will need to focus on the identification of new nodes and connections in a context - sensitive manner , where cell type and tissue environment are taken into account . finally , identified mutations and their contributions to adverse manifestations need be the catalogued and mapped into the bn models in a consistent manner . this is essential to systems biology in general and systems toxicology in particular and will require the coordination of efforts that can easily dwarf the human genome project . the ability to predict the potential health risk of long - term exposure to biologically active substances is an ultimate goal in systems toxicology . however , disease or population - level responses might take decades to manifest , at which point changes in therapeutic regime , life style , or exposure would not prevent disease onset . it is therefore necessary to develop methods that are predictive and quantitative for risk assessment . it is well - understood that exposure to biologically active substances leads to modifications in the abundance of molecular entities within a living system , and , in some cases , modifications in the very nature of these entities , such as splice variants and post - translational modifications of proteins , lead to bn perturbations , which in turn can later lead to tissue - level changes and adverse events or disease . as described earlier , causal computable bn models are designed to describe these causal links between biological entities and thus provide a possible quantitative framework for biological impact assessment . indeed , these models can be used to derive a data - driven quantitative assessment of the perturbation of a given bn . recent toxicogenomics approaches have proven the potential of mechanism - based assessment methods by using a simple scoring method to evaluate the overall gene set - level expression changes to profile biologically active substances . although the use of gene sets provides a first assessment based on gene lists derived from , for example , kegg and other pathway databases , bn models will provide a more accurate and detailed analysis of the mechanistic effects of the exposure as well as a quantitative evaluation of the perturbation of each model under consideration and an overall pan - model biological impact . by way of example , we have published a five - step approach to derive such a biological impact factor ( bif ) for a given exposure or stimulus . this approach employs causal computable bn models as the substrate to analyze high - throughput data sets derived from in vitro and in vivo dose response and time - course experiments . briefly , quality - controlled measurements generated in vitro or in vivo constitute systems response profiles ( srps ) for each given exposure in a given experimental system . these srps express the degree to which each individual molecular entity is changed as a consequence of the exposure of the system . these srps are then analyzed in the context of a collection of bn models designed to cover the molecular mechanisms perturbed by the exposure . this analysis is designed to answer questions about the completeness of the models ( i.e. , whether additional nodes and edges need to be added to the model to cover all of the exposure effects and to identify any new mechanisms not yet reported in the literature ) . this analysis employs rcr , by which downstream measurements ( e.g. , gene expression ) are causally mapped as effects of an activity of individual elements in the model . once this step is completed , we apply specific algorithms to derive the specific network perturbation amplitude ( npa ) scores . specifically , the npa algorithm translates gene fold - changes ( or other downstream measurements ) into the perturbation of individual elements in the model and finally aggregates these perturbations into a bn - specific score . by providing a measure of biological network perturbation , npa scores allow correlation of molecular events with phenotypes that characterize the network at the cell , tissue , or organ level . finally , the overall biological impact of a perturbing agent can be estimated by aggregating the npa scores from several bn models into one holistic value , the bif , that expresses the overall impact on the entire biological system . this bif can be readily correlated with other measurable end points ( e.g. , collected at a higher level of biological organization through phenotypic changes ) or used to generate testable hypotheses that could provide new insight into mechanisms of disease onset or progression . the bn models described above provide either a static or a computable environment to contextualize experimental data in the framework of current knowledge . although these approaches enable a detailed understanding of molecular processes and the identification of new nodes and edges to supplement existing bn in a context - sensitive manner ( e.g. , particular cell type in a specific tissue environment ) , they do not exhibit the simulation capabilities necessary to understand the dynamic response of a molecular system to an exposure or more importantly to predict the effect of new exposures a priori . key to this transition from descriptive to executable biology is the development of mathematical models that represent the dynamic properties of biological processes and their corresponding computational models that can be executed in silico . therefore , the ultimate objective of computational systems toxicology is to develop dynamic ( executable ) mathematical models of these bns in such a way that their simulation correctly represents the observed behavior of the biological process ( figure 3 ) . such models will eventually permit true predictions , such as the emergence of new network behaviors following in silico perturbations . the development of such models is currently still in its infancy and represents a major frontier in systems biology and systems toxicology . epa and aims to build a virtual embryo modelin silico to predict the potential for environmental chemicals to affect embryonic development . the predictive power of the model will be tested with a selection of well - characterized chemicals with known health effects in animal studies . another example of predictive modeling is provided by the physiolab platform developed by entelos ( foster city , ca , usa ) . several large - scale dynamic mathematical models were developed and include both human and mouse models of diseases : ( 1 ) virtual nod mice ( type 1 diabetes physiolab ) , ( 2 ) virtual apoe mice ( apoe mouse physiolab ) , ( 3 ) virtual humans in both rheumatoid arthritis and ( 4 ) the skin sensitization physiolab . all of these physiolab platforms are large multiscale biological models where molecular and cellular interactions are generally represented by ordinary differential equations , the numerical solution of which simulates the dynamic response of the biological system across several layers of organization . although most of these computational systems are primarily designed to plan and understand , for example , clinical studies , two of them are sample applications for systems toxicology . for instance , the apoe physiolab was designed to understand the toxicological effects of cigarette smoke and cessation on atherosclerotic plaque growth . the objective was to build a framework to translating experimental apoe mouse results to the human setting despite the uncertainty around the mechanisms underlying disease etiology , the relative importance of these mechanisms as drivers of progression , and how these roles change in response to lifestyle changes . this model may be used to optimize in vivo experiments and to pave the way for a similar modeling approach for human disease . the second example is the dilisym modeling software , a multiscale representation of drug - induced liver injury ( dili ) that is associated with simpops ( see http://dilisym.com/products-services/simpops.html ) , a collection of simulated individuals that match a particular range and distribution of parameters and patient characteristics , allowing the exploration of the interindividual variability in response to potential dili - causing drugs . the third example is driven by recent changes in european union legislation , prohibiting the use of in vivo experimental animal models for testing cosmetics ( eu cosmetics regulation ) . as a consequence , nonanimal approaches to provide the data for skin sensitization risk assessment need to be further developed . in this context , maxwell and mackay have developed an in silico model of the induction of skin sensitization to characterize and quantify the contribution of each pathway to the overall biological process . the resulting skin sensitization physiolab is thus an important attempt to integrate data derived from different forms of nonanimal hazard data to inform new skin sensitization risk - assessment decisions and demonstrates how computational systems toxicology can contribute to hazard evaluation in a new framework for consumer safety risk assessment . predictive model development has also been started in nonhumans , namely , fish , and focuses on the reproductive effects resulting from disturbance of the endocrine axis by environmental chemicals in a research program led by the u.s . the development of dynamic ( executable ) mathematical models of aops linking exposure to individual and population - wide adverse outcomes is the grand challenge of computational systems toxicology . although new computational methods and mathematical tools are certainly needed , the major challenge in building such models is our limited understanding of how bn perturbations are linked to the kes in the aops at a higher level of biological organization . although this obviously requires a deep understanding of the dynamic behavior of the causal link between bn perturbations and kes , it also requires a far deeper understanding of the causal link between these kes and the adverse outcome for the individual and how these translate to the population . although the dynamic models described above are a step toward understanding the links between bn perturbations and individual adverse outcomes , the last step could be achieved by creating large sets of mechanistically distinct virtual humans that , upon simulation , statistically match the prevalence of phenotypic variability reported in human studies . the recently reported algorithm termed mechanistic axes population ensemble linkage ( mapel ) , which utilizes a mechanistically based weighting method to match clinical trial statistics , may be an approach enabling such population - based risk assessment and expands on the concept of simpops . similar model can be developed for other organisms , such as fish or plants . such dynamic models will eventually allow for simulating the behavior of biological systems at both the individual and population levels to generate new hypotheses and insights into the potential effect of an exposure a priori . the needs of risk assessment are context - dependent and can vary from simple classification of a substance for hazard ( e.g. , is it genotoxic or not ) to prioritization by the nature and severity of hazard for further investigation to quantitative estimates of risk to determine the urgency and nature of any risk - management action . although existing approaches have served society well , there is increasing concern , some real and some hypothetical , that there may be important weaknesses in the assumptions used in current risk assessment . these include the shape of the dose response relationship under human - relevant exposures , whether biological thresholds exist and for what end points , the extent of population variability in toxicological response , and the influence of factors such as life stage on response . despite a substantial amount of research , some in large groups of animals , it has not been possible to resolve these concerns . this shortcoming is due to the intrinsic limitation of all experimental observations by the power of the study . as discussed above , systems toxicology has real potential to provide the data as well as the quantitative mechanistic models to address these issues . in this sense , systems toxicology can provide a deep mechanistic understanding of toxicological effects , permitting prediction of responses to chemicals . if adequately described , a systems description should enable prediction of responses for which experimental data were not available ( i.e. , the system will exhibit emergent properties entailing novel patterns and properties arising from the inherent structure of the system ) . hence , it will be some time before they can be used in risk assessment . furthermore , risk assessors are not often well - trained in relevant mathematical procedures and hence there is inherent reluctance to adopt computationally intensive approaches with which they are poorly familiar . one way in which this might be achieved is by adapting the more familiar mode of action / adverse outcome pathway paradigm . a moa / aop comprises a series of quantifiable key events that are necessary but individually usually not sufficient for a toxicological response to a chemical . it should be possible to use a modular approach to the development of systems toxicology , where one starts with a systems - based model of a single key event and incorporates that into a quantitative biological model with dose response data for the other key events at an operational level . in this way , one could progressively add systems - based modules describing each key event until a full systems - based description of the response is achieved . this will also allow incorporation of systems toxicologically in a progressive fashion into risk assessment , while providing risk assessors the opportunity to become familiar and comfortable with evaluating such data . major efforts are underway to develop nonanimal test methods , such as the high - throughput toxcast program discussed earlier . however , in themselves , these platforms are unlikely to meet the needs of risk assessment beyond hazard identification . taking a further step will require additional quantitative approaches , for which systems toxicology would be ideally suited . however , development of suitable models will be a complex and time - consuming undertaking . mode of action can provide a translational bridge , enabling the incorporation of information from in vitro and other approaches into a systems - based description of key events , thereby enabling the stepwise development of a full systems toxicology characterization of the organ and eventually the organism we stand at the edge of an unprecedented transformation in the conduct of toxicological evaluations . a central tenant of the new toxicology involves applying modern molecular analysis techniques to elucidate mechanisms of toxicity and is being enabled by several factors . the first is the increasing power and availability of molecular measurement tools able to probe the functioning of biological networks inside organisms , organs , tissues , and cells . the second is the increasing affordability of high - throughput and high - content characterization approaches that can be applied to thousands of chemicals in short time periods rather than the chemical - by - chemical approach of the past 4 decades that involves thousands of animals and perpetual high costs and years of duration . the third enabler is the increasing computational power , data - storage capacity , and information - management tools now available to the scientific community that has facilitated the ability to employ complicated systems biology models . the fourth enabler is the acceleration in the development of adequate in vitro test systems to complement and gradually replace animal models . the fifth enabler is that of significant resource investment by governments throughout the world in funding efforts to develop the scientific foundation of systems toxicology . for example , darpa ( the defense advanced research projects agency , usa ) recently invested $ 70 million to develop up to 10 organs on chips within the next 5 years . likewise , the eu has invested several hundred million euros in fp7 ( see http://www.axlr8.eu/ ) for various supporting projects such as seurat-1 ( safety assessment ultimately replacing animal testing , see http://www.seurat-1.eu ) , with prospects to continue the investment in the european funding framework , horizon 2020 . ultimately , these international efforts need to be combined to produce a real shift in the risk - assessment paradigm that not only assures the highest levels of protection of public health and the environment but also enables economic growth and global trade . finally , a key requirement in addressing the challenge is that contributors and stakeholders , whether from government , academia , industry , ngos , regulatory bodies , or the public at large , reach a consensus that new approaches based on systems toxicology reach the standards they demand .

systems toxicology is the integration of classical toxicology with quantitative analysis of large networks of molecular and functional changes occurring across multiple levels of biological organization . society demands increasingly close scrutiny of the potential health risks associated with exposure to chemicals present in our everyday life , leading to an increasing need for more predictive and accurate risk - assessment approaches . developing such approaches requires a detailed mechanistic understanding of the ways in which xenobiotic substances perturb biological systems and lead to adverse outcomes . thus , systems toxicology approaches offer modern strategies for gaining such mechanistic knowledge by combining advanced analytical and computational tools . furthermore , systems toxicology is a means for the identification and application of biomarkers for improved safety assessments . in systems toxicology , quantitative systems - wide molecular changes in the context of an exposure are measured , and a causal chain of molecular events linking exposures with adverse outcomes ( i.e. , functional and apical end points ) is deciphered . mathematical models are then built to describe these processes in a quantitative manner . the integrated data analysis leads to the identification of how biological networks are perturbed by the exposure and enables the development of predictive mathematical models of toxicological processes . this perspective integrates current knowledge regarding bioanalytical approaches , computational analysis , and the potential for improved risk assessment .

Introduction The Exposome: A Systems Approach to Understanding Exposure Large-Scale Molecular Measurement Data and Information Management Computational Platforms for Systems Toxicology Systems Toxicology and Risk Assessment Outlook

PMC5155131

matsuba and shimamura s report on the exercise physiology and performance testing of racehorses ( fig . 1fig . this report was a large book of 294 pages ( b5 size ) consisting of 2 parts ; the first part was entitled exercise physiology of the racehorse , and the second part was entitled performance testing of the racehorse . ) was a large book of 294 pages ( b5 size ) consisting of 2 parts ; the first part was entitled exercise physiology of the racehorse , and the second part was entitled performance testing of the racehorse . research results obtained from a large - scale project during the period of 1928 to 1932 , which was conducted at the shimofusa imperial farm and the koiwai farm , the two greatest farms for racehorses at that time , and included a total of 92 thoroughbred racehorses , were reported in this book . matsuba and shimamura of the university of tokyo played major roles in this study , and were assisted by 25 students . this project was performed as a commissioned research project from the teikoku keiba kyokai ( the imperial racing association ) . because this report was written using an archaic form of japanese language characters nevertheless , it was a valuable contribution because the raw numbers for every variable measured were cited and it contained a large number of data tables ( table 1table 1.raw numbers and percentages for pulse rate obtained at the koiwai farm during the period of july 29th to august 7th , 1931horse nameraw numberpercentageprepost1 hr2 hr3 hrprepost1 hr2 hr3 hrdai 4 rachidia34106403434100.0310.3117.7100.0100.0shining star3398413536100.0297.0123.4106.1109.1dai 6 shian mor34112393637100.0329.4109.5105.9108.8dai 4 shian mor36116423836100.0322.2113.8105.5100.0dai 2 shian mor3494403937100.0276.4117.7114.7108.8dai 3 shian mor30102373434100.0340.0123.3113.3113.3dai 5 shian mor34104443634100.0305.8129.4105.9100.0dai 7 shian mor3698363232100.0272.2100.088.188.1dai 19 koiwai3094373330100.0313.3123.2110.0100.0average33.4102.739.635.234.4100.0307.5118.6105.4103.0trot 800 m , canter 1,600 m , gallop 800 m. the person in charge of experiment : shigeo matsuba . this report was a large book of 294 pages ( b5 size ) consisting of 2 parts ; the first part was entitled exercise physiology of the racehorse , and the second part was entitled performance testing of the racehorse . trot 800 m , canter 1,600 m , gallop 800 m. the person in charge of experiment : shigeo matsuba . cited from a report by matsuba and shimamura . the first chapter of the first portion of the book began with a sentence regarding the factors necessary for exercise including healthy internal organs and tough skeletal muscles , especially for racehorses for which high speeds must be attained within a short time . matsuba and shimamura stated that the reason why this project was performed was that while there were many papers on the exercise physiology of the warhorse at that time , there were few reports on the exercise physiology of the racehorse . as an overall research plan , the first and second years were considered to be preparatory periods for conducting research on the exercise physiology of the racehorse , and the third to fifth years were to be intensive periods for research on exercise physiology and for evaluating performance testing of racehorses . they stated that they decided to measure many variables because most previous studies on the exercise physiology of racehorses had measured only a couple of variables . the first seven variables studied were defined as being clinical variables , and the eighth to twentieth variables were defined as blood variables as follows : 1 ) body temperature , 2 ) respiratory frequency , 3 ) pulse rate , 4 ) blood pressure , 5 ) cardiac function ( auscultation of cardiac sound ) , 6 ) state of conjunctiva , 7 ) state of perspiration , 8) specific gravity of blood , 9 ) erythrocyte count , 10 ) leukocyte count , 11 ) hemoglobin concentration , 12 ) packed cell volume ( pcv ) , 13 ) viscosity of blood , 14 ) blood glucose concentration , 15 ) plasma co2 concentration , 16 ) plasma ph , 17 ) serum freezing point depression , 18 ) serum refractive index , 19 ) erythrocyte fragility , and 20 ) serum spectrogram . fourteen of these variables were measured during the first year , and all 20 variables were measured in the fourth year , while 10 variables were measured in the fifth year . measurements were made before exercise ( pre ) , just after exercise ( post ) , 1 hr after exercise ( 1 hr ) , 2 hr after exercise ( 2 hr ) , and 3 hr after exercise ( 3 hr ) . a summary of surveys and actual performance protocols is provided in table 2table 2.summary of surveysnumberdateplacehorseexercise protocol1st1928 ( aug . 14th21st)shimofusa farm 6 ( 6 four - yo : 6 colts)c ( 11,200 m)2nd1928 ( nov . 12th20th)shimofusa farm 7 ( 3 two - yo & 4 four - yo : 4 colts & 3 fillies)c ( 6,400 m)3rd1929 ( jul . 16th26th)shimofusa farm10 ( 7 two - yo & 3 three - yo : 3 colts , 2 fillies & 5 geldings)c ( 5,000 m ) , w ( 200 m ) , or t ( 200 m)4th1929 ( jul . 16th24th)shimofusa farm10 ( 2 two - yo , 6 three - yo & 2 four - yo ; 10 geldings)c ( 5,000 m ) , w ( 200 m ) , or t ( 200 m)5th1930 ( mar . 22nd31st)shimofusa farm 9 ( 9 two - yo : 9 colts)t ( 800 m ) + c ( 1,600 m ) + g ( 800 m)6th1931 ( mar . 16th31st)shimofusa farm15 ( 15 two - yo : 7 colts & 8 fillies)t ( 800 m ) + c ( 1,600 m ) + g ( 800 m)7th1931 ( jul . 7th)koiwai farm 9 ( 9 two - yo : 9 colts)t ( 800 m ) + c ( 1,600 m ) + g ( 800 m)8th1932 ( jun . 18th24th)shimofusa farm16 ( 16 two - yo : 8 colts & 8 fillies)t ( 800 m ) + c ( 1,600 m ) + g ( 800 m)9th1932 ( aug . 3rd6th)koiwai farm10 ( 10 two - yo : 3 colts & 7 fillies)t ( 800 m ) + c ( 1,600 m ) + g ( 800 m)although various exercise protocols were used in the 1st and 2nd years ( 1st to 4th surveys ) , the same exercise protocol was used from the 3rd to 5th years ( 5th to 9th surveys ) . w , walk ; t , trot ; c , canter ; and g , gallop . two - yo , 2-year - old - horse ; three - yo , 3-year - old - horse ; and four - yo , 4-year - old - horse . cited from a report by matsuba and shimamura .. the total number of thoroughbred racehorses used in this study was 92 ( 71 two - year - old , 9 three - year - old , and 12 four - year - old ; 49 colts , 28 fillies , and 15 geldings ) , and all were raised at either the shimofusa imperial farm or the koiwai farm . the total duration of each survey was long , and a total of 61 days were used for the surveys . although a variety of exercise protocols were used during the first and second years ( the first to fourth surveys ) , the same exercise protocol ( trot , 800 m , canter , 1,600 m , gallop , 800 m ) was used from the third to fifth years ( the fifth to ninth surveys ) . in this article , the authors showed changes in selected variables . although data were plotted individually in the original report ( fig . . 2.pulse rate measured at the koiwai farm in 1931 ( july 29th august 7th ) . data obtained from each horse were plotted individually . cited from a report by matsuba and shimamura . ) , the authors generated graphs ( mean sd ) using data from tables obtained during the seventh survey on july 29 , 1931 , at the koiwai farm . although various exercise protocols were used in the 1st and 2nd years ( 1st to 4th surveys ) , the same exercise protocol was used from the 3rd to 5th years ( 5th to 9th surveys ) . w , walk ; t , trot ; c , canter ; and g , gallop . two - yo , 2-year - old - horse ; three - yo , 3-year - old - horse ; and four - yo , 4-year - old - horse . cited from a report by matsuba and shimamura . pulse rate measured at the koiwai farm in 1931 ( july 29th august 7th ) . data obtained from each horse were plotted individually . cited from a report by matsuba and shimamura . it was showed that average body temperature rose from 38.1c to 39.5c from pre to post and returned to pre values within 2 hr after exercise . they mentioned that the temperature increase was larger for a canter than for a walk and trot and was larger during a canter of longer distance than for a shorter canter . respiratory frequency ( rf ) was measured based on counting movements of the ribcage or the upper flank with the naked eye . average rf increased from 12.4 breath / min at pre to 71.3 breath / min at post and decreased to 21.4 breath / min at 1 hr and 14.7 breath / min at 3 hr ( fig . the authors generated this graph using data from a table in a report by matsuba and shimamura . ) . they noted that because rf was very changeable just after exercise , rapid measurements would be required to accurately measure it . in 1944 , okabe and sugiyama measured rf continuously during exercise at a walk , trot , and canter by recording expiratory sound with a microphone attached near a nostril and reported that rf immediately after exercise differed completely from rf during exercise because a very rapid decrease in rf occurred just after finishing exercise . the authors generated this graph using data from a table in a report by matsuba and shimamura . the mean pulse rate at pre was 33.4 beat / min and increased to 102.7 beat / min at post , after which it decreased to 39.6 beat / min at 1 hr after exercise and 34.4 beat / min at 3 hr after exercise ( fig . the authors generated this graph using data from a table in a report by matsuba and shimamura . ) . although it was considered that heart rate ( hr ) measurement was preferable to that of pulse rate by auscultation , recording of electrocardiograms ( ecgs ) of horses to calculate hr was impossible at that time , even at rest . moreover , although pulse rate immediately after exercise was also measured by auscultation , because direct measurement of hr during exercise was impossible at the time , pulse rate immediately after exercise was considered to indicate hr during exercise . the first ecg recordings of horses in japan were carried out in 1944 by yasuda et al . , and the first ecg recording in the world of a horse during field exercise that included fast galloping was carried out in 1964 by nomura et al . the authors generated this graph using data from a table in a report by matsuba and shimamura . blood pressure was measured in these studies using the tycos sphygmomanometer at the point of the radial artery , and systolic pressure was recorded by measuring pulses of the digital artery . average blood pressure increased from 156 mmhg at pre to 238 mmhg at post and then decreased to 176 mmhg at 1 hr and 158 mmhg at 3 hr after exercise ( fig . 5fig . the authors generated this graph using data from a table in a report by matsuba and shimamura . ) . it was noted that blood pressure increased even at lower intensity exercise , but the magnitude of the increase was much larger with higher intensity exercise . although the average value of 156 mmhg at pre was considered to be relatively high , it is unknown if the values had been converted to account for hydrostatic pressure differences to the values at the height of the heart or not . considering the difference in height between the height of the radial artery and the heart , the reported values might be reasonable , even if the values were not converted . the authors generated this graph using data from a table in a report by matsuba and shimamura . the average erythrocyte count at rest of thoroughbred racehorses was noted to be markedly higher than those of half - blooded horses . erythrocyte counts increased by about 2030% with exercise , and it returned to the pre value by 1 hr after exercise ( fig . the authors generated this graph using data from a table in a report by matsuba and shimamura . ) . the average pcv at rest was approximately 40% and was higher than that of other types of horses . pcv increased to about 50% at post and decreased to almost the pre value 1 hr after exercise . matsuba and shimamura speculated that one of the main reasons for these increases after exercise might be water loss caused by exercise . approximately 40 years later , the reason for these changes was clarified by persson et al . . they performed splenectomies on standardbred trotting horses and found that the equine spleen can release erythrocyte - rich blood into the systemic circulation during exercise , increasing the erythrocyte count and pcv . the authors generated this graph using data from a table in a report by matsuba and shimamura . the average hemoglobin ( hb ) concentration at pre was 14.2 g / dl , increased to 20.0 g / dl at post , and then decreased gradually to 16.0 g / dl at 1 hr and 14.6 g / dl at 3 hr ( fig . 7fig . 7.changes in hemoglobin concentration ( [ hb ] ) at different times relative to exercise . the authors generated this graph using data from a table in a report by matsuba and shimamura . ) . they stated that the reason for the increase in hb concentration was due to the increased erythrocyte count because hb exists inside of red blood cells . when blood viscosity was defined as its ratio to the viscosity of water ( water viscosity=1.000 ) , it was 5.06.0 at rest . the average blood viscosity was 6.4 at pre and increased to 10.6 with exercise , and returned to the pre value at 1 hr after exercise . changes in hemoglobin concentration ( [ hb ] ) at different times relative to exercise . the authors generated this graph using data from a table in a report by matsuba and shimamura . when matsuba and shimamura looked at all data on changes in variables induced by exercise that were measured in a single horse during the first and second years of the study ( 1928 and 1929 ) , they noticed there was a tendency for many variables measured in horses considered to be fitter to recover to their pre values more rapidly than those of other horses ; that is , it appeared that an excellent horse could recover from fatigue caused by intense exercise more rapidly than other horses . accordingly , they decided to evaluate the recovery status of each variable by calculating the percentage of its pre value at different times during recovery . in the case of body temperature , when recovery state was evaluated , the numbers used for calculation were the raw temperature data minus 30 , because the rate of increase in body temperature sometimes did not exceed 5% . for the same reason , data used for specific gravity of blood measurements were the raw data minus 1.000 . as mentioned above , measurements of 20 variables were carried out a total of five times before exercise ( pre ) , just after exercise ( post ) , 1 hr after exercise ( 1 hr ) , 2 hr after exercise ( 2 hr ) , and 3 hr after exercise ( 3 hr ) . later , the number of variables was decreased to 11 , as shown in table 3table 3.raw numbers and percentages for yamaougi in the 5th survey at the shimofusa imperial farm in march 1930examines itemraw numberpercentageprepost1 hr2 hr3 hrprepost1 hr2 hr3 hrbody temperature3838.738.438.438.6100.0108.8105.0105.0107.5respiratory rate1538262018100.0253.3173.3133.3130.0pulse rate3672413636100.0200.0113.9100.0100.0blood pressure164194174178174100.0118.4106.1108.5106.1specific gravity of blood1.0541.0541.0541.0301.054100.0100.0100.098.1100.0blood viscosity4.84.54.94.94.8100.093.8102.1102.1100.0[hemoglobin]9.510.510.610.09.7100.0109.7111.3104.8101.6erythrocyte count7,7609,4409,1768,7448,640100.0121.6118.2112.7111.3leukocyte count10,10010,90011,55010,55511,220100.0107.9114.4104.5111.1blood glucose10790909595100.084.184.189.089.0co2 concentration59.455.555.556.559.4100.093.493.495.1100.0percentages in boldfaced type indicate that the parameter recovered to 100 5% . cited from a report by matsuba and shimamura .. the percentage of the pre value for each variable at 1 hr , 2 hr , and 3 hr was calculated . when the percentage reached within 100 5% ( 95105% ) compared with the pre value , the variable was considered to be recovered . then the percentage of the total number of recovered variables at each time ( rr at 1 hr [ rr@1hr ] , rr at 2 hr [ rr@2hr ] , and rr at 3 hr [ rr@3hr ] ; where rr is recovery rate ) was calculated , and an overall average was calculated from these ( ( rr@1hr + rr@2hr + rr@3hr)/3 ) ) ; the authors proposed calling this overall average the recovery rate of each horse . percentages in boldfaced type indicate that the parameter recovered to 100 5% . cited from a report by matsuba and shimamura . they found a tendency for horses in good condition to have a lower increase in pulse rate after constant exercise , and the blood pressure of these horses tended to be higher . based on these observations , they thought that the d / p ratio ( where d is the blood pressure difference between pre and post and p is the pulse rate difference between pre and post ) might become larger as training advanced . they named the d / p ratio the pulse pressure ratio and considered that it might be a valuable index for evaluating fitness of racehorses in future research . additionally , measurement of pulse rate and blood pressure was thought to be useful because they could be obtained noninvasively . the recovery rate and d / p ratio were obtained from every horse for all surveys from the fifth through the ninth . table 3 shows results from a single horse , yamaougi , collected during the fifth survey in march 1930 . percentages in boldfaced type show that this variable had recovered to within 100 5% . as shown in table 4table 4.calculation of the recovery rate for yamaougi1 hr2 hr3 hrnumber of examined items111111number of recovered items375recovery rate0.270.640.45the recovery rate was 0.27 ( 3/11 ) at 1 hr , 0.64 ( 7/11 ) at 2 hr , and 0.45 ( 5/11 ) at 3 hr . the authors generated this table using data from a table in a report by matsuba and shimamura . , the recovery rate was 0.27 ( 3/11 variables ) at 1 hr , 0.64 ( 7/11 ) at 2 hr and 0.45 ( 5/11 ) at 3 hr , and the overall average was 0.45 ( ( 0.27 + 0.64 + 0.45)/3 ) . table 5table 5.summary of results of the 5th survey conducted during march 1930 at the shimofusa imperial farmnumbernamerecovery raterecovery rate at each timed / p1 hr2 hr3 hr1yamayoshi0.820.640.910.912.212yamanari0.700.730.730.642.163yamahisa0.640.550.730.641.814yamahide0.600.450.640.731.475yamataka0.580.550.640.551.736yamamichi0.580.450.550.731.527yamamune0.510.450.640.451.308yamanobu0.480.500.450.451.509yamaougi0.430.200.600.501.83d / p : ratio of d ( blood pressure difference between pre and post ) and p ( pulse rate difference between pre and post ) . cited from a report by matsuba and shimamura . the recovery rate was 0.27 ( 3/11 ) at 1 hr , 0.64 ( 7/11 ) at 2 hr , and 0.45 ( 5/11 ) at 3 hr . the authors generated this table using data from a table in a report by matsuba and shimamura . d / p : ratio of d ( blood pressure difference between pre and post ) and p ( pulse rate difference between pre and post ) . cited from a report by matsuba and shimamura . a follow - up survey was conducted on the relationship between recovery rate and racing performance of 25 horses whose records could be traced over their entire racing careers . a horse s winning percentage was selected as its index of performance , and the relationship between winning percentage and recovery rate was found to be positively correlated ( fig . 8fig . the authors generated this graph using data from a table in a report by matsuba and shimamura . ) . however , matusba and shimamura pointed out that although the recovery rate could indicate the fitness level and capacity for performance at the time when the recovery rate was measured , because this index was obtained from a single measurement at the time a horse was 2 years old , recovery rate would not be particularly useful for evaluating performance after a horse had undergone training and started racing . it was recommended that it would be necessary to make multiple recordings of the recovery rate in order to evaluate a horse s racing performance exactly . finally , they mentioned that since there are numerous factors that can influence racing performance , it would be difficult to predict performance by using only the single variable of recovery rate . they proposed that both talent and training are important factors that have an influence on a horse s performance and emphasized the importance of training and of the need for research on the effects of training . the authors generated this graph using data from a table in a report by matsuba and shimamura . kimata , who was a student of matsuba , wrote interpretive articles in a journal for veterinarians on the exercise physiology of animals , which at that time was a new concept in veterinary medicine . in those articles , he described in detail the effects of exercise on body temperature , respiration , pulse rate , and blood temperature . matsuba and shimamura established new methods for performance testing of racehorses and indicated that studies on the effects of training would become an important area of research in the future . accordingly , a series of experiments on the effects of training were conducted at the nakayama race club ( now nakayama racecourse of the japan racing association ) over a total of four years . a research report entitled studies on the performance and training of the racehorse , which contained results of those experiments , this report was a large book of 492 pages ( a5 size ) and mainly dealt with the effects of training ; the report covered physical changes of blood characteristics with training . all of the results were published as five papers in the journal of the central society for veterinary medicine in 1938 . the series of experiments was carried out over a total of four years during the periods of 19331934 and 19361937 . this project was carried out as research commissioned by the teikoku keiba kyokai ( the imperial racing association ) . although it had become possible to evaluate the performance of racehorses by using the new index , the recovery rate , the study that introduced the recovery rate had been performed on 2-year - old racehorses trained on training farms , and the horses had not yet raced . therefore , matsuba decided to perform a series of experiments on racehorses trained at racecourses in order to better understand their exercise physiology and performance . however , he had been thinking that because it was not easy to evaluate performance even on training farms where it was possible to conduct standardized and regulated exercise tests under controlled experimental conditions , it would be difficult to perform standardized exercise tests under nonexperimental situations , e.g. , during actual training at a racecourse . accordingly , as the first experiment , he decided to evaluate the training method that racehorses actually used at the nakayama race club in order to understand its effects on exercise physiological variables and create a database for future studies matsuba dubbed the training method that was being used at nakayama race club the present method of training . training was carried out from the end of july to the beginning of december in 1933 . table 6table 6.feeding regimens in september at the nakayama race clubseptembermorningnooneveningnightoats ( g)630840840wheat bran ( g)220220220chaff ( g)2,2602,2602,260grass ( g)3,000salt ( g)22.622.622.6fresh forage ( g)15,000 ( per day)cited from a report by matsuba horses were trained with practical methods and ran several times at speeds assumed to be maximal during racing . the first stage began on july 25th , and a maximal speed run was carried out on october 14th . in the second stage , which began on october 15th , after a short period of rest , training was carried out , and a maximal speed run was then conducted on november 21st . in the third stage , which began on november 22nd , after a short period of rest , training was again started , and a maximal speed run was carried out on december 9th . table 7table 7.training protocol for a horse during the period of september to octobertrot ( m)canter ( m)total time ( sec)gallop ( m)total time ( sec)sep . 253,2001,600136263,2001,600136271,6001,600119282,4001,600136291,6001,600136301,6001,60013180052.3oct . 13,20022,40080013631,6001,60013641,6001,60011253,2001,60013661,6001,60012671,6001,60010883,20092,400800126101,6001,600126111,6001,600112123,2001,600136131,6001,600136141,6001,600108the authors generated this table using data from a table in a report by matsuba . the distance of an intensive gallop workout was 1,600 m in most cases , and the time required to complete it was 110 sec ; the horse s average speed was 14.5 m / sec ( 13.8 sec/200 m ) . the canter distance on a typical day was also 1,600 m , and its average speed was 11.111.7 m / sec ( 1718 sec/200 m ) , while the distance trotted was 1,6003,200 m. in general , the distance trotted was longer and the distance cantered on a typical day was shorter compared with protocols used currently . cited from a report by matsuba . the authors generated this table using data from a table in a report by matsuba . five clinical variables ( body temperature , respiratory frequency , pulse rate , blood pressure , and cardiac sounds ) were measured once each week . three variables ( erythrocyte count , blood glucose concentration , and blood co2 concentration ) were measured about once every two weeks as an examination of blood characteristics . urine analysis included eleven variables ( color , turbidity , ph , specific gravity , viscosity , glucose concentration , protein concentration , phosphoric acid concentration , total nitrogen concentration , creatinine concentration , and indican ) that were measured every 10 days . six variables ( height , body weight , chest circumference , cannon circumference , chest cross - sectional area , and cannon cross - sectional area ) were measured every two weeks during a physical examination . seven variables ( body temperature , respiratory frequency , pulse rate , blood pressure , erythrocyte count , blood glucose concentration , and blood co2 concentration ) were measured four times at the time of mock races on september 30th , november 21st , november 28th , and december 9th . the results of monitoring clinical variables that were ovserved with the progress of training were as follows : 1 ) body temperature was unchanged , 2 ) respiratory frequency decreased , 3 ) pulse rate decreased , 4 ) blood pressure increased , and 5 ) cardiac sounds became clearer . matsuba suggested that changes observed in pulse rate , blood pressure , and cardiac sounds were related to improved cardiac function and that the decrease in pulse rate was associated with an increased stroke volume . in addition , because pulse rate was increased by a short period of rest from november 22nd , it was suggested that there would be interesting relationships between training and pulse rate . changes in blood variables with the progression of training were as follows : the erythrocyte count and blood glucose increased , and the blood co2 concentration initially increased but later decreased . changes in urine variables occurred irregularly and appeared not to be useful as an index . most variables associated with the physical examination , including body weight , did not change during the training period . changes in the recovery rate throughout the training period were small , and in some cases , the recovery rate tended to decrease when training intensified due to conduct of maximal speed runs . moreover , considerable variability occurred in both the recovery rate and pulse pressure ratio . matsuba suggested that the cause of these results was that the same exercise protocol was not used . based on these results , he concluded that the present method of training was not the optimal training method to improve racehorse performance and that there was room for improvement . the second experiment was performed by using a new training method that was a modified version of the present method of training . matsuba named the training method used for this study the conservative method of training . four 2-year - old racehorses were studied , and training was conducted from april to october in 1934 . the training intensity was lower and the trotting distance was shorter compared with the present method of training , and the training intensity was gradually increased . clinical , blood , urine , feces , and physical examinations were all performed once per week , and a performance test was conducted once every two weeks . the clinical , blood , and urine variables examined were identical to those in the previous experiment . the summary of the results of this study was approximately the same as that for the previous study . because respiratory frequency and pulse rate decreased and blood pressure increased , matsuba suggested that improvement in cardiovascular function occurred due to the training . on the other hand , because the erythrocyte count and blood glucose concentration increased only slightly , but the blood co2 concentration did not change , he concluded that blood characteristics may have properties that are not affected by excessive training . in this study , although the running distance and dates of the performance tests were designed to be held constant , it proved to be difficult to set a constant running speed due to weather , track condition , and the occurrence of various diseases . therefore , although variations in the results were observed , he concluded that relatively constant data were obtained compared with the previous experiment . however , since changes in the recovery rate and pulse pressure ratio were small , he considered that there was no improvement in performance . matsuba concluded that the training method used in this study was safe because all horses finished the study able to participate in a race ; however , he considered this method was slightly inferior to the present method of training that was being used and that future studies would be needed to evaluate positive effects of training on racehorse performance . matsuba tried to use a similar protocol for the performance test to calculate the recovery rate , because the recovery status of each variable was largely influenced by the exercise intensity of the performance test . if the performance test was performed on only a single occasion on several horses to evaluate their fitness at that time , it would be easy to set a constant exercise intensity for all the horses by making all the horses run simultaneously in the same group . however , when the effects of training on recovery rate were to be evaluated , the exercise intensities of the performance tests performed multiple times during the training period would need to be set as identical . moreover , in the field training study , it was difficult to complete the entire training plan without any of the horses experiencing musculoskeletal disorders . in addition , the number of horses matsuba used in these studies was small . taking these factors into consideration , the authors thought it unavoidable that the expected results were not obtained in these experiments . matsuba also evaluated the effects of administration of various kinds of medicaments on the performance of racehorses using four 2-year - old racehorses . the training protocol used in this study was a modified version of the conservative method with the trotting distance increased . administration of nine medicaments ( 5% glucose , 3% calcium iodide , 20% benzoic acid locke s solution , radium ringer s solution , oxygen inhalation , cam - phenal , vitacamphor , deigalen , and adrenalin locke s solution ) was performed , after which the effects of the drugs on running speed and degree of recovery from fatigue were evaluated . administration tests were carried out once per week from the middle of september to the end of october ( the first and final tests were control tests ) . for each treatment , the 5% recovery rate ( 100 5% ) , 10% recovery rate ( 100 10% ) , and pulse pressure ratio were reported . when evaluation was performed by using the 10% recovery rate , the best result was obtained with administration of 3% calcium iodide . however , because the second - best result was obtained in the control tests , matsuba concluded that the recovery rate tended to decrease simply due to the administration of a medicament . in terms of training regimens , because relatively good results were obtained by increasing the trotting distance , matsuba suggested that increasing the trotting distance was more essential than increasing the canter distance . in the final experiment , changes in the blood s physical characteristics were investigated , and the utility of that information for use in regulating training and evaluating performance was examined . four racehorses ( 2 two - year -old , 1 five - year - old , and 1 six - year - old ) were studied . training regimens used in this study were similar to the protocols used in previous experiments . five clinical variables , twelve blood variables ( erythrocyte count , maximal erythrocyte fragility , minimal erythrocyte fragility , specific gravity , viscosity , erythrocyte sedimentation rate ( esr ) , pcv , freezing point depression , surface tension , ph , electrical conductivity , and blood co2 concentration ) , and seventeen urine variables were measured once per week , and a fecal examination was also performed . because erythrocyte fragility , blood viscosity , esr , freezing point depression , and ph changed with the progression of training , matsuba suggested that these five variables might be useful indices to study further in the future . both the maximal and minimal fragility of erythrocytes increased as training advanced ; furthermore , blood viscosity increased , and esr decreased as training progressed . the relationships of esr with training and condition were later followed up by sakurai , who had studied under matsuba . the history of research on the exercise physiology of the horse in japan began in the 1920s during a time when the warhorse was still the major focus of equine interest , and studies later began to be performed that were focused on racehorses . matsuba and shimamura of the university of tokyo played major roles in initiating studies of racehorses during the 1930s . their first study , published in 1933 , was the single most important study ever published in the field of equine exercise physiology in japan . it is remarkable that they were doing research to evaluate performance of racehorses in the 1930s at the dawning of the era of modern horse racing in japan . another interesting point about their study is that data were collected from elite thoroughbred racehorses such as wakataka , the winner of the first japanese derby held in 1932 at meguro racecourse . wakataka won seven of 11 races in which he ran , including the emperor s cup in the autumn of 1932 at meguro racecourse . in addition to wakataka , data were collected from a number of elite racehorses : happy chapel , winner of the emperor s cup in the spring meeting at meguro racecourse in 1932 ; hakusetsu , winner of the emperor s cup in the autumn meeting at yokohama racecourse in 1933 ; and ascot , winner of the emperor s cup in the spring meeting at meguro racecourse in 1933 . later , ascot participated in eventing at the berlin olympics in 1936 with baron nishi , who was the gold medalist in individual show jumping at the los angeles olympics in 1932 . data have also been collected from several contemporary japanese derby winners such as tanino gimlet ( 69th derby in 2002 ) , deep impact ( 72nd derby in 2005 ) , meisho samson ( 73rd derby in 2006 ) , and logi universe ( 76th derby in 2009 ) , making it all the more remarkable to realize that detailed data from the first winner of the japanese derby had been measured . in recent times , it has become possible to measure cardiopulmonary functions during exercise , such as oxygen uptake , carbon dioxide production , minute ventilation , cardiac output , blood gases , and blood pressures , due to the introduction of equine high - speed treadmills and flow - through gas collection systems . these technological advances have been used for measuring oxygen uptake since 1992 and for evaluating the effects of training on the oxygen transport system , cardiopulmonary function [ 1,2,3,4 , 32 ] , and skeletal muscle [ 26 , 49 , 50 ] . at the present time , one of a number of variables that have been measured in field exercise tests , the v200 ( running speed at which the hr reaches 200 beat / min ) of numerous young horses trained at training farms has been recorded several times during their training periods [ 5 , 33 , 34 ] , and the data indicate that this variable may be useful for evaluating fitness and the effects of training . however , it has been impossible to predict future racing performance using only this variable . when the values for the variable vhrmax ( running speed at maximal hr ) measured in jra racehorses were compared , it was found that stronger horses ( open class in the jra classification ) had higher vhrmax values than other horses ( newcomer or maiden class ) . however , it was still impossible to determine the performance of a racehorse by using only this variable . even if maximal oxygen uptake , the best indicator of aerobic work capacity of a racehorse , was measured , it would still not be possible to predict overall performance . the pioneering work of matsuba and shimamura created a legacy that continues today as researchers attempt to better understand the factors that determine , and might be used to predict , racehorse performance .

abstractthe history of research on the exercise physiology of racehorses in japan dates back to the 1930s . a research report entitled studies on exercise physiology and performance testing of the racehorse , published in 1933 by shigeo matsuba and torao shimamura of the university of tokyo , was epoch - making and the most important study in the history of equine exercise physiology in japan . research results were reported from 92 thoroughbred racehorses in a large - scale project during the period of 1928 to 1932 at the shimofusa imperial farm and the koiwai farm , which were the two greatest racehorse farms at that time . a total of 20 physiological variables were measured to evaluate the fitness of thoroughbred racehorses before exercise ( pre ) , just after exercise ( post ) , 1 hr after exercise ( 1 hr ) , 2 hr after exercise ( 2 hr ) , and 3 hr after exercise ( 3 hr ) in order to calculate their recovery rates as an index of fitness and performance . the percentage of the pre value at 1 hr , 2 hr , and 3 hr was calculated . when the percentage of a variable reached 95105% of the pre value , the variable was considered to be recovered . the percentage of the total number of variables that were recovered for each time period was calculated , and an overall average was calculated from them ; matsuba and shimamura proposed calling this overall average the recovery rate , which could then be applied to evaluate each horse . the effects of training on racehorses were subsequently evaluated by measuring the various physiological variables and the recovery rate .

Exercise Physiology of the Racehorse Performance Testing Using Recovery Rate as an Index Effect of Training on Performance of the Racehorse Conclusions

PMC5154714

rugby union is a field based team sport , requiring repeated bouts of moderate to high intensity running and activity separated by low intensity running or walking ( 1 , 3 , 15 ) . a rugby union match involves two teams of 15 athletes competing in two 40-min halves . players are separated into positional groups with forwards tending to possess greater strength / power and backs tending to possess greater speed / agility . not only have the physical characteristics of rugby union athletes been studied ( 3 , 10 ) , but so have their movement patterns during competitive matches through the use of global positioning systems ( gps ) ( 1 , 2 ) . gps systems have allowed for coaches to track the amount of distance covered during a rugby union match and categorize distance covered into varying velocity zones . previous research has shown that 64% of the distance covered during a rugby union match is spent walking / jogging , while only 24% is covered while cruising / striding , and 11% while running / sprinting ( 1 ) . however , little is known as to physical or cognitive abilities that are associated with movement patterns during rugby . agility , defined as the ability to change direction or velocity rapidly , is considered an important skill in the sport of rugby due to the intermittent , rapid changes in direction , in a high - intensity sport ( 12 ) . previous research in professional rugby league matches has shown that change of direction speed did not correlate to attacking or defensive performance , while perceptual skills did correlate with attacking performance ( 6 ) . the ability to objectively quantify perceptual skills , such as spatial awareness , may provide insight into athlete movement patterns . spatial awareness , in the form of 3-dimensional multiple - object - tracking speed ( 3dmots ) testing , has been shown to distinguish between athletes of different competitive levels ( 4 ) and is related to sport - specific performance measures ( 11 , 16 ) . a common defensive tactic in rugby is to minimize the time and space in which the opponent can advance towards scoring ( 13 ) . while physically constraining space may be most effective , psychologically constraining space by positioning the defenders to alter the opposition s perception of space is commonly utilized ( 13 ) . an athlete s ability to make a decision and act in this space is affected by their perception of their individual ability , the task requirements , and the environment ( 14 ) . currently , the only studies to assess 3dmots and athletes have been in soccer ( 16 ) and basketball ( 11 ) . mangine and colleagues ( 11 ) correlated 3dmots threshold scores with basketball statistics ; while romeas and colleagues observed improved passing accuracy decision - making when training with 3dmots . the use of 3dmots testing in rugby union athletes has not yet occurred due to the relative novelty of the testing methodology . therefore , there needs to be a better understanding of 3dmots and how it is related to movement patterns during rugby union matches . once established , future research could examine the effect of training on in - game performance . the purpose of the current study is to assess the relationship between spatial awareness , agility performance , and movement patterns during a competitive rugby match . we hypothesize that spatial awareness will be related to the in - game movement patterns of rugby union athletes . twelve male american championship - level collegiate rugby union athletes ( age : 21.21.4 y ; weight : 85.016.0 kg ; 7 forwards and 5 backs ) volunteered for this study . spatial awareness was assessed via a 3dmots test ( neurotracker ; cognisens athletic , inc . , the procedures for the 3dmots test have been previously described by mangine and colleagues ( 11 ) . each participant wore 3d glasses and sat on a stool placed 7 ft from an 88 ft projection screen inside of a simulator cave for one core session . one core session consisted of 20 trials in which participants were required to track 4 yellow balls , while ignoring 4 distractors , as they all moved through space at a set speed . each participant was provided 5 practice trials as a familiarization . at the start of each trial , a 3d transparent cube was presented on the screen containing 8 identical yellow balls ( figure 1a ) . four of the 8 balls were randomly illuminated for 2 seconds to indicate which balls were to be tracked ( figure 1b ) . all balls returned to yellow , then randomly moved around the transparent cube at a set speed for 8 seconds ( figure 1c ) . after 8 seconds , the balls stopped moving and were numbered 18 ( figure 1d ) . after each trial , the speed of the objects was adjusted to either increase or decrease depending on the success of the previous trial . this pattern of trial , speed adjustment , and subsequent trial was repeated until 20 trials were completed . at the completion of 20 trials , each participant was given a threshold score ( table 1 ) , which was considered to be the threshold for effective perception and processing of visual information sources ( 4 ) . the threshold scores obtained from the 3dmots has been correlated to sport - specific ability in professional basketball players ( 11 ) . to assess agility , 11 of the 12 participants ( one held out due to injury ) completed the pro agility and t drill . all agility testing was completed on the same day , on a synthetic turf field . the pro agility and t drill have been previously described by hoffman ( 8) and are depicted in figure 2a and 2b , respectively . all timing for the agility drills was conducted using the same stopwatch by the same timer . the best time for each drill was used for later analysis ( table 1 ) . to track movement patterns during a single rugby match , each participant was outfitted with a 10-hz gps receiver / transmitter ( minimaxx 4.0 , catapult systems , victoria , australia ) during an official usa rugby match . both teams agreed to allow the gps receiver / transmitters to be worn and for data to be collected . in addition to a gpa receiver / transmitter , the units also contained a 100-hz tri - axial accelerometer . the procedures for gps tracking were similar to those utilized by wells and colleagues ( 17 ) . after the pre - game warm - up , the units were placed into a harness which was positioned between the shoulder blades and under the participant s jersey . participants were tracked in real - time during the course of a competitive rugby match using a laptop computer with catapult sprint software ( sprint v5.1 ) and antenna . following the game , data was downloaded from the gps units and exports for analysis . distance covered was divided into three velocity zones as previously described by cunniffe and colleagues ( 1 ) ( table 2 ) . analysis of variance was used to compare the relative distance covered in each velocity zone and pearson product moment correlations were used to examine the relationship between distance covered in each velocity zone , agility performance , and spatial awareness . in the event of a significant main effect of velocity zone , bonferroni - adjusted post hoc tests were run to compare the three speed zones against each other . twelve male american championship - level collegiate rugby union athletes ( age : 21.21.4 y ; weight : 85.016.0 kg ; 7 forwards and 5 backs ) volunteered for this study . spatial awareness was assessed via a 3dmots test ( neurotracker ; cognisens athletic , inc . , the procedures for the 3dmots test have been previously described by mangine and colleagues ( 11 ) . each participant wore 3d glasses and sat on a stool placed 7 ft from an 88 ft projection screen inside of a simulator cave for one core session . one core session consisted of 20 trials in which participants were required to track 4 yellow balls , while ignoring 4 distractors , as they all moved through space at a set speed . each participant was provided 5 practice trials as a familiarization . at the start of each trial , a 3d transparent cube was presented on the screen containing 8 identical yellow balls ( figure 1a ) . four of the 8 balls were randomly illuminated for 2 seconds to indicate which balls were to be tracked ( figure 1b ) . all balls returned to yellow , then randomly moved around the transparent cube at a set speed for 8 seconds ( figure 1c ) . after 8 seconds , the balls stopped moving and were numbered 18 ( figure 1d ) . after each trial , the speed of the objects was adjusted to either increase or decrease depending on the success of the previous trial . this pattern of trial , speed adjustment , and subsequent trial was repeated until 20 trials were completed . at the completion of 20 trials , each participant was given a threshold score ( table 1 ) , which was considered to be the threshold for effective perception and processing of visual information sources ( 4 ) . the threshold scores obtained from the 3dmots has been correlated to sport - specific ability in professional basketball players ( 11 ) . to assess agility , 11 of the 12 participants ( one held out due to injury ) completed the pro agility and t drill . all agility testing was completed on the same day , on a synthetic turf field . the pro agility and t drill have been previously described by hoffman ( 8) and are depicted in figure 2a and 2b , respectively . all timing for the agility drills was conducted using the same stopwatch by the same timer . the best time for each drill was used for later analysis ( table 1 ) . to track movement patterns during a single rugby match , each participant was outfitted with a 10-hz gps receiver / transmitter ( minimaxx 4.0 , catapult systems , victoria , australia ) during an official usa rugby match . both teams agreed to allow the gps receiver / transmitters to be worn and for data to be collected . in addition to a gpa receiver / transmitter , the units also contained a 100-hz tri - axial accelerometer . the procedures for gps tracking were similar to those utilized by wells and colleagues ( 17 ) . after the pre - game warm - up , the units were placed into a harness which was positioned between the shoulder blades and under the participant s jersey . participants were tracked in real - time during the course of a competitive rugby match using a laptop computer with catapult sprint software ( sprint v5.1 ) and antenna . following the game , data was downloaded from the gps units and exports for analysis . distance covered was divided into three velocity zones as previously described by cunniffe and colleagues ( 1 ) ( table 2 ) . analysis of variance was used to compare the relative distance covered in each velocity zone and pearson product moment correlations were used to examine the relationship between distance covered in each velocity zone , agility performance , and spatial awareness . in the event of a significant main effect of velocity zone , bonferroni - adjusted post hoc tests were run to compare the three speed zones against each other . the average playing time for the twelve participants was 65.921.6 minutes ; while the average spatial awareness score was 1.350.59 cm.sec . a significant difference ( f=142.99 , p<0.001 , 2=0.929 ) was noted in distance covered per minute between the three velocity zones . post hoc tests revealed that the distance covered during the low intensity walking / jogging ( 39.54.5 m / min ) was significantly greater than the distance covered during the moderate intensity cruising / striding ( 20.96.5 m / min ; p<0.001 ) and the high intensity running / sprinting ( 4.93.5 m / min ; p<0.001 ) . furthermore , the distance covered during moderate intensity cruising / striding was greater ( p<0.001 ) than distance covered during the high intensity running / sprinting . when assessing the relationship between movement patterns and spatial awareness , distance covered during walking / jogging and running / sprinting did not correlate to spatial awareness scores ( r=0.106 , p=0.740 and r=0.175 , p=0.590 , respectively ) . however , distance covered during cruising / striding had a significant positive relationship with spatial awareness scores ( r=0.798 , p<0.001 ) . in terms of agility performance , there was no significant relationship between the pro agility ( 5.060.28 secs ) and walking / jogging ( r=0.217 , p=0.522 ) , cruising / striding ( r=0.321 , p=0.336 ) , and running / sprinting ( r=0.081 , p=0.812 ) . furthermore , there was no significant relationship between the t drill ( 10.620.39 secs ) and walking / jogging ( r=0.114 , p=0.738 ) , cruising / striding ( r=0.095 , p=0.781 ) , and running / sprinting ( r=0.038 , p=0.913 ) . neither the pro agility ( r=0.073 , p=0.830 ) nor t drill ( r=0.052 , p=0.878 ) significantly correlated to spatial awareness . the results of this study showed that during a competitive collegiate rugby match , athletes covered the most distance per minute of play while walking / jogging and the least amount of distance per minute of play while running / sprinting . these findings are consistent with previous research conducted during rugby union matches ( 1 ) . furthermore , only cruising / striding had a significant positive relationship with spatial awareness scores , indicating that players who covered the most distance at cruising / striding had higher spatial awareness scores . interestingly , no relationships were found between the other velocity zones and spatial awareness or between agility and in - game performance . the primary finding of this study was that spatial awareness scores only correlated to distance covered while cruising / striding . to the authors knowledge this is the first study to compare movement patterns and spatial awareness from 3dmots in the sport of rugby union , or any field - based sport . previous research using 3dmots , revealed most likely and likely positive correlations with assists , steals , turnovers , and assists - to - turnovers ratio in professional basketball players ( 11 ) . however , no study has assessed the relationship between spatial awareness and movement patterns in any athletic population . cruising / striding speed previous research has shown that attention focus switches for dissociative ( external stimuli ) to associative ( internal stimuli ) as the intensity of exercise increases ( 9 ) . the ability to track teammates and opponents while cruising / striding speed may be result of the processing of external and internal stimuli while generally attempting to navigate open space on the pitch . future investigations should examine the differences in spatial awareness amongst different positions in rugby union and whether those values still correlate to cruising / striding for each position the lack of correlation between agility and distance covered in the three velocity zones is in agreement with previous findings from other sports . gabbert and colleagues ( 6 ) noted that change of direction did not correlate to attacking or defensive performance in professional rugby league players . however , reactive agility did correlate to attacking performance ( 6 ) , which could be viewed as skills that require both perceptual and physiological abilities . future research should examine the relationship between reactive agility and distance covered at different velocity zones . furthermore , no study has utilized 3dmots in rugby league athletes or rugby union athletes of different competitive levels . future research should compare the relationship between spatial awareness and moderate intensity running amongst different field - based sports and different levels of competition within those sports . while spatial awareness was correlated with distance covered while cruising / striding , we can not deduce if there is a cause and effect relationship between the spatial awareness and movement patterns . furthermore , there is limited research assessing the trainability of spatial awareness and what effect increased awareness has on match play performance . previous research has assessed the effectiveness of perceptual training on match performance and decision making in soccer athletes ( 7 , 16 ) , but no perceptual training studies have been assessed in rugby union athletes . as the current study has established the relationship between spatial awareness and moderate intensity movement patterns , it would be prudent to determine how cognitive training alters rugby competition movement patterns . future studies should assess the usefulness of training interventions on spatial awareness and how an increased spatial awareness effects match performance in rugby union athletes . spatial awareness , as determined using 3dmots , appears to be related with distance covered while cruising / striding during a competitive rugby match , while agility performance did not correlate to distance covered at any speed . this is the first study to observe a relationship between spatial awareness and moderate intensity movement during a rugby match . while the existence of the correlation is interesting , future research should investigate the trainability of spatial awareness and the effect on rugby match performance . spatial awareness as assessed by 3dmots may be an important skill to develop for rugby athletes .

the purpose of the present study was to evaluate the relationship between spatial awareness , agility , and distance covered in global positioning system ( gps ) derived velocity zone classifications during a collegiate rugby match . twelve american collegiate rugby union players ( meansd ; age : 21.21.4 y ; weight : 85.016.0 kg ; 7 forwards & 5 backs ) on a single team volunteered to participate in this investigation . the distances travelled at low ( walking / jogging ; < 2.7 m/s ) , moderate ( cruising / striding ; 2.75.0 m / s ) , and high intensities ( running / sprinting ; > 5.0 m / s ) were measured for each player using gps sensors and normalized according to playing time during an official usa rugby match . spatial awareness was measured as visual tracking speed from one core session of a 3-dimensional multiple - object - tracking speed ( 3dmots ) test ( 1.350.59 cmsec-1 ) . agility was assessed utilizing the pro agility ( 5.050.28 sec ) and t drill ( 10.620.39 sec ) . analysis of variance revealed that athletes travelled the greatest distance during walking / jogging ( 39.54.5 mmin-1 ) and least distance during running / sprinting ( 4.93.5 mmin-1 ) . pearson product moment correlations revealed that only distance covered while cruising / striding ( 20.96.5 mmin-1 ) was correlated to spatial awareness ( r=0.798 , p=0.002 ) . agility did not correlate to distance covered at any velocity zone or spatial awareness . spatial awareness , as determined by 3dmots , appears to be related to the moderate intensity movement patterns of rugby union athletes .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

PMC3604851

it is characterized by a complex of neuropathological , biochemical , and behavioral symptoms , that gradually impair memory and the ability to learn , carry out daily activities and behavior . in particular , ibotenic acid - induced lesions of the nbm in rats produce impairment in acquisition and retention phase of passive avoidance tasks . studies have shown that nbm has been widely involved in the pathogenesis of ad and is accompanied with cognitive deficits . a number of studies . suggested that exercise in aged rats improves learning and neurogenesis . so it may slow the onset and progression of learning and memory deficit in ad . it is proved that regular exercise attenuates motor deficits , increases formation of new neurons , and ameliorates neurological impairment in several neurodegenerative diseases.[1214 ] some studies on transgenic mouse models of ad have showed beneficial effects of exercise on general activity , spontaneous alternation , object recognition memory and , spatial learning performance . some animal studies have revealed that treadmill running improves spatial and passive avoidance learning in nbm - lesion rats . in previous studies , transgenic and nbm - lesion models , they investigated the effects of physical activity after onset of ad in these models and , there have been no basic researches performed to estimate the preventive effects of exercise . the aims of this study were to investigate preventive effects of treadmill running in nbm - lesion rats on learning and memory deficit by passive avoidance task in order to examine whether treadmill running can prevent learning impairment in this model of ad . male wistar rats ( 250 - 300 g ; n = 54 ) were obtained from jondishapour university , ahwaz , iran ) . the animals were kept in animal house and provided with food and water ad libitum and experienced a 12:12-h light - dark cycle ( 07:00 - 19:00 ) in a temperature controlled environment ( 22 2c ) . this study was approved by the ethics committee for animal experiments at isfahan university approved the study , and all experiments were conducted in accordance with the international guiding principles for biomedical research involving animals , which was revised in 1985 . rats were randomly allocated to the following groups : control group ( co ; n = 11 ) : no injection , no exercise.sham operation ( sh ; n = 10 ) : saline ( drug solvent ) was injected in nbm.alzheimer group ( a ; n = 11 ) : ibotenic acid was injected in nbm.exercise before alzheimer group ( e - a ; n = 12 ) : ibotenic acid was injected bilaterally in nbm , and then rats exercised 21 days.exercise group ( e ; n = 10 ) : rats exercised 21 days . . sham operation ( sh ; n = 10 ) : saline ( drug solvent ) was injected in nbm . alzheimer group ( a ; n = 11 ) : ibotenic acid was injected in nbm . exercise before alzheimer group ( e - a ; n = 12 ) : ibotenic acid was injected bilaterally in nbm , and then rats exercised 21 days . exercise group ( e ; n = 10 ) : rats exercised 21 days . rats were anesthetized with chloral hydrate ( 450 mg / kg , i.p ) and then placed in a stoelting stereotaxic apparatus ( incisor bar 3.3 mm , ear bars positioned symmetrically ) . the scalp was cleaned and incised on the midline and a burr hole was drilled through the skull and ibotenic acid ( cat number 12765 , sigma ) was injected at coordinates of : ap = 1.2 , ml = 3.2 , dv = 7.5 mm from surface skull . 10 g/l of ibotenic acid was injected ( 5 g/l each sides ) with microinjection pump at the speed of 120 lit / h . instead of ibotenic acid solution , post - operatively , the rats were given special care until spontaneous feeding was restored . behavioral tests were conducted four weeks after the surgery and were evaluated blind to the treatments by the observer . rats in the exercise group run on a treadmill at a speed of 20 - 21 m / min for 60 min daily ( 6 days a week ) , for 3 weeks at inclination . to familiarize animals with the experimental set up , the treadmill was switched on and the speed was increased from 5 to 21 m / min and over the course of 6 days , the duration was increased from 10 to 60 min . when exercising rats moved back on the treadmill , electric shocks were sparingly used to impel the animal to run . from week 2 onwards , speed and duration were kept constant at 20 - 21 m / min , 60 min per run after warm up . the non - runners groups were not put on the treadmill for the same duration of running as runners did . after 21 days , rats in all exercise groups , were subjected to passive avoidance learning ( pal ) test . the training apparatus had two compartments comprising a small chamber ( 25 25 20 cm ) and a large dark compartment ( 50 25 20 cm ) . electric shocks were delivered to the grid floor by an isolated stimulator . at the beginning of the test , each rat was placed in the apparatus for 5 min to become habituated . on the second day , an acquisition trial was performed ; rats were placed individually in the illuminated chamber . after a habituation period ( 1 min ) then after the rat entered the dark chamber , the door was lowered and an inescapable scrambled single electric shock ( 75v , 0.2 ma , 50hz ) was delivered for 3 second . latency to cross the dark compartment ( i.e. , pre - shock latency ) was recorded . after exposure to the foot shock , the rat was removed from the passive avoidance apparatus to its home cage . the rat was placed in the lighted ( safe ) compartment again with access to the dark compartment without any shock . the latency to enter the dark compartment was measured ( i.e. , testing latency ) up to a maximum of 300 seconds . in the passive avoidance test , all results were compared using a kruskal - wallis nonparametric one - way analysis of variance corrected for ties , followed by a two - tailed mann - whitney u test . the comparisons of retention time 24 h ( within groups ) were analyzed by friedman test , followed by a wilcoxon signed ranks test . after the completion of behavioral tests , the rats were sacrificed and brains were removed and fixed in formalin , and then were sectioned to verify ibotenic acid injection site . the lesions were reconstructed on standardized sections of the rat brain [ figure 1 ] . male wistar rats ( 250 - 300 g ; n = 54 ) were obtained from jondishapour university , ahwaz , iran ) . the animals were kept in animal house and provided with food and water ad libitum and experienced a 12:12-h light - dark cycle ( 07:00 - 19:00 ) in a temperature controlled environment ( 22 2c ) . this study was approved by the ethics committee for animal experiments at isfahan university approved the study , and all experiments were conducted in accordance with the international guiding principles for biomedical research involving animals , which was revised in 1985 . rats were randomly allocated to the following groups : control group ( co ; n = 11 ) : no injection , no exercise.sham operation ( sh ; n = 10 ) : saline ( drug solvent ) was injected in nbm.alzheimer group ( a ; n = 11 ) : ibotenic acid was injected in nbm.exercise before alzheimer group ( e - a ; n = 12 ) : ibotenic acid was injected bilaterally in nbm , and then rats exercised 21 days.exercise group ( e ; n = 10 ) : rats exercised 21 days . . sham operation ( sh ; n = 10 ) : saline ( drug solvent ) was injected in nbm . alzheimer group ( a ; n = 11 ) : ibotenic acid was injected in nbm . exercise before alzheimer group ( e - a ; n = 12 ) : ibotenic acid was injected bilaterally in nbm , and then rats exercised 21 days . exercise group ( e ; n = 10 ) : rats exercised 21 days . rats were anesthetized with chloral hydrate ( 450 mg / kg , i.p ) and then placed in a stoelting stereotaxic apparatus ( incisor bar 3.3 mm , ear bars positioned symmetrically ) . the scalp was cleaned and incised on the midline and a burr hole was drilled through the skull and ibotenic acid ( cat number 12765 , sigma ) was injected at coordinates of : ap = 1.2 , ml = 3.2 , dv = 7.5 mm from surface skull . 10 g/l of ibotenic acid was injected ( 5 g/l each sides ) with microinjection pump at the speed of 120 lit / h . instead of ibotenic acid solution , post - operatively , the rats were given special care until spontaneous feeding was restored . behavioral tests were conducted four weeks after the surgery and were evaluated blind to the treatments by the observer . rats in the exercise group run on a treadmill at a speed of 20 - 21 m / min for 60 min daily ( 6 days a week ) , for 3 weeks at inclination . to familiarize animals with the experimental set up , the treadmill was switched on and the speed was increased from 5 to 21 m / min and over the course of 6 days , the duration was increased from 10 to 60 min . when exercising rats moved back on the treadmill , electric shocks were sparingly used to impel the animal to run . from week 2 onwards , speed and duration were kept constant at 20 - 21 m / min , 60 min per run after warm up . the non - runners groups were not put on the treadmill for the same duration of running as runners did . after 21 days , rats in all exercise groups , were subjected to passive avoidance learning ( pal ) test . the training apparatus had two compartments comprising a small chamber ( 25 25 20 cm ) and a large dark compartment ( 50 25 20 cm ) . electric shocks were delivered to the grid floor by an isolated stimulator . at the beginning of the test , each rat was placed in the apparatus for 5 min to become habituated . on the second day , an acquisition trial was performed ; rats were placed individually in the illuminated chamber . after a habituation period ( 1 min ) , the guillotine door was lifted . then after the rat entered the dark chamber , the door was lowered and an inescapable scrambled single electric shock ( 75v , 0.2 ma , 50hz ) was delivered for 3 second . latency to cross the dark compartment ( i.e. , pre - shock latency ) was recorded . after exposure to the foot shock , the rat was removed from the passive avoidance apparatus to its home cage . the rat was placed in the lighted ( safe ) compartment again with access to the dark compartment without any shock . the latency to enter the dark compartment was measured ( i.e. , testing latency ) up to a maximum of 300 seconds . in the passive avoidance test , all results were compared using a kruskal - wallis nonparametric one - way analysis of variance corrected for ties , followed by a two - tailed mann - whitney u test . the comparisons of retention time 24 h ( within groups ) were analyzed by friedman test , followed by a wilcoxon signed ranks test . after the completion of behavioral tests , the rats were sacrificed and brains were removed and fixed in formalin , and then were sectioned to verify ibotenic acid injection site . the lesions were reconstructed on standardized sections of the rat brain [ figure 1 ] . the latency was measured in pre foot shock ( acquisition time ) and 24 h post foot shocks ( retention time ) . results indicated that the pre - shock latency was the same among all groups , but compared to group alzheimer ( a ) as well as between group a and group exercise - alzheimer ( e - a ) , acquisition time was longer in control ( co ) group ( p < 0.01 , p < 0.05 respectively ) . there was a significant difference between group exercise ( e ) and a ( p < 0.01 ) , as well as group sham ( sh ) and a [ p < 0.05 , figure 2 ] . comparison of latency to enter the dark chamber before receiving foot shock ( acquisition time ) . there were significant differences between control ( n= 11 ) and a ( n= 11 ) group ( p < 0.01 ) , as well as a and e ( n= 12 ) group ( p < 0.01 ) . there were significant differences between a and others ( sh and e - a groups , p < 0.05 ) . all results were analyzed by kruskal - wallis test followed by mann - whitney u test however , the retention time during testing ( i.e. , testing latency carried out 24 h after receiving foot shock ) was significantly decreased in group a when compared to other groups ( co , sh , e and groups e - a ; p < 0.001 , p < 0.01 , p < 0.001 , p < 0.001 respectively ) . the retention time changes were not significant between group e and group sh . also , there were not significant differences in retention time between control group , as well as sh and e - a groups [ figure 3 ] . comparison of latency to enter the dark chamber 24 h after receiving foot shock ( the retention time ) . the retention time was significantly decreased in the a group compared to others ( control , sh and e - a group , p < 0.001 , p < 0.01 , p < 0.001 respectively ) . all results were analyzed by kruskal - wallis test followed by mann - whitney u test the results of pre foot shock and post foot shock latency were analyzed by a paired sample to evaluate changes within groups . in this part , our data showed that there were significant differences in pre and post foot shock latency among all groups [ p < 0.01 , figure 4 ] . there were significant differences in pre and post foot shock latency in all groups ( p < 0.01 ) the latency was measured in pre foot shock ( acquisition time ) and 24 h post foot shocks ( retention time ) . results indicated that the pre - shock latency was the same among all groups , but compared to group alzheimer ( a ) as well as between group a and group exercise - alzheimer ( e - a ) , acquisition time was longer in control ( co ) group ( p < 0.01 , p < 0.05 respectively ) . there was a significant difference between group exercise ( e ) and a ( p < 0.01 ) , as well as group sham ( sh ) and a [ p < 0.05 , figure 2 ] . comparison of latency to enter the dark chamber before receiving foot shock ( acquisition time ) . there were significant differences between control ( n= 11 ) and a ( n= 11 ) group ( p < 0.01 ) , as well as a and e ( n= 12 ) group ( p < 0.01 ) . there were significant differences between a and others ( sh and e - a groups , p < 0.05 ) . all results were analyzed by kruskal - wallis test followed by mann - whitney u test however , the retention time during testing ( i.e. , testing latency carried out 24 h after receiving foot shock ) was significantly decreased in group a when compared to other groups ( co , sh , e and groups e - a ; p < 0.001 , p < 0.01 , p < 0.001 , p < 0.001 respectively ) . the retention time changes were not significant between group e and group sh . also , there were not significant differences in retention time between control group , as well as sh and e - a groups [ figure 3 ] . comparison of latency to enter the dark chamber 24 h after receiving foot shock ( the retention time ) . the retention time was significantly decreased in the a group compared to others ( control , sh and e - a group , p < 0.001 , p < 0.01 , p < 0.001 respectively ) . all results were analyzed by kruskal - wallis test followed by mann - whitney u test the results of pre foot shock and post foot shock latency were analyzed by a paired sample to evaluate changes within groups . in this part , our data showed that there were significant differences in pre and post foot shock latency among all groups [ p < 0.01 , figure 4 ] . there were significant differences in pre and post foot shock latency in all groups ( p < 0.01 ) in the present study , the passive avoidance retention and retrieval function was impaired by ibotenic acid nbm - lesions . our results showed that treadmill exercise training significantly improves passive avoidance performance in normal and nbm - lesion rats [ figures 2 and 3 ] . chopin et al . demonstrated significant performance deficits in both passive avoidance and morris water maze tests in bilateral nbm - lesion rats . they showed that regular exercise significantly attenuated lesion - associated decrease in brain functions . in these studies , animals exercised after onset ad in this animal model . in this research , relationship between treadmill running and preventive effects on learning and memory deficits observed in nbm - lesion rats were investigated . our results indicated that in comparison to nbm - lesion rats , learning and memory in groups e - a were improved that is , exercise has preventive effects on acquisition and retention time impairment in passive avoidance test in nbm - lesion [ figures 2 and 3 ] . furthermore , it will postpone memory impairments that may result from some exercise mechanisms such as increase in dopamine and muscarinic receptor density , acetylcholine level , neurotransmitter release in the hippocampus , brain derived neurotrophic factor , bdnf gene expression , neuron proliferation and survival in the animal 's brain . similarly , van praag et al . also , other researchers suggested that exercise training ( swimming ) increased memory of rats in passive avoidance test , but such increase was temporary after stopping exercise because swimming requires physical effort and compared to treadmill running in rats , was known as an effective stressor . according to our results , treadmill running had beneficial effects on encountering learning and memory deficits with nbm - lesion as animal model of ad and probably can reduce dementia in patients . exercise effects might be the result of structural and biological changes in brain , which enhance neuron numbers . a cell proliferation increase or cell death decrease increases the length and number of dendrites connection between neurons , as well as synaptic plasticity in hippocampus , which is involved in learning and memory.[253234 ] it has been suggested that mechanisms for some mentioned effects of exercise including gene expression , increment of neurotrophic factors such as brain derived neurotrophic factor and insulin - like growth factor i , which are important for neuronal survival and differentiation , as well as synaptic plasticity.[3537 ] hence , all of the aforesaid factors may have a pivotal role in learning and memory enhancement by exercise . our results at the behavioral level ( passive avoidance test ) emphasize the role of treadmill running in the prevention of learning and memory impairments in nbm - lesion rats . memory deficit caused by nbm lesion was also reversed by treadmill running , suggesting enhancement of learning and memory functions through physical activity .

background : alzheimer 's disease was known as a progressive neurodegenerative disorder in the elderly and is characterized by dementia and severe neuronal loss in the some regions of brain such as nucleus basalis magnocellularis . it plays an important role in the brain functions such as learning and memory . loss of cholinergic neurons of nucleus basalis magnocellularis by ibotenic acid can commonly be regarded as a suitable model of alzheimer 's disease . previous studies reported that exercise training may slow down the onset and progression of memory deficit in neurodegenerative disorders . this research investigates the effects of treadmill running on acquisition and retention time of passive avoidance deficits induced by ibotenic acid nucleus basalis magnocellularis lesion.methods:male wistar rats were randomly selected and divided into five groups as follows : control , sham , alzheimer , exercise before alzheimer , and exercise groups . treadmill running had a 21 day period and alzheimer was induced by 5 g/l bilateral injection of ibotenic acid in nucleus basalis magnocellularis.results:our results showed that ibotenic acid lesions significantly impaired passive avoidance acquisition ( p < 0.01 ) and retention ( p < 0.001 ) performance , while treadmill running exercise significantly ( p < 0.001 ) improved passive avoidance learning in nbm - lesion rats.conclusion:treadmill running has a potential role in the prevention of learning and memory impairments in nbm - lesion rats .

INTRODUCTION METHODS Animals Exercise protocol Passive avoidance learning test Statistical analysis Histology RESULTS Passive avoidance test in rats DISCUSSION CONCLUSION

PMC2266970

the immense and diverse potential of phthalocyanines ( pc ) in a variety of technical ( chemical sensors , liquid crystals , electrocatalysis , and nonlinear optics ) and medicinal ( primarily photodynamic therapy [ 58 ] ) applications has generated a great deal of interest in these macrocyclic compounds . this , together with the extraordinary stability of these complexes , has resulted in considerable research being carried out on the phthalocyanine complex upon incorporation of nearly all metals in the periodic table into the pc core . complexes with metalloids and nonmetals of groups iva and va are of particular interest due to the two different valence / oxidation states available to the central atom . triazatetrabenzcorroles ( tbc ) are phthalocyanine - like compounds which have lost one of the bridging nitrogen atoms . the synthesis of ge , si , ga , and al tbc complexes have been reported [ 10 , 11 ] . were the first to report on the synthesis and unusual electronic spectra of the pcp compared to pcp [ 9 , 13 ] . it became apparent that upon complexation with the trivalent phosphorous , a bridging nitrogen was lost to form the phosphorous oxide tetrabenzcorroles ( ptbc ) . since then , a number of octa- and tetrasubstituted ptbc derivatives have been synthesized [ 1419 ] . high fluorescence quantum yields and singlet oxygen quantum yields have been reported for the complexes . the water - soluble tetrasulphonated ptbc showed good photodynamic therapy towards hela cells . for applications in pdt , high triplet lifetimes and yields are desired ; however there are no reports on the triplet - state behavior of the ptbc complexes . in this work , we report on ptbc complexes tetra- or octasubstituted with chloride and bromide ring substituents ( see schemes 1 and 2 ) , since the halogens are expected to enhance intersystem crossing , resulting in high triplet yields due to the heavy atom effect . a ptbc derivative containing a mixture of butoxy and chloro ring substituents has been reported , but no photophysical data has been reported for halogenated ptbcs . dimethylsulfoxide ( dmso ) , methanol ( meoh ) , phosphorous tribromide ( pbr3 ) , deuterated chloroform ( cdcl3 ) , deuterated dimethylsulfoxide ( dmso - d6 ) , deuterated pyridine ( pyr - d5 ) , 4,5 dichlorophthalonitrile , 1,8-diazabicyclo undec-7-ene ( dbu ) , 4-nitrophthalonitrile , copper ( i ) bromide , copper ( i ) chloride , hydrobromic acid , hydrochloric acid , palladium on carbon ( pd / c ) , and dicyanobenzene were purchased from sigma - aldrich ( miss , usa ) and used as received . pyridine and 1-pentanol were obtained from sigma - aldrich and dried prior to use . ground - state electronic absorption spectra were recorded on a varian cary 500 uv - vis - nir spectrophotometer . h and c nmr spectra were obtained using a bruker amx 400 mhz and a bruker avance ii+ 600 mhz nmr spectrometer . fluorescence emission and excitation spectra were recorded on a varian cary eclipse spectrofluorimeter , while ft - ir spectra ( kbr pellets ) were recorded on a perkin - elmer spectrum 2000 ft - ir spectrometer . triplet absorption and decay kinetics were recorded on a laser flash photolysis system , the excitation pulses were produced by an nd : yag laser ( quanta - ray , 1.5 j/90 ns ) pumping a dye laser ( lambda physic fl 3002 , pyridin 1 in methanol ) . the analyzing beam source was from a thermo oriel xenon arc lamp , and a photomultiplier tube was used as detector . signals were recorded with a two - channel digital real - time oscilloscope ( tektronix tds 360 ) where the kinetic curves were averaged over 256 laser pulses . triplet lifetimes were determined by exponential fitting of the kinetic curves using originpro 7.5 software . unsubstituted ptbc ( 7 ) was synthesized from unmetallated phthalocyanine ( 6 ) and pbr3 in a 16.4% yield , and characterized as reported in the literature [ 9 , 13 , 19 ] . 4-aminophthalonitrile ( 2 ) and 4-bromophthalonitrile ( 4 ) were synthesised following literature procedures [ 20 , 21 ] with some minor modifications . 4-chlorophthalonitrile ( 5 ) was synthesised by modifying the procedure used for compound 4 . 4-nitrophthalonitrile(1 ) ( 1.00 g , 5.78 mmol ) was placed in a round bottom flask and 100 ml of ethanol added to obtain a suspension . the catalyst pd / c ( 55 mg ) was added to the flask , the apparatus evacuated and then filled with hydrogen and the mixture vigorously stirred at room temperature until the absorption of hydrogen had completely stopped . the reaction mixture was subsequently filtered over celite and the solution evaporated in vacuo . h nmr ( 400 mhz , dmso - d6 ) ppm 7.39 ( dd , j = 8.66 , 1.34 hz , 1h ) , 6.93 ( d , j = 2.19 hz , 1h ) , 6.81 ( dd , j = 8.67 , 2.18 hz , 1h ) , 6.36 ( br s , 2h ) . compound 2 ( 500 mg , 3.5 mmol ) was taken up in a mixture of water ( 4 ml ) and hydrobromic acid ( 4 ml , 48% ) and the solution cooled to 0c using an ice - salt bath . a solution of sodium nitrite ( 276 mg , 4 mmol ) in water ( 2 ml ) was then added dropwise to the acid mixture to form the diazonium salt 3a as an intermediate . copper ( i ) bromide ( 1.00 g , 6.97 mmol ) was dissolved in hbr ( 4 ml , 48% ) and cooled to 0c . the cold diazonium salt solution was then added dropwise to the cubr reagent and the solution stirred for 1 hr at 0c after an hour , the solution was left to stand at room temperature overnight . the aqueous solution was then extracted with ethyl acetate ( 3 15 ml ) , the combined organic extracts washed with brine , dried over magnesium sulphate and concentrated to give 248 mg of 4 . h nmr ( 400 mhz , cdcl3 ) ppm 7.96 ( d , j = 1.78 hz , 1h ) , 7.89 ( dd , j = 8.37 , 1.89 hz , 1h ) , 7.68 ( d , j = 8.37 hz , 1h ) . c nmr ( 75 mhz , cdcl3 ) ppm 136.6 ( d ) , 136.3 ( d ) , 134.4 ( d ) , 128.1 ( s ) , 117.3 ( s ) , 114.7 ( s ) , 114.5 ( s ) , 114.0 ( s ) . a similar procedure to the one above was used to prepare compound 5 . compound 2 ( 500 mg , 3.5 mmol ) was dissolved in a water ( 4 ml)/hydrochloric acid ( 5 ml , 32% ) mixture , and the sodium nitrite ( 276 mg , 4 mmol ) in water ( 2 ml ) reagent was added to the amine hydrochloride solution to form the diazonium salt ( 3b ) . copper ( i ) chloride ( 1.00 g , 10.1 mmol ) the diazonium salt was then added to the cucl reagent and treated in the same manner as reported above . h nmr ( 400 mhz , cdcl3 ) ppm 7.80 ( d , j = 1.88 hz , 1h ) , 7.75 ( s , 1h ) , 7.73 ( d , j = 1.87 hz , 1h ) . c nmr ( 75 mhz , cdcl3 ) ppm 140.2 ( s ) , 134.5 ( d ) , 133.7 ( d ) , 133.5 ( d ) , 117.4 ( s ) , 114.6 ( s ) , 114.2 ( s ) , 114.1 ( s ) . complex 9 was synthesized from the unmetallated derivative ( 8) . the first step was the synthesis of 8 , following established methods as follows : 4 ( 200 mg , 0.97 mmol ) was reacted with dbu ( 0.36 ml ) in 1-pentanol under reflux for 12 hours under a nitrogen atmosphere . the dark green mixture was cooled down to room temperature , then methanol ( 5 ml ) and water ( 2 ml ) were added and the mixture precipitated out and centrifuged . this green solid complex 8 , confirmed to be a phthalocyanine molecule by its uv spectrum , was not purified any further and was employed as is for the synthesis of complex 9 . complex 9 was synthesized by heating ( under reflux ) a mixture of complex 8 ( 100 mg , 0.12 mmol ) and pbr3(0.34 ml , 3.60 mmol ) in pyridine at 90 to 100c for 2 hours . the reaction was then allowed to cool to room temperature and poured carefully into water and allowed to stand overnight . the green product obtained was then centrifuged and washed with copious amounts of water . upon drying , the precipitate was chromatographed on a silica gel column using pyridine as the eluant . the complex was collected as a dark green band , while the unmetallated pc remained at the top of the column . uv / vis ( dmso ) , maxnm ( log ) ] : 662 ( 4.86 ) , 630 ( 4.56 ) , 602 ( 4.29 ) , 578 ( 3.79 ) , 448 ( 5.19 ) , 439 ( 4.94 ) , 418 ( 4.70 ) . [ ( kbr ) vmax/cm ] : 3063 , 1717 , 1608 , 1506 , 1451 , 1396 , 1326 , 1298 ( p = o ) , 1271 , 1151 , 1109 , 1077 , 1043 , 967 , 956 , 832 , 813 , 758 , 723 , 691 , 578 . h nmr ( 600 mhz , pyr - d5 ) ppm 10.159.92 ( m , 4h ) , 9.839.57 ( m , 4h ) , 8.598.41 ( m , 4h ) . p nmr ( 162 mhz , pyr - d5 ) ppm 198.6 . maldi complex 11 was synthesized as explained above for 9 , except that complex 5 ( 200 mg , 1.23 mmol ) was reacted with dbu ( 0.24 ml ) in 1-pentanol instead of compound 4 to obtain the 4-chloro h2pc analogue ( complex 10 ) . the presence of the metal - free pc was again confirmed by its uv spectrum . complex 10 was then employed for the synthesis of complex 11 ( 100 mg , 0.15 mmol ) by reacting it with pbr3(0.42 ml , 4.50 mmol ) in hot pyridine . uv / vis ( dmso ) , max/nm ( log ) ] : 661 ( 4.56 ) , 630 ( 4.26 ) , 603 ( 3.99 ) , 447 ( 4.82 ) , 438 ( 4.59 ) , 417 ( 4.39 ) . [ ( kbr ) vmax/cm ] : 2922 , 1717 , 1609 , 1505 , 1456 , 1399 , 1328 , 1297 ( p = o ) , 1143 , 1068 , 973 , 910 , 814 , 769 , 724 , 690 , 580 . h nmr ( 600 mhz , pyr - d5 ) ppm 9.979.66 ( m , 8h ) , 8.458.26 ( m , 4h ) . 682.9 , found 682.0 { cl4(tbc)p(o)}. the first step was the synthesis of metal - free complex 12 , using the same procedure as outlined above for the synthesis of 8 , except that 4,5-dichlorophthalonitrile ( 300 mg 1.53 mmol ) was reacted with dbu ( 0.12 ml ) instead of complex 4 . confirmation of the formation of the h2pc ( 12 ) was provided by the uv spectrum . complex 13 was then synthesized as described for complexes 9 and 11 using 12 ( 100 mg , 0.128 mmol ) and pbr3 ( 0.36 ml , 3.84 mmol ) . a paucity of proton signals was expected and observed in the h nmr spectrum of this octasubstituted complex . uv / vis ( dmso ) , max/nm ( log ) ] : 667 ( 3.47 ) , 635 ( 3.19 ) , 606 ( 2.94 ) , 452 ( 3.83 ) , 442 ( 3.61 ) , 422 ( 3.46 ) , 411 ( 3.42 ) . [ ( kbr ) vmax/cm ] : 2927 , 1726 , 1586 , 1521 , 1349 , 1298 ( p = o ) , 1248 , 1197 , 1138 , 948 , 908 , 837 , 743 , 602 . h nmr ( 600 mhz , pyr - d5 ) ppm 8.07 ( m , 8h ) . maldi m / z : not observed . the comparative method was used to determine the fluorescence quantum yields ( f ) according to the following equation , utilizing unsubstituted znpc in dmso as the standard ( f = 0.18 ) : ( 1)f=f(std ) f astd 2fstd a std2 , where f and fstd are the areas under the fluorescence curve of the sample and the standard , respectively . similarly , a and astd are the absorbance of the compound and the standard at the excitation wavelength , and std are the refractive indices of solvents used for the sample and the standard , respectively . natural or radiative lifetimes ( n ) were estimated using photochemcad program which uses the strickler - berg equation . the fluorescence lifetimes ( f ) were evaluated using the following equation : ( 2)f=fn . the rate constants for intersystem crossing from the excited singlet state to the triplet state ( kisc(st ) ) were estimated using the following equation : ( 3)kisc(st)=(1/f)(1/f0 ) , where f and f are the excited singlet - state lifetimes for the halogenated derivatives and unsubstituted ptbc , respectively . similarly , the rate constants for intersystem crossing from the triplet state to the ground state ( kisc(ts ) ) were estimated using the following equation : ( 4)kisc(ts)=(1/t)(1/t0 ) , where t and t0 are the excited triplet - state lifetime for the halogenated derivatives and unsubstituted ptbc , respectively . solutions of the ptbc complexes were bubbled with argon in a 1 cm pathlength spectrophotometric cell , irradiated at the q band of the respective ptbc complexes , with the triplet quantum yields ( t ) determined by the triplet absorption method . the comparative method was applied as in the following equation , using znpc in dmso as the standard : ( 5)t=tstdat tstdatstd t . changes in the triplet - state absorbances of the ptbc derivative and the standard are represented by at and atstd , respectively ; while t and tstd are the triplet - state molar extinction coefficients for the ptbc derivative and the standard , respectively ; while tstd is the triplet quantum yield for the standard ( t = 0.65 for znpc in dmso ) . triplet lifetimes ( t ) were determined by exponential fitting of the kinetic curves using originpro 7.5 software . quantum yields of internal conversion ( ic ) were obtained from the following equation , which assumes that only three processes ( fluorescence , intersystem crossing , and internal conversion ) jointly deactivate the excited singlet state of ptbc derivatives : ( 6)ic=1(f+t ) . 2(3 ) , 9(10 ) , 16(17 ) , 23(24)-tetrasubstituted phthalocyanines can be synthesized from 4-substituted phthalonitriles , while octasubstituted phthalocyanines can be synthesized from 4,5-dichlorophthalonitrile . in the case of tetrasubstituted derivatives , a mixture of four possible structural isomers are obtained , which can be designated by their molecular symmetry as c4h , c2 , cs , and d2h . in this study , synthesized tetrasubstituted a variety of halogenated phosphorous oxide triazatetrabenzcorroles ( complexes 9 , 11 , and 13 ) were prepared by treatment of unmetallated phthalocyanines with pbr3 in pyridine according to literature procedures . the products thus obtained were then subjected to silica gel column chromatography using pyridine as an eluant . generally , phthalocyanine complexes are insoluble in most organic solvents ; however introduction of substituents to the ring increases the solubility . the halogenated complexes ( particularly 9 and 11 ) exhibited excellent solubility in organic solvents such as pyridine and dmso . for comparative purposes , the unsubstituted ptbc was synthesized and found to be soluble in dmso , but only sparingly in pyridine . the new compounds were characterized by uv - vis , ir , mass , and nmr spectroscopies ( including p nmr ) and the analyses were consistent with the predicted structures as shown in section 2 . however , mass spectral data proved to be difficult to obtain for complex 13 . the p = o vibrations were observed at ~1295 cm(in accordance with ) in the ir spectra , confirming the presence of o coordinated to the phosphorous atom . this was corroborated by the p nmr shifts obtained , that is , ~ 198 ppm , which is typical of a p = o bond . h nmr investigations of 9 and 11 gave the characteristic chemical shifts , with three proton signals integrating for a total of 12 for each complex . for complex 13 , a multiplet due to the nonperipheral protons , was observed in the h nmr spectrum . tbc complexes have distinct uv - vis spectra with a sharp peak at ~450 nm [ 1619 ] , which can be employed in their characterization . the formation of the tbc complexes occurs when the mpc molecules no longer retains a pc moiety as they no longer have the fourth azomethine nitrogen ( see schemes 1 and 2 ) . it is believed that in the presence of excess metal halide , the bridge nitrogen of the pc is eliminated , forming tbc . in this study , the unmetallated pc derivatives ( 8 , 10 and 12 ) were formed first , which , upon reaction with pbr3 , resulted in the formation of ptbc derivatives . this was judged spectroscopically by the collapse of the sharp q band in the visible region of unmetallated pcs to three bands ( in the q band region ) , together with the formation of the sharp soret band at 440 nm ( see figure 1 ) . the spectra of unmetallated pcs 6 , 8 , 10 , and 12 in dmso and pyridine showed a single q band ( see figure 1 ) which is uncharacteristic of unmetallated pcs . typically , unmetallated pcs show a split q band due to lack of symmetry . solvation in polar aprotic solvents ( such as dmso and pyridine ) occurs through their unshared electrons . thus , in dmso ( see figure 1 ) and pyridine , the spectra of 8 , 10 , and 12 did not show the normal splitting of the q band that is typical of free - base phthalocyanines , showing instead a single sharp q band . the nonsplit q band is a result of the basicity of the solvents . it has been documented that in strongly basic solvents , the inner pyrrole hydrogens are acidic enough to dissociate resulting in a charged system ( pc ) which becomes symmetric and thus possesses an unsplit q band . figure 2 compares the spectra of the complexes synthesized in this work , while table 1 lists the q and b band maxima . it is clear in table 1 and figure 2 that the presence of bromines and chlorines shifts the spectra to the red region . the red shift of spectra on halogenation has been observed before for znpc derivatives . aggregation in phthalocyanines and related complexes is usually depicted as a coplanar association of rings progressing from monomer to dimer and higher - order complexes . it is dependent upon the concentration of the complex , the nature of the solvent , as well as the nature of the substituents and the complexed metal ions . in this study , the aggregation behavior of the tbc complexes ( 7 , 9 , 11 , and 13 ) was investigated in dmso ( see figure 3 ) . the complexes did not show aggregation at concentrations less than 8 10 mol dm . the shapes of the excitation spectra for the tbc complexes were similar to the absorption spectra ( see figure 4 ) . however , these spectra were not mirror images of the fluorescence spectra for the ptbc derivatives , in that the emission spectra showed only a single band , while the q band of the absorption spectra has a split q band . the observation of a single - emission band in the q band region is typical of unsymmetric phthalocyanine complexes such as unmetallated derivatives [ 3133 ] . metal - free pcs are known to fluoresce with only one main peak in non - aqueous media which has been assigned as the 00 transition of the fluorescence . the emission spectra were slightly red - shifted with stokes shifts ranging from 5 to 9 nm , suggesting no change in nuclear configurations following excitation . the largest shifts were observed for the pocltbc ( 13 ) and ptbrtbc ( 9 ) , while the smallest shift was observed for the ptbc derivative ( 7 ) . the fluorescence quantum yields ( f ) of the ptbc derivatives are given in table 2 . these values are much lower than reported for mpc complexes , except for the unsubstituted ptbc complex , which gives f values in the range for mpc complexes . the low values obtained for the halogenated derivatives are most likely due to the heavy atom effect of the halide functional group , which encourages intersystem crossing to the triplet state . halogenation of znpc has been reported to give a remarkable decrease in fluorescence quantum yields and lifetimes , since incorporation of a halogen into the photosensitizer increases the level spin - orbit coupling . fluorescence lifetimes ( f , table 3 ) were calculated using the strickler - berg equation . using this equation , a good correlation has been found between experimentally and theoretically determined lifetimes for the unaggregated molecules as found in this work . thus , we believe that the values obtained using this equation are a good measure of fluorescence lifetimes . halogenation is expected to decrease fluorescence quantum yields and lifetimes , increase triplet - state formation , and shorten triplet lifetime . thus , as expected , the f values of the halogenated derivatives were lower than for unsubstituted ptbc and lower than generally observed for mpc complexes . octasubstitution with chlorines increased the f values compared to tetrasubstituted derivatives when comparing complexes 11 and 13 . it is also interesting to note that a decrease in fluorescence lifetime was observed upon contraction of the ring in tin tetrasulphonated ,,-tetrabenzcorroles compared to tin tetrasulphonated phthalocyanines . the acquisition of f values allowed us to determine the rate constants for various processes . the rate constant for fluorescence ( kf ) , table 3 , was highest for complex 9 , which also had the lowest triplet quantum yield as will be discussed below . likewise , the rate constants for intersystem crossing from the singlet state to the triplet state ( kisc ( st ) ) was the highest for complex 9 , table 3 . the transient absorption spectra were recorded in argon - degassed solutions by exciting the photosensitizer ( in dmso ) in the q band region and recording the transient absorption spectra point by point from 400 to 750 nm ( see figure 5 ) . the q and the soret bands showed a negative absorption ( bleaching ) and the transient spectra showed a broad positive absorption ~500 nm ( see figure 5 ) . the triplet lifetimes for the ptbc derivatives , ranging from 256 to 1740 microseconds , are listed in table 2 . the latter value was observed for the unsubstituted ptbc ( table 2 ) and it is an unusually high triplet lifetime . values in the millisecond range have been reported for alpc derivatives , however they are still not as high as the value observed here for unsubstituted ptbc . the presence of the halogens was expected to lower the triplet lifetimes ( when compared to unhalogenated ptbc ( 7 ) ) , as observed in table 2 ; with the octasubstituted complex , 13 , giving the lowest triplet lifetime . contrary to the heavy atom effect , which results in the decrease in triplet lifetimes with an increase in the size of the halogen , this work shows an increase in lifetime on going from the chlorinated ( 11 ) to brominated ( 9 ) ptbc derivatives , with the octachlorinated ( 13 ) complex showing the lowest triplet lifetime . the latter ( 13 ) could have a lower triplet lifetime than the tetrachlorinated derivative ( 11 ) due to the plurality of chlorine atoms and the heavy atom effect . we have recently shown that upon contraction of the ring in tin tetrasulphonated ,,-tetrabenzcorrole compared to tin tetrasulphonated phthalocyanine , there was a decrease in t values and the triplet lifetimes . however , the lifetimes reported here for ptbc derivatives are high compared to mpc complexes in general . in general , there is an increase in t values upon halogenation with a striking exception of the brominated complex ( 9 ) . the octachlorinated derivatives ( 13 ) gave the highest triplet quantum yield ( t ) , while surprisingly the bromo substituted complex 9 gave the lowest value . the t value for 9 is almost ten - fold lower than for 11 . again , the larger value of t for 13 could be due to the plurality of chlorines and the heavy atom effect . the low t value for the brominated derivative compared to the chlorinated one contradicts the heavy atom effect . the increased t value for the chlorinated derivatives 11 and 13 compared to 9 will result in shorter triplet lifetime for the former , and this is the case in table 2 . quantum yields of internal conversion ( ic ) were calculated using ( 6 ) and are high due to low t values . using the triplet lifetimes , the rate constants for intersystem crossing from the triplet state to the ground state ( kisc ( ts ) ) were determined and are shown in table 3 . the lowest ( kisc ( ts ) ) value obtained is for complex 9 ( table 3 ) yet this complex has the lowest triplet quantum yield . in conclusion , we have synthesized halogenated ptbc derivatives ( complexes 9 , 11 , and 13 ) and compared their photophysical data with that of the unsubstituted ptbc complex . the latter complex shows a very high triplet lifetime value , higher than that for mpc complexes . complexes 11 and 13 show reasonably high triplet lifetimes and yields , making them possible candidates for pdt . complex 9 having bromine substituents showed a behavior different from the other halogenated complexes ( 11 and 13 ) in that it gave a very low triplet quantum yield .

the synthesis of phosphorous oxide triazatetrabenzcorroles ( tbc ) tetra- ( 9 , 11 ) or octa- ( 13 ) substituted on the ring with halogenated functional groups is reported . the complexes are not aggregated in dimethylsulfoxide ( dmso ) and show solubility in solvents such as pyridine . the q band absorption spectra of the complexes are red - shifted compared to unsubstituted ptbc . the latter complex shows a large triplet lifetime ( 1.7 milliseconds ) , higher than for mpc derivatives . the chlorinated derivatives show good triplet yields ( t 0.46 and 0.36 ) and relatively long lifetimes ( 256 and 452 microseconds ) , respectively , for 11 and 13 .

1. INTRODUCTION 2. EXPERIMENTAL PROCEDURES 3. RESULTS AND DISCUSSION 4. CONCLUSIONS

PMC3238386

the macromolecular crystallography ( mx ) experiment lends itself perfectly to high - throughput technologies , automation and remote experimentation . the experiment comprises a series of distinct steps , beginning in the wet laboratory with protein expression , purification , crystallization and crystal mounting using flash - cooling in liquid nitrogen , and progressing through to the screening of crystals for diffraction quality , the collection of diffraction data , data processing and structure determination . most of these steps have been fully automated , and in many cases it is now possible to go from expressed protein to fully determined three - dimensional structure with only minimal intervention . however , several steps still require expert human intervention , including the choice of crystal for data collection . since the ultimate goal of the experiment is to produce a high - quality high - resolution structure of the protein in question , this relies heavily upon the choice of the best possible crystal for data collection and the most appropriate data - collection strategy . in this regard , the careful training and education of students and novices is of fundamental importance to these aspects of the process and can not be overlooked , however much automation and remote access are involved in the experiment . some of the most important developments in the automation of protein expression , purification and crystallization have taken place under the auspices of the nih - funded protein structure initiative ( burley et al . with regard to high - throughput crystal screening and data collection , many facilities and groups worldwide have developed automated sample changers , including abbot laboratories in illinois , usa ( muchmore et al . , 2000 ) , doris in hamburg , germany ( karain et al . , 2002 ; pohl et al . , 2004 ) , the spring8 synchrotron in japan ( ueno et al . , 2004 ) , the european synchrotron radiation facility in grenoble , france ( ohana et al . , 2004 ; cipriani et al . , 2006 ) and the advanced light source ( als ) in berkeley , california , usa ( snell et al . , 2004 ) . in an effort to produce a true high - throughput crystal - screening and data - collection facility , and to improve the efficiency of the synchrotron radiation resource , the stanford synchrotron radiation lightsource ( ssrl ) structural molecular biology ( smb ) group and the structure determination core of the joint center for structural genomics ( jcsg ) ( lesley et al . , 2002 ) worked together to develop the stanford auto - mounting ( sam ) system ( cohen et al . in addition to complete automation of the experiment , ssrl has also implemented fully remote access to the mx beamlines ( soltis et al . notwithstanding the obvious increase in throughput and efficiency , the advent of automation and remote access at the ssrl mx beamlines has generated substantial spinoffs for the scientific user community by providing increased opportunities for collaboration between research groups and allowing scientists who might not typically have had access to a national user facility to obtain valuable beam time . it has also introduced many young scientists to synchrotron radiation science by providing educational and training opportunities for graduate students and postdoctoral researchers in user laboratories . the scientific staff at ssrl offer in - house training workshops and attending one of these workshops is strongly encouraged before taking part in remote - access beamtime . furthermore , often the most effective training is from the experiences gained during remote - access beamtime , when new researchers conduct their own experiments under the advice and encouragement of other members of the home laboratory and of ssrl user support scientists , who are readily available via cellular telephone , email and a chat feature ( instant messaging ) in the blu - ice / dcs beamline control system . ssrl has a long history of excellence in structural biology research , including some of the first reports of x - ray absorption spectra from a biological sample ( kincaid et al . , 1975 ) , the first published report of single - crystal diffraction from protein crystals using synchrotron radiation ( phillips et al . , 1976 ) , fundamental studies of what would become the multiple - wavelength anomalous diffraction phasing experiment ( phillips et al . , 1977 , 1978 ; phillips & hodgson , 1980 ; templeton et al . , 1980 ) and the development of insertion devices as sources of high - intensity radiation ( doniach et al . , 1997 ) . ssrl is a national user facility funded by the us department of energy office of basic energy science , the national institutes of general medical sciences ( nigms ) and the national center for research resources , the latter two being components of the us national institutes of health ( nih ) . ssrl provides extremely bright x - ray and uv photon beams produced by the third - generation 3 gev spear3 storage ring , for applications in materials science , environmental science , chemistry and structural biology research , utilizing scientific techniques including photoelectron spectroscopy , small - angle x - ray scattering ( saxs ) , x - ray absorption spectroscopy ( xas ) , total x - ray reflection fluorescence and mx . the smb group at ssrl ( http://smb.slac.stanford.edu ) operates and maintains ten beamlines , seven for mx ( bl1 - 5 , bl7 - 1 , bl9 - 1 , bl9 - 2 , bl11 - 1 , bl12 - 2 and bl14 - 1 ) , two for biological xas ( bl7 - 3 and bl9 - 3 ) and one for biological saxs ( bl4 - 2 ) . all seven mx beamlines at ssrl are fully automated , employing the sam system which has been integrated into the blu - ice / dcs beamline control system and graphical user interface developed earlier at ssrl ( mcphillips et al . to 288 crystals can be screened in a matter of hours without manual intervention using this reliable and robust robotic system . the use of the sam system has not only seen an increase in throughput by research groups but also an improvement in the overall quality of the diffraction data being collected . researchers are now able to screen all their crystals reliably and take advantage of the automated image - analysis tools developed at ssrl , prior to choosing the best quality crystals for subsequent diffraction data collection . these tools include the crystal analysis server , which will automatically analyze test images and feed relevant parameters and statistics back to the researcher via blu - ice , and the browser - based web - ice interface ( gonzlez et al . , 2008 ) , where diffraction and video images of the samples can be viewed , crystals ranked and data - collection strategies calculated . the seven ssrl mx beamlines are all very similar , in that the experimental table , front - end beam - conditioning system , kappa goniometer , cryosystem and detector positioner are nearly all identical . the undulator micro - focus beamline ( bl12 - 2 ) differs somewhat in design to meet the more demanding hardware requirements for microbeam and microcrystal experiments , but is still compatible with the sam system and standard beamline control software . every aspect of beamline control inside the experimental hutch , and also on the upstream optics elements ( mirrors , monochromators and slits ) , is motorized to the extent that it is unnecessary to enter the hutch to change any of the experimental parameters ( x - ray energy , beam size , x - ray detector position , fluorescence detector position , beamstop position , attenuation and lighting ) , to mount or dismount samples , or to anneal or wash ice from samples . this degree of automation of the beamlines is absolutely critical to the implementation of fully remote access ; if there remains a single task that requires human intervention inside the hutch during the normal course of crystal screening and data collection then remote access is not practical . automated sample mounting was made available to general experimenters during the first spear3 run of 2004 on three beamlines . since its inception , use of the sam system has accelerated such that , during the last scheduling period ( 2009 ) , 110 out of 121 research groups ( 91% ) were using sam during their experiments . , 2002 ; smith & cohen , 2008 ; soltis et al . , 2008 ) . during the first year of operation ( 2004 ) , 30 research groups used the automated mounter and over the course of 60 experimental starts mounted over 3500 crystals . the jcsg , one of the original sam test user groups , mounted an additional 2000 or more crystals from 125 target proteins that year , and were successful in solving 30 new structures from 36 unique proteins ( smith & cohen , 2008 ) . the number of crystals mounted using the sam system has also increased dramatically since it was first introduced , such that currently well over 300 000 crystals have been screened by researchers ( fig . 1 ) . fully remote access was made available to research groups during the 2005 scheduling period . during the first two years the number of research groups choosing to conduct their experiments remotely rose from 24 to 44% , and has continued rising each year ( fig . 2 a ) until the last scheduling period , which saw 105 of the 121 groups ( 87% ) screening their crystals and collecting their data using remote - access tools . most noticeably , the total number of remote starts saw an almost exponential growth in 2007 ( fig . 2 b ) , which can be primarily attributed to an increase in beamline efficiency ( fewer beam - hours per start ) as the coupled use of the sam system and remote access became more popular . this increase in beamline efficiency can also be seen in the total number of crystals mounted via the sam system since its inception , which also experienced a dramatic rise in 2007 ( fig . 1 ) . the remote - access experiment at ssrl has been described previously ( smith & cohen , 2008 ; soltis et al . scientists ship their cryo - cooled samples to ssrl in 96-port cassettes custom - designed at ssrl for use with the sam system , or in 16-port uni - pucks ( http://smb.slac.stanford.edu/robosync/universal_puck ) . the cassettes have been designed such that two can be shipped in a standard dry shipper ( 192 crystals in total ) . up to seven uni - pucks ( 112 crystals in total ) may be shipped in a standard dry shipper . the uni - pucks have been designed as part of a collaboration between developers at synchrotrons throughout the united states , allowing research groups to take advantage of automated sample - mounting systems at different synchrotron facilities ( http://smb.slac.stanford.edu/robosync/ ) . the uni - pucks are based upon the als - style puck , and are currently used with the sam robot at ssrl , with many als - style robots at the three other large doe - funded synchrotrons in the us ( als , the advanced photon source and the national synchrotron light source ) , with the actor robot ( rigaku , usa ) , and with various other sample - mounting robots in europe , australia and asia . at ssrl , four uni - pucks are mounted in an adaptor cassette such that the sample pins can be accessed by the sam system in the same way as it accesses sample pins in an ssrl cassette . during their allotted beam time , the remote researchers connect to the beamline computers via an nx server / client application ( http://www.nomachine.com ) . the nx client is downloaded for free onto the researchers home computers , and they can then connect to an nx server running on an ssrl computer . the client uses minimal cpu and memory resources on the host computer , with the entire computational load on the ssrl server . 3 a ) , identical in all aspects to the environment they would see on a computer at the beamline . they can then use the blu - ice control interface ( mcphillips et al . , 2002 ) and/or the web - ice interface ( gonzlez et al . , 2008 ) to screen their crystals and obtain results directly back into the blu - ice screening interface ( fig . 3 b ) , collect monochromatic diffraction data , measure absorption edges prior to multiple- ( mad ) or single - wavelength anomalous diffraction ( sad ) data collection , monitor all aspects of the experiment , and connect to user support staff and collaborators via a real - time chat feature . in fact , everything that a crystallographer would typically do during a synchrotron data - collection visit can be achieved in the remote - access experiment . the remote desktop also gives researchers access to all the crystallographic software installed on the ssrl computers , for data processing , structure solution and analysis . although experimental control , decision making and strategy calculation are carried out in the home laboratory by the researchers and their students , research associates , postdoctoral fellows and/or collaborators ( soltis et al . , 2008 ) , ssrl user support staff are available to troubleshoot experiments , help analyze the screened crystals or advise on data - collection strategy if required . fedex crystallography , whereby researchers send their cryo - cooled samples to the synchrotron but the decision making and data collection are carried out solely by beamline staff ( robinson et al . , 2006 ) , or the more limited telepresence described for a small - molecule crystallography beamline at daresbury ( warren et al . , 2008 ) . based upon feedback from recent ssrl remote - access workshops , remote - access demonstrations at national and international meetings and conferences , anecdotal evidence from informal discussions with research groups , and a recent remote - access survey sent to research groups who regularly use ssrl , the remote - access capabilities have not only revolutionized the way in which diffraction data at synchrotrons are collected but also changed the way in which graduate students and postdoctoral researchers , new to crystallography or synchrotron data collection , are introduced to the area and trained . the general consensus is that the remote - access capabilities at ssrl are a useful tool in training graduate students and postdoctoral fellows in the collection of good quality diffraction data . prior to automation and remote access , a research group comprising , on average , three laboratory members ( perhaps one or two experienced people and some graduate students ) would undertake a synchrotron data - collection trip and spend 4872 h continuously screening crystals and collecting diffraction data . since the first beamlines were developed and made available to the general scientific community , a synchrotron data - collection trip has almost been viewed as a rite of passage for scientists , young postdoctoral fellows and graduate students . it is quite likely that most , if not all , synchrotron beamline users can remember the first time they set foot in one of these laboratories . in recent years , with the increased pressure on funding , the use of research grants to take a large group of scientists to a synchrotron beamline has become uneconomical , particularly given the trend towards increased numbers of crystals being produced in some laboratories , which necessitates more and more access to beamlines . although the use of a national user facility such as ssrl has no direct cost associated with it ( it is mandated that such facilities give free access to us and international scientists at academic institutions ) , there are still significant costs involved with travel and accommodation ( table 1 ) . with the advent of remote - access data collection , new students or other laboratory members who would not normally be sent on a data - collection trip are now exposed to the synchrotron resource , and this access provides valuable experience for their future careers in science . fatigue from travel and prolonged presence at the beamline form a hurdle which has , on occasion , given rise to errors and mistakes during mounting of the crystals , analysis of the diffraction or determination of the optimum collection strategy . prior to the incorporation of the robotic sample mounter , the screening of flash - cooled crystals typically involved manual mounting using cryo - tongs pre - cooled in liquid nitrogen , which enclose the crystal ( mounted in a fiber loop at the end of a sample pin ) inside a hollow cavity ( parkin & hope , 1998 ; rodgers , 2001 ; pflugrath , 2004 ; smith & cohen , 2008 ) to maintain the crystal at cryogenic temperatures during transfer into the experimental hutch and onto the goniometer . although this method has proved to be very reliable since its inception in the 1990s ( pflugrath , 2004 ) , it becomes laborious and tedious when repeated many times . the skill and patience of the experimenter , rather than the number of samples available , have often dictated the quality of the crystal selected for data collection ; crystals were screened manually until a crystal deemed good enough to collect a complete diffraction data set was found . in cases like this , other crystals from the same project would go unscreened ; if a better quality crystal were among those which were unscreened , it would go undetected and uncollected . the process of crystal screening , crystal selection and data - collection strategy determination has become significantly easier with the implementation of the sam system , the crystal analysis server and web - ice . as noted above , useful crystal parameters and statistics [ including the bravais lattice , the unit - cell parameters , the estimated mosaicity , the predicted resolution , the r.m.s . fit from mosflm ( leslie , 1992 ) and an overall score ] are continually fed back into the blu - ice spreadsheet ( fig . 3 b ) , and these are also accessible through web - ice , where researchers can also inspect the diffraction images and crystal video images . the availability of screening results and the crystal analysis have provided a new resource for training novice crystallographers during the experiment . researchers can easily access and compare diffraction images , video images of each crystal and the results of the crystal analysis server to decide how best to proceed . for example , a crystal may need to be rescreened because the best part of the crystal was not in the beam , or perhaps the crystal may need washing as it was covered with surface ice ( visible on the crystal images and as strong ice rings on the diffraction images ) , or the automated strategy may be confirmed as a good approach for subsequent data collection . access to all this information through web - ice makes it easier to teach novice crystallographers when to use automated results and when to question them . it is undeniable that hands - on experience with the control systems of a synchrotron beamline , and the ability to analyze and monitor the data as they come off the detector in real time , are vital not only to the collection of the best possible diffraction data ( which will ultimately lead to the best possible structures ) but also in the training of the next generation of synchrotron beamline users . our contention , which is thoroughly backed up by the feedback we have received over the past five years , is that the training being received by students and novices via ssrl user support staff and the ssrl remote - access tools is fully comparable with the on - site training they would have received had they made an actual trip to ssrl or other synchrotron facilities . in most cases this is a guided participation approach , whereby an experienced researcher , principal investigator ( pi ) or ssrl user support person will demonstrate the fundamental aspects of the system to perhaps a small group of students or novice group members , and then guide them through the experiment as they take control of the blu - ice or web - ice interface . it is well understood that people learn by different methods , whether it be through observation , analysis , discussion or activity , or a combination of these . the remote - access tools available to the ssrl user groups offer something to all types of learner and therefore provide a very effective method of teaching the new user the best possible ways in which to collect the highest quality diffraction data , this being the ultimate goal of any x - ray diffraction experiment . direct contact with ssrl user support staff is strongly emphasized as being the important first step in remote training for any research group . the user support staff have a vast amount of knowledge and expertise with the ssrl beamline systems , the sam robot and the remote - access capabilities , and can direct researchers to the appropriate information and resources to make their group training , and ultimately their valuable beam time , a most effective and efficient process . moreover , ssrl user support staff can effectively facilitate remote training with a research group over the telephone , employing all the remote - access tools available to the research group . these tools include ( i ) access to the ssrl user guide , ( ii ) access to a number of video tutorials which illustrate various steps in a remote - access data - collection experiment , ( iii ) connection to a simulated beamline , facilitated through ssrl user support staff , ( iv ) information on software packages installed and supported on ssrl computers ( http://smb.slac.stanford.edu/public/facilities/software/ ) , ( v ) access to test images and data sets so that the processing software and structure - solution software and scripts can be tested by or demonstrated to students and novices , ( vi ) use of the chat feature in blu - ice , and ( vii ) use of the shared desktop capabilities of the nx server / client interface , whereby ssrl support staff can demonstrate the blu - ice or web - ice interfaces while a remote research group follows on their local computers . the full capabilities of the nx desktop - sharing tools are described on the developer s website ( http://www.nomachine.com ) . the ssrl user support staff are a group of expert crystallographers and engineers who are available before , during and after beam time for consultation and practical help . typically , one staff member is responsible for a given beamline for a specified period , and research groups can determine who their particular support person will be from the online user support schedule ( http://smb.slac.stanford.edu/schedule/sch_staff.cgi ) . as noted above , research groups are strongly encouraged to contact the responsible staff member by either telephone or email prior to upcoming remote - access beam time to discuss beamline characteristics , sample preparation , and experimental design and strategy , to gain access to the simulated beamlines , to test connectivity through the nx server / client system , and to organize either pre - beam remote training or training once their beam time starts . the use of remote training as a teaching tool in research laboratories assumes the presence in the research group of an experienced user of the ssrl beamlines and the blu - ice or web - ice interfaces who can facilitate this training . if the research group is new to ssrl then this may not be the case , and under these circumstances we strongly recommend that the group send at least one representative to either an on - site or a remote ssrl workshop to gain hands - on experience with blu - ice and web - ice , the ssrl computing systems , and in the use of the cryo - tools associated with the sam system , the storage and transport options available , and the proper sample preparation techniques . sample preparation is absolutely critical to the success of the experiment , irrespective of whether it is on - site or remote . these trained scientists can then return to their laboratories and facilitate the training of group members in the use of these systems , with the assistance of ssrl user support staff . a comprehensive description of the tools and their use , along with correct sample - pin selection and preparation , is also available through the smb website ( http://smb.slac.stanford.edu/public/users_guide/manual/using_ssrl_automated_mounti.html ) . once screening and data collection are underway , staff are also on hand to help with connectivity problems or beamline troubleshooting , to give blu - ice or web - ice help , and to give direct experiment - related advice regarding crystal selection , data - collection strategy determination , processing software help and data backup . staff can contact remote scientists by telephone , by email or using the chat feature in blu - ice , and researchers can contact staff using the same methods . ssrl user support staff contact details are available on the smb website ( http://smb.slac.stanford.edu/public/staff/index.shtml ) . 4 ; http://smb.slac.stanford.edu ) contains up - to - date information for research groups on the state of the mx beamlines , the beamline schedule and the spear accelerator status , with links to the computing and software resources available ( through the facilities tab ) , and to the user guide ( http://smb.slac.stanford.edu/public/users_guide/index.shtml ) . the user guide is available online to all users at any time , irrespective of whether they have beam time , and can be downloaded as a pdf file . the guide gives a detailed description of all aspects of mx experiments at ssrl , from becoming an ssrl user , to detailed instructions on the use of the sam system and the preparation of samples , and how to use the blu - ice and web - ice interfaces effectively to set up and carry out a crystal - screening and data - collection experiment . the differences between an on - site and a remote experiment are clearly defined , such that novices and first - time remote - access users have all the information at hand prior to the start of their beam time . information specific to the collection of mad data and high - resolution monochromatic data are presented , and the data - processing software packages available to researchers are described , along with short tutorials on the most effective use of these programs . a set of detailed answers to frequently asked questions ( faqs ) is also included at the end of the user guide to aid users in their experiments , and to help with programs and with questions should they arise . the video tutorials can be accessed from the user guide page of the smb website as given above , or via the link http://smb.slac.stanford.edu/public/users_guide/tutorials/ ) . this project is constantly being developed and updated as new beamline capabilities and tools become available . current tutorials include those that give information on tasks that can be carried out prior to beam time , such as ( i ) downloading and installing the nx client software , ( ii ) the best ways to fill in the excel spreadsheet with crystal information for a remote - access or on - site sam - assisted experiment , and ( iii ) instructions on how to upload the completed spreadsheet to the crystal database prior to or at the beginning of the user beam time . three additional videos describe ( iv ) the sam - assisted remote - access experiment in detail , demonstrating how to use the sam system to screen crystals in a cassette , ( v ) how to interpret the screening results subsequently to select crystals for data collection and ( vi ) a simulated web - ice strategy calculation . a strategy calculation for a mad or sad data collection is also demonstrated . prior to the start of beam time , the members of a research group can connect to the ssrl computers and gain access to a the seven ssrl beamlines each have a simulated counterpart which can be accessed in exactly the same way as the access is only possible by contacting one of the ssrl user support staff beforehand and asking for authorization on one of the simulated beamlines . following authorization , the remote user connects to the simulated beamline through a blu - ice interface indistinguishable from the one that will be used later to screen crystals and collect data . all the motors that control experiment variables , such as beam size , detector distance , x - ray energy and the beamstop position , can be moved . since the remote user is not actually connected to a real beamline , this does not affect experiments currently being carried on the real counterpart of the simulated beamline . the cassette spreadsheet can be uploaded and new users can then be taken through the steps involved in crystal screening by the experienced users in the group . the simulated beamlines are an extremely valuable resource for a research group that may be new to remote - access data collection , the ssrl beamlines or synchrotron data collection in general . the best use of these simulated beamlines involves the inclusion of a member of the ssrl user support staff in the remote training exercise , whereby the use of the blu - ice interface on the simulated beamline can be fully described and discussed in detail with all members of the group . this can be facilitated by a telephone call or by use of the desktop - sharing tools available with the nx server / client software ( http://www.nomachine.com ) . in most remote - access experiments , there are generally several experienced people in the home laboratory responsible for the data collection . because the nx client system allows multiple connections with the same user account , experienced users can passively monitor the screening and data collection being carried out by students or postdoctoral researchers , which still allows the students their independence and involvement in the decision - making process , yet allows for the correction of mistakes or the suggestion of alternative strategies . this capability also makes it easy for ssrl user support staff to monitor the screening and data collection , and to step in if they see a potential problem . multiple connections under the same user account can have the name and telephone number of the scientist associated with each one in the users tab of blu - ice , making it easy to identify who is currently active should user support staff wish to contact the researcher . this can be extended beyond the home laboratory to the laboratories of collaborators , who can also connect during active beam time , again with a name and telephone number associated with the connection on the users tab , either passively to monitor the data collection , or actively to play a role in the screening , analysis and choice of crystals , or the data collection . the general consensus amongst ssrl research groups is that providing beamline access to collaborators under the auspices of their proposals has given these collaborating scientists and their group members exposure to synchrotron beamlines that they would never have been able to obtain without remote access . in some cases , this exposure has led to these collaborating scientists writing their own successful proposals for synchrotron beam time . a prime example of this is the beam - time proposal submitted by the center for molecular structure ( cmols ) at the california state polytechnic university pomona campus , which is part of the california state university ( csu ) . this was not a single - user proposal , as are the majority of proposals , but a wide - ranging one encompassing at least five csu campuses and several different co - pis . the csu campuses are traditionally undergraduate institutions which have not typically had access to synchrotron resources in the past , either because of a lack of funding or because it was not something that was ever thought of as being a possibility . remote connection to the mx beamlines at ssrl is now giving these researchers and their undergraduate students continued access to state - of - the - art facilities , and is having a positive impact on their approach to science and research . scientific staff from the smb group not only are regularly involved in one - to - one user support via email and telephone ( before , during and after the experiment ) , but also facilitate remote - access workshops to train new researchers in the use of blu - ice and web - ice , and in the practical aspects of sample mounting and cryo - cooling , synchrotron data collection , and data processing . several of these remote - access workshops have been held locally at ssrl , and scientists from the group have also traveled both nationally and internationally to hold remote - host workshops ( table 2 ) . they are usually scheduled at the start of the user run , or more often , depending on demand . occasionally , these workshops also take place in conjunction with the annual ssrl users meeting ( see table 2 ) . a typical workshop lasts half a day and includes a thorough introduction to the experimental facilities for mx users , including hands - on tutorials on the optimal use of the sam robot tools , data collection with blu - ice , analysis and strategy calculations with web - ice , and data processing with the available locally installed software packages . the remote - host locations have included the hauptmann woodward medical institute ( hwi ) in buffalo , new york , usa ( august 2006 ) , the university of melbourne , australia ( february 2007 ) , the university of pittsburgh , pennsylvania , usa ( october 2008 ) , and the california institute of technology ( caltech ) , pasadena , usa ( june 2009 ) . during the university of melbourne workshop , one of the participants screened crystals that had previously been shipped to ssrl , identified the best quality crystal , collected a mad data set and solved a novel protein structure ( schmidberger et al . , 2008 ) , completely remotely , fully utilizing the computational resources made available to researchers at ssrl . ssrl remote access has also been incorporated into two workshops sponsored by the center for workshops in chemical sciences ( http://chemistry.gsu.edu/cwcs ) at cmols , which were aimed at faculty from predominantly undergraduate institutions . additional workshops at which ssrl staff have presented the remote - access tools and capabilities are listed in table 2 . another important method of disseminating information regarding the ssrl remote - access tools to the user community is through seminars and live remote - access demonstrations at conferences and meetings ( table 2 ) . this turns out to be a perfect test of the capabilities of the nx client system , because generally at conference locations the wireless internet access can be somewhat intermittent and with variable speed or bandwidth , particularly as conference participants continually connect and disconnect to the system . since the nx client system is designed to run on only 20 kbps of network bandwidth , good performance is generally maintained in the seminar locations , even on a busy wireless network . the use of a remote - access connection to either an ssrl mx beamline or a simulated beamline , when combined with conference lectures or seminars , workshop presentations , or in a formal university teaching environment , is a powerful pedagogical tool . we strongly encourage and support such use of the ssrl systems by the scientific community . the sam system has been used to screen a total of over 300 000 crystals for diffraction quality in the past seven years , and has most certainly proved its worth . when coupled with the remote - access capabilities that have been available to scientific user groups ( general users ) for the past five years , this system has led to the mx beamlines at ssrl becoming a true high - throughput facility . the efficiency of the research groups who use remote access has increased remarkably , which has in turn given synchrotron access to more user groups than ever before and resulted in a surge in the number of user starts at ssrl . researchers are now easily able to screen all crystals being grown in the laboratory , in order to choose the best possible crystals for data collection , whereas before they may have limited themselves to the crystals that simply appeared to be the best , or else spent innumerable hours on a home source screening crystals . it has become increasing clear that many user groups are forgoing in - house screening , and simply cryo - cooling as many crystals as they can fit into a cassette or uni - pucks and letting the robust efficient sam system do the work for them . this is exactly the vision the developers of the stanford auto - mounter had in mind for the system : to provide a true high - throughput platform for the screening of large numbers of protein crystals . the ways in which remote access to the ssrl beamlines can facilitate training and collaboration have most certainly not gone unnoticed by the scientific community . all research groups who collect their data remotely use the available tools provided by ssrl to train and educate their laboratory members in the most effective ways to collect the best possible diffraction data . approximately 60% of researchers with active proposals and current beam time have at some point had collaborators participate in remote - access data collection , where they take either a passive or an active role , and in some cases have even used the time to train or educate members of their own laboratory . the way in which remote access to ssrl beamlines serves to bring collaborators together is one of the most fundamental examples of what has been described as a cultural community , as noted by the director of the nsf report cyberinfrastructure vision for 21st century discovery ( nsf cyberinfrastructure council , 2007 ) . this idea is something that we at ssrl will continue to foster and promote . at ssrl we are dedicated to making the remote - access experience as easy , efficient and instructive as possible , and making a synchrotron beamline accessible to anyone in the scientific community who has a need for a high - intensity x - ray beam and expects high - quality diffraction data .

the ultimate goal of synchrotron data collection is to obtain the best possible data from the best available crystals , and the combination of automation and remote access at stanford synchrotron radiation lightsource ( ssrl ) has revolutionized the way in which scientists achieve this goal . this has also seen a change in the way novice crystallographers are trained in the use of the beamlines , and a wide range of remote tools and hands - on workshops are now offered by ssrl to facilitate the education of the next generation of protein crystallographers .

Introduction Synchrotron radiation research at SSRL Training and collaboration Education and outreach Conclusions

PMC4703417

clinical research is the type of scientific research that involves human subjects and includes patient - oriented research , epidemiologic and behavioral studies , and outcomes research and health services research . clinical studies evaluate the effect of interventions or exposures on biomedical or health - related outcomes that include prevention , diagnosis and treatment of diseases . clinical trials ( cts ) or interventional studies are clinical studies in which participants are assigned by researchers / investigators to receive one or more interventions to assess their safety and efficacy . in observational studies , phase 0 studies are exploratory studies involving very limited human exposure to an investigational new drug ( ind ) for example screening studies and microdose studies . the primary aims of phase 0 studies are identifying , early in the process of drug development , viable candidates and eliminating those lacking promise with a potential reduction in costs and time - to - first - in - human testing . the endpoints include evaluation of analogs for lead selection , modulation of a molecular target in vivo , whole - body imaging for tissue distribution / target binding affinity , and agent pharmacokinetics [ ] . phase 1 studies aim to find out the drug s most frequent and serious adverse events and the drug metabolism and excretion . phase 3 studies gather more information about safety and effectiveness . the randomized controlled trial ( rct ) phase 4 studies occur after marketing approval of a drug by authorities and aim to gather additional information about a drug s safety , efficacy and optimal use . participation in clinical trials is a voluntary action after subjects are fully informed of the research and give their consent . without participants , no ct can conclude . clinical trial registries ( ctrs ) facilitate the prospective registration of cts and the accessibility of their information by patients , physicians , researchers and other interested stakeholders . this enhances transparency , increases participation in cts and can eliminate publication bias that arises from publishing positive trial results more than the negative ones , . some countries mandate ct registration ; others do not ask for registration , but often strongly encourage it . federal law , declaration of helsinki , european union clinical trials directive , who clinical trials directive , international committee of medical journal editors ( icmje ) . currently , there are many ctrs with a scale being global ( e.g. who international clinical trials registry platform [ ictrp ] ) , continental / regional ( e.g. eu clinical trials register [ eu - ctr ] and pan - african clinical trials registry [ pactr ] ) , country - specific ( e.g. us clinicaltrials.gov ) or companies and associations ( e.g. international federation of pharmaceutical manufacturers associations [ ifpma ] ) . clinicaltrials.gov registry ( ctgr ) is a trial registry hosted by the us national institute of health ( nih ) . it is a governmental website . in february 2000 , it was made available to the public as a registry of clinical trials information on federally and privately funded trials conducted under investigational new drug ( ind ) applications to test the effectiveness of experimental drugs for serious or life - threatening diseases or conditions . in september 2008 , more information on study participants and a summary of study outcomes , including adverse events were made available . by 17th of march 2014 , ctgr contained 163,090 studies . the focus then shifted to establishing the two key elements of the platform : the international clinical trials registry platform ( the ictrp network ) and a single point of access ( the ictrp clinical trials search portal ( ctsp ) ) . ctsp provides a single point of access for the identification of trials in many contributing registries . ctsp was launched in may 2007 and initially contained trial records provided by three ctrs : the australian new zealand clinical trials registry ( anzctr ) , ctgr and the international standard randomised controlled trial number ( isrctn ) registry . in addition to the above registries , the ictrp registry network includes registries based in brazil , china , cuba , eu - ctr , germany , india ( ctri ) , iran , korea , japan , the netherlands , pactr , sri lanka , thai and uk . by 17th of march 2014 , ictrp contained 271,811 records for 229,638 trials . in early 2007 , the pan - african clinical trials registry ( pactr ) was established by the south african cochrane centre , in partnership with the european and developing countries clinical trials partnership and the cochrane infectious disease group . in the initial phase , pactr registered trials in hiv / aids , tuberculosis and malaria to demonstrate proof of concept . once established , the goal of pactr is to become the registry of choice for any clinical trial conducted in africa . on 25th of september 2009 , pactr was officially launched as a member of the who primary registry network in abuja , nigeria . pactr is presently the only african member of the who network of primary registers and transfers all trial information to the who ctsp on a quarterly basis . as of 17th of march 2014 the aim of this work is to study the profile of clinical trials in egypt in three clinical trial registries with different scopes : the global ictrp registry , the regional / continental pactr registry and the us ctgr . the us clinical trial registry clinicaltrials.gov ; ctgr was searched for trials conducted in egypt . in the advanced search http://clinicaltrials.gov/ct2/search/advanced , egypt was searched in the countries 1 , 2 and 3 and the search yielded 548 entries . clinical trials search portal ( ctsp ) ( http://apps.who.int/trialsearch/advsearch.aspx ) was searched for trials from egypt and the search yielded 686 entries . the pan - african clinical trials registry pactr ( http://www.pactr.org/ ) in the advanced search and 73 entries for 56 studies were retrieved . all searches were done on the same day ( 10th of march , 2014 ) . the ictrp was selected because it is an international registry that accepts studies from several registries including country - specific , regional and continental ones . the clinicaltrials.gov was chosen because it is the most mature registry and it preceded the ictrp and thus it may contain studies not listed in the latter registry . the pactr was chosen because it is the only registry in the continent where egypt lies ( africa ) . studying this latter registry as of 10th march 2014 , the total numbers of studies included in the ictrp , ctgr and pactrp were 229,638 , 163,090 and 645 , respectively . within ictrp , egypt ranked the 55th with a total of 686 studies representing 0.30% of ictrp total . within ctgr , egypt ranked the 115th with a total of 548 studies representing 0.34% of its total . within pactrp , egypt ranked the 2nd ( following south africa ) with a total of 73 entries for 56 studies representing 11.3% of pactrp total ( table 1 ) . ctgr and pactrp are primary registries that receive data directly from registerers while ictrp receives trial information from many primary registries . main sources of egypt s studies in ictrp were ctgr ( 72.3% ) , anzctr ( 13.6% ) , eu - ctr ( 7.7% ) , isrctn ( 4.2% ) and ctri ( 2% ) . comparing study titles registration of egypt s clinical studies to both ctgr and ictrp started in 1999 and peaked in 2012 ( fig . 1 , fig . active recruiting status was encountered in 27.4% in ctgr , 19.1% in ictrp and 26% in pacrp . completed status was encountered in 53.8% in ctgr and 33.9% in pactrp and not mentioned in ictrp . study results were only available in ctgr and only 8% of egypt s studies have results ( table 1 ) . in pactrp and ctgr , most studies were sponsored by universities / organizations followed by industry in ctgr or individual researcher in pactrp . the most common egyptian sites were cairo university , mansura university , ain shams university , assiut university and alexandria university ( fig . the most common industrial sponsors were novartis , hoffman - la roche , sanofi , pfizer , eli lilly and bayer ( fig . interventional studies were the most common type of studies in both ctgr and ictrp ( 84% ) followed by observational studies . treatment was the most common study purpose ( 69% ) in ctgr and pactrp followed by prevention ( table 2 ) . in ctgr , combined safety / efficacy was the commonest endpoint ( 49.1% ) followed by efficacy alone ( 19.5% ) . in the three registries , inclusion of both genders and adults was the most common practice . the median number of enrolled subjects was 155 in ctgr , 150 in ictrp and 63 in pactrp . for interventional studies in ctgr and pactrp , the most common intervention was drug use ( 61.9% ) followed by procedure ( 12.8% ) . in the three registries , the commonest study phase was phase 3 followed by phase 4 and phase 2 ( table 3 ) . the commonest allocation method was the randomized method ; blinding was implemented in many of trials and was mostly double - blind . in ctgr and ictrp , observational studies constituted almost 15% of the studies and were mostly prospective and cohort studies ( table 4 ) . the most researched conditions were digestive system diseases , cancers / other neoplasms , symptoms / general pathology ( fig . the most common conditions were cancer , hepatitis and diabetes mellitus ( fig . 6 ) . in pactrp , diseases and conditions being researched were listed as individual conditions rather than grouped items . in pactrp , pregnancy and infertility were the commonest conditions ( n = 12 ; 21.4% ) followed by surgical procedure ( n = 9 ; 16.1% ) , cancer ( 12.5% ) , pains ( 10.7% ) , and others ( 39.3% ) . egypt is the land of civilizations and is reputed worldwide for its distinct 7000-year - old record of civilization and immense wealth of knowledge . he performed surgery , practiced some dentistry , extracted medicine from plants and knew the position and function of the vital organs . surgical instruments and surgical procedures such as circumcision are documented on the paintings on the temples , . with its 82-million population , however , it contributes only 0.34% of records in us clinical trial registry ( clinicaltrials.gov ; ctgr ) and 0.30% in the who registry ( ictrp ) . there are 24 faculties of medicine with up to 34 departments in each faculty , 18 faculties of dentistry with up to 12 departments in each faculty , 34 faculties of pharmacy with up to 9 departments in each faculty , 12 faculties of nursing with up to 7 departments in each faculty and 6 faculties of physical therapy with up to 8 departments in each faculty . in egypt , there are more than 42,000 faculty members and 344,000 post - graduate students , , 140,000 physicians , 18,200 dentists , 37,500 pharmacists 176,000 nurses and 35,000 physical therapists . clinical research including clinical trials is an essential mandate for getting master and doctorate degrees . thus , the output of about 94 health - related faculties , 1400 departments and the huge number of health care professionals and faculty members is definitely more than the registered studies . the apparent low contribution of egypt in trial registries may be related to multiple factors . there is no egyptian national trial registry that hosts all trials running in the country . many trials are not recorded in trial registries whether global e.g. who ictrp , regional / continental e.g. pactrp or other e.g. ctgr . . the reasons why some egyptian trials are registered can be imputed from examining the trial sponsorship where most trials are sponsored by either pharmaceutical industry or academic universities . many of the pharmaceutical industrial sponsors include an egyptian site in addition to other non - egyptian sites . in addition to innovation , great majority of research conducted in the egyptian universities aims at publication in renowned international journals that both increases author citation h - index and advances the university ranking in the university index , . based on icmje , most renowned journals ask author to provide details for trial registration prior to publication . otherwise , many egyptian investigators may be unaware of the presence of trial registries or the mandate of registration they may miss the support from administrative research offices / facilities that can carry out this job on their behalf . of the three studies registries , the most mature registry is ctgr followed by the ictrp and there are many similarities between search fields in the three registries ( table 5 ) . ctgr allows searching by intervention , study phase , gender , age groups , date of last update , sponsors / collaborators , results , completion status . it displays the total number of studies and allows further work on the results within the website . pactrp allows further search by study purpose , dates of start and end and principle investigator name and country . however , interface was less user - friendly and total results are not shown and could not be worked within the website . the most common diseases researched in the registries were digestive system diseases , cancers / neoplasms , hepatitis , dm and urogenital conditions . while most of these conditions are included in the top egyptian biomedical research topics , yet similarly , tumors were the 7th research priority but came first in the registered trials . reasons for the difference between the country s research priorities and the actual research done can be many universities are not under the portage of the ministry of health ( moh ) and thus are not mandated to comply with its set priorities . industry enforces research topics that are within their main interest e.g. to get a drug s approval . additionally and as mentioned above , trials in the registries do not provide a comprehensive picture of the biomedical research in egypt . most of the studies were funded by either academic universities / organizations ( 3854% ) or industry ( 3843% ) and most of the industries are multinational ones . unfortunately , we could not get information on the amount of funding and the share of different stakeholders . in developed countries such as us , investment in health research comes mainly from industry ( 64.4% ) , federal government ( 32.6% ) and universities ( 3.0% ) . industry focuses on research with the best return on investment ( roi ) . this may , or may not , coincide with the national priorities . thus , in developing countries such as egypt , governmental sponsorship should predominate to enforce the national priorities . while the mentioned registries were set to host clinical trials , yet up to 17% of the entries refer to observational studies i.e. non - trial studies . with time , the number of observational studies in clinicaltirals.gov increased and the scope of registration widened . for example , the clinicaltrials.gov was initially set to host clinical trials on ind for life - threatening conditions , then it expanded to include other types of studies as well as their results . this was because much of the rationale for the prospective registration of clinical trials applies to the registration of observational studies . observational studies could provide valuable information not achieved even by the well conducted phase iii clinical trials particularly related to drug safety . making both observational and interventional studies available in a single registry will provide researchers and others with a more comprehensive view of the growing evidence base [ http://www.ncbi.nlm.nih.gov/pmc/articles/pmc2952011/#!po=10.7143 ] . however , this relates to the source registries and the way they were designed ( table 5 ) . for example , some registries ask specifically for some information not requested by the others making comparison unfeasible . also , some of the information requested may be either optional or may not have exhaustive quality checks for what was entered . even , if some data were entered there were no exhaustive checks that read , understand and advise authors for the necessary changes . . however , clinical trials from egypt in three trial registries are apparently low and do not accurately reflect clinical research conducted in egypt or its potential . we recommend that egyptian authorities immediately start a national egyptian clinical research registry ( egcrr ) that matches the well - developed registries particularly ctgr and hosts clinical trials as well as observational studies ( table 5 ) . we propose that egcrr be hosted by the egyptian ministry of health , the ministry of higher education or the ministry of scientific research . we recommend that all egyptian clinical research mandatorily get registered to this egcrr immediately following ethical approval or exemption by the ethical committees , and that investigators and institutions receive appropriate training on research registration . in addition to the several advantages of registration e.g. transparency , enhanced participation and bias elimination , such egyptian registry ( egcrr ) would ensure that research is not repeated and that the country s research activity can be assessed at any time . to ensure compliance with registration this can be done at least at two levels ; first funding may be linked to registration and second there should be some legislation to mandate registration . we are to see the first egyptian clinical research law that will regulate and advances clinical research as well as protect research participants rights , safety and well - being . this law should address registration . to protect the interests of sponsors and investigators , they are highly encouraged to patent and copy - right their investigational agents and intellectual work prior to embarking on experimentation in humans and hence registration . the registry should not reveal the sensitive and secret aspects of an investigational agent e.g. its chemical structure . we also recommend setting of research support offices / facilities that can carry out the job of registration or at least provide the support and guidance . it is highly recommended that egypt sets a clear and frequently updates national research priorities . these priorities should be decided on with the involvement of all stakeholders including , but not limited to , moh , academic institutions , politicians , researchers , funding agencies , pharmaceutical companies and community representatives . research funding should be linked to compliance with these priorities . to avoid skewing research toward the best interest of international pharmaceutical industry , governmental funding of research

registering clinical trials ( cts ) in public domains enhances transparency , increases trust in research , improves participation and safeguards against publication bias . this work was done to study the profile of clinical research in egypt in three ct registries with different scopes : the who international ct registry platform ( ictrp ) , the continental pan - african ct registry ( pactr ) and the us clinicaltrials.gov ( ctgr ) . in march 2014 , ictrp , pactr and ctgr were searched for clinical studies conducted in egypt . it was found that the number of studies conducted in egypt ( percentage ) was 686 ( 0.30% ) in ictrp , 56 ( 11.3% ) in pactr and 548 ( 0.34% ) in ctgr . most studies were performed in universities and sponsored by university / organization , industry or individual researchers . inclusion of adults from both genders predominated . the median number of participants per study in the three registries ranged between 63 and 155 . the conditions researched differed among the three registries and study purpose was mostly treatment followed by prevention . endpoints were mostly efficacy followed by safety . observational : interventional studies ( i.e. clinical trials ) represented 15.5%:84.5% in ictrp , 0%:100% in pactr and 16.4%:83.6% in ctgr . most interventions were drugs or procedures . observational studies were mostly prospective and cohort studies . most cts were phase 3 and tested drugs or procedures . parallel group assignment and random allocation predominated . blinding was implemented in many of trials and was mostly double - blind . we conclude that cts from egypt in trial registries are apparently low and do not accurately reflect clinical research conducted in egypt or its potential . development of an egyptian ct registry is eagerly needed . registering all egyptian cts in public domains is highly recommended .

Introduction Methods Results Discussion Conclusions Recommendations Conflict of Interest Compliance with Ethics Requirements

PMC1469486

despite the recent declines in environmental lead exposure in the united states and mexico , the potential for delayed toxicity from bone lead stores remains a significant public health concern . some evidence indicates that mobilization of lead from bone may be markedly enhanced during the increased bone turnover of pregnancy and lactation , resulting in lead exposure to the fetus and the breast - fed infant . we conducted a cross - sectional investigation of the interrelationships between environmental , dietary , and lifestyle histories , blood lead levels , and bone lead levels among 98 recently postpartum women living in mexico city . lead levels in the patella ( representing trabecular bone ) and tibia ( representing cortical bone ) were measured by k x - ray fluorescence ( kxrf ) . multivariate linear regression models showed that significant predictors of higher blood lead included a history of preparing or storing food in lead - glazed ceramic ware , lower milk consumption , and higher levels of lead in patella bone . a 34 micrograms / g increase in patella lead ( from the medians of the lowest to the highest quartiles ) was associated with an increase in blood lead of 2.4 micrograms / dl . given the measurement error associated with kxrf and the extrapolation of lead burden from a single bone site , this contribution probably represents an underestimate of the influence of trabecular bone on blood lead . significant predictors of bone lead in multivariate models included years living in mexico city , lower consumption of high calcium content foods , and nonuse of calcium supplements for the patella and years living in mexico city , older age , and lower calcium intake for tibia bone . low consumption of milk and cheese , as compared to the highest consumption category ( every day ) , was associated with an increase in tibia bone lead of 9.7 micrograms pb / g bone mineral . the findings of this cross - sectional study suggest that patella bone is a significant contributor to blood lead during lactation and that consumption of high calcium content foods may protect against the accumulation of lead in bone.imagesfigure 1.figure 1.figure 2.figure 2 .

Images

PMC2602909

drosophila stocks , culture conditions the sod1 and ry strains are described in ref , 10 . hsod1 was expressed using lines carrying uas - hsod1 and the gal4 driver line , dagal4 as described ( 14 ) . stocks were maintained at 25 c on standard cornmeal and agar medium unless otherwise stated . the copper chaperone coding region was amplified by pcr ( primers ccs5 + ccs7 ) using dna extracted from a drosophila canton s adult cdna library ( stratagene catalog number 936603 ) . the 930-bp pcr product was blunt - ended and subcloned into the smai site of pbluescriptiisk ( stratagene ) to generate the vector pbcc . the transformation vector pucc was constructed by subcloning the ecori - noti fragment of pbcc into puast ( 15 ) . yeast strains , growth conditions , plasmids yeast strains used in this study were derived from eg103 ( mat , leu2 - 3,112 , his31 , trp1 - 289 , ura3 - 52 ) ( 16 ) and include ks107 ( sod1 ) ( 17 ) and ls101 ( sod1 ccs1 ) ( 18 ) . cells were propagated at 30 c either in enriched yeast extract , peptone - based medium supplemented with 2% glucose ( ypd ) , or in synthetic complete ( sc ) medium ( 19 ) . solid medium was supplemented with 15 mg / liter ergosterol and 0.5% tween 80 to enhance growth in anaerobic conditions . yeast expression plasmids for dsod1 and dccs were produced using cdna clone re52090 ( open biosystems ) for dsod1 and plasmid pucc for dccs . the coding sequences for dsod1 and dccs were pcr0-amplified introducing 5 bglii and 3 snabi sites in dsod1 and 5 mlui and 3 rsrii sites in dccs . the dsod1 pcr product was digested with the appropriate enzymes and ligated into pls108 ( 18 ) cut with bglii / snabi , replacing the yeast sod1 coding sequence with that of dsod1 resulting in plasmid plj373 . the digested dccs pcr product was inserted into plj366 ( a derivative of pls113 ( 18 ) with an mlui site just prior to the yccs1 start codon and an rsrii site immediately after the stop codon ) , replacing the yccs1 coding sequence with that of dccs , generating plasmid plj375 . both dsod1 and dccs were under control of their corresponding s. cerevisiae regulatory sequences . the sequence integrity of these plasmids was ensured by double - stranded dna sequencing ( dna analysis facility , the johns hopkins university ) . yeast ccs1 plasmids phal-413 , pls008 ( c229s , c231s ) , and pls010 ( k136e , g137e ) have been described previously ( 18 ) . domain i mutation in yccs1 ( c17s , c20s ) was introduced using the quikchange mutagenesis kit ( stratagene ) using phal-413 as a template ( 4 ) resulting in plasmid pps031 . sod1 in - gel activity assay to analyze sod1 activity from drosophila , five males were homogenized in 50 l of 1% triton x-100 . 10 l of supernatant was transferred to a tube containing an equal volume of loading buffer ( 125 mm tris - hcl , ph 6.8 , 20% glycerol , 0.01% bromphenol blue ) . the gel was incubated in nitro blue tetrazolium ( 2 mg / ml ) for 5 min followed by incubation in 36 mm k2hpo4 , 0.035 mm riboflavin , 0.3% temed for 7 min and then washed several times in h2o as described ( 20 ) . sod enzymatic activity was assayed from yeast grown shaking in selected sc medium to an a600 of 1.5 . yeast lysates were generated by glass bead homogenization in a buffer containing 0.6 m sorbitol , 10 mm hepes , ph 7.2 , and protease inhibitors . analysis of sod activity by nondenaturing gel electrophoresis using 12% pre - cast gel ( invitrogen ) and staining with nitro blue tetrazolium was performed as described previously ( 21 ) except that 10 mm edta was added to the native running buffer . sod1 spectrophotometric assay2448-h - old males were homogenized in b1 ( 50 mm sodium phosphate , ph 7.4 , 0.1 mm edta ) . the supernatant was extracted twice with chloroform ( 0.15 v ) , ethanol ( 0.25 v ) with a 15-min incubation time for each extraction . sod1 activity was determined spectrophotometrically by monitoring the autooxidation of 6-hydroxydopamine ( 6-hd ) at 490 nm at 37 c in 500 l of b1 containing 0.1 mm 6-hd ( 22 ) . aconitase activity assay30 adult males were homogenized in 120 l of extraction buffer ( 0.6 mm mncl2 , 2 mm citric acid , 50 mm tris - hcl , ph 8.0 ) and centrifuged at 13,000 g. samples were electrophoresed on sepraphore iii membranes ( pall corp . ) . aconitase was detected by incubation of the membrane in 100 mm potassium phosphate , ph 6.5 , 1 mm nadph , 2 mm cis - aconitic acid , 1.2 mm 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide , 0.3 mm phenazine methosulfate , 25 mm mgcl2 , and 5 units / ml isocitrate dehydrogenase ( 23 , 24 ) . western blotting for analysis of sod1 from drosophila , males were homogenized in 1% triton x-100 . the extracts were centrifuged at 13,000 g for 5 min at 4 c , and the supernatant was transferred to a tube containing an equal volume of loading buffer ( 125 mm tris - hcl , ph 7.2 , 4% sds , 10% mercaptoethanol , 20% glycerol , 0.01% bromphenol blue ) . samples were boiled for 5 min , and 10 g of total protein was separated on a sds - polyacrylamide gel ( 4% stacking , 15% separating ) . the protein was transferred to nitrocellulose membrane ( ecl ) and probed with sheep anti - human cu , zn superoxide dismutase ( cedarlane catalog number k9007c ) and rabbit anti - sheep igg horseradish peroxidase conjugate ( chemicon catalog number ap147p ) . the ecl western blotting detection reagents ( rpn 2109 , amersham biosciences ) was used for detection . immunoblot analysis of cu , zn - sod expressed in yeast was carried out with cell lysates prepared as described above and a polyclonal antibody that displays strong reactivity with cu , zn - sods from several species ( jh765 ) at a 1:5000 dilution ( 8) . detection utilized an alexa fluor 680 goat anti - rabbit secondary antibody ( invitrogen ) , and immunoblots were visualized using the odyssey infrared imagining system ( licor biosciences ) . a , genomic architecture of ccs , the drosophila copper chaperone for superoxide dismutase 1 ( fly base annotation cg17753 , fbgn0010531 ) . 29e is the mutant chromosome containing a 1907-bp deletion extending upstream from bp 124 of the 2nd exon through the first intron , the first exon and 1717 bp of the 5-noncoding region . drosophila ccs . a , genomic architecture of ccs , the drosophila copper chaperone for superoxide dismutase 1 ( fly base annotation cg17753 , fbgn0010531 ) . 29e is the mutant chromosome containing a 1907-bp deletion extending upstream from bp 124 of the 2nd exon through the first intron , the first exon and 1717 bp of the 5-noncoding region . drosophila life span determination024-h - old males were collected , 25 males per vial , and kept on cornmeal food containing tegosept . drosophila paraquat resistance024-h - old males were collected and kept on cornmeal food for 24 h , 10 males in each vial . they were then transferred to vials containing 3-mm paper filter disks saturated with 250 l of 1% sucrose , 1% sucrose containing 2 mm paraquat ( sigma ) or 1% sucrose and 5 mm paraquat . the vials were stored at 25 c in the dark , and flies were enumerated after 24 h as originally described ( 10 ) . dccs , the copper chaperone of sod1 gene of drosophila the drosophila genomic homologue of yeast and mammalian ccs was initially identified by isolation of a sod1-like sequence from a drosophila genomic library , later verified by sequence annotation in flybase ( cg17753 , fbgn0010531 , and confirmed by amplification of a ccs cdna from a d. melanogaster cdna library ( fig . the architecture of the native drosophila gene includes a 795-bp coding sequence interrupted by two small introns of 51 and 61 bp , respectively . the nearest open reading frame ( cg11867 ) is 5 kb upstream from the 5-transcription start site . the encoded sequence contains the expected region of high homology to sod1 ( 2 ) in the central domain ii of ccs ; the domain iii cxc is conserved . unexpectedly , the domain i cxxc copper - binding motif is absent ( fig . a comparison to all known ccs - like molecules in the data base revealed that the absence of a cxxc motif in drosophila ccs is unique . of 20 ccs sequences analyzed from fungi to humans , only d. melanogaster and anopheles gambiae lack the mxcxxc copper - binding site ( table 1 ) . however , all 20 ccs molecules retain the cxc copper site in domain iii . table 1drosophila ccs lacks domain 1 mtcxxcthe presence of the domain i mxcxxc and domain iii cxc sequences was examined in ccs proteins from fungi , plants , and animals . drosophila ccs lacks domain 1 mtcxxc the presence of the domain i mxcxxc and domain iii cxc sequences was examined in ccs proteins from fungi , plants , and animals . ccs deletion mutation a loss of function mutation of ccs ( ccs ) , generated by an apparent imprecise excision of a p element located near ccs in the autosomal enhancer - detector parent strain , h340 ( 25 ) , was identified by recovery of a strain , 29e , with reduced sod1 activity and a set of phenotypes exhibited by known sod1 deficiency mutants ( see below ) . sequence analysis of ccs , restriction analysis by southern hybridization , and sod1 activity all indicated that ccs is functionally and structurally intact in h340 . the genomic deletion in ccs includes 1617 bp of 5-upstream region and 1907 bp downstream from the transcription start site , including all of the first exon , the first intron , and 124 bp of the second exon ( fig . are relatively rare throughout normal development in h340 and are undetectable by northern blot analysis in 29e ( data not shown ) . loss of sod1 enzymatic activity in ccs - null flies because the only known assay for ccs function is via its role in the activation of sod1 , we assayed sod1 activity in extracts of h340 and 29e using two functionally different methods , the in - gel with nitro blue tetrazolium reduction interference assay and the spectrophotometric 6-hydroxydopamine reduction interference assay ( fig . 2 , a and b ) . using both methods , we were unable to detect significant sod1 activity in 29e . loss of ccs in 29e appears to have no effect on the activity of the mitochondrial manganese - containing sod2 ( see fig . 4a ) . these results by themselves would suggest that ccs in drosophila , as in yeast and mammals , is specifically required for activating sod1 . however , analysis of sod1 protein levels revealed that the steady state level of sod1 polypeptide in 29e is reduced to about one - quarter of its normal level in h340 ( fig . 2c ) . thus , the loss of sod1 activity in 29e probably arises primarily from the reduced level of sod1 protein in the absence of ccs . further analysis confirmed that the lack of sod1 activity in 29e is not because of a mutation in the sod1 gene.4 deficiency of ccs reduces the functional level of specific iron - sulfur protein sentinels the cytosolic and mitochondrial aconitases both contain cubane [ 4fe-4s ] centers that are sensitive targets of inactivation by superoxide ( 23 , 26 ) . steady state levels of these activities in drosophila are significantly reduced in the absence of sod1 and sod2 , respectively ( 24 ) . assay of cacon and macon activities in 29e shows that cacon activity is selectively depleted by about 50% ( h340 1.6 29e ) , with no detectable affect on the activity of macon ( fig . these data indicate that at the biochemical level , the absence of dccs is equivalent to a deficiency in sod1 . the important question then arises , does this parallel extend to whole organism phenotypes ? genetic ablation of ccs elicits broad phenotypic consequences at a minimum , the mutational loss of ccs should generate a set of phenotypes predicted by the loss of sod1 , including reduced adult life span with little or no overt effect on pre - adult development and hypersensitivity to applied oxidative stress . 3a shows that the absence of ccs confers early onset adult mortality with an 30% reduction in the median adult life span . in contrast , 29e displays the extreme toxic hypersensitivity to the redox cycling agent , paraquat , exhibited by sod1-null ( fig . based on previous studies with rna interference - mediated knockdown of sod1 ( 27 ) , and transgenic rescue of sod1-null mutants ( 28 ) , which show that paraquat toxicity is a more sensitive indicator of sod1 deficiency than life span , the phenotype of 29e is consistent with the presence in 29e of a very small residual level of sod1 activity which , although below the level of detection by conventional assays of whole - fly extracts , is sufficient to extend median adult life span ( 30 days ) beyond the 10-day median life span of sod1-null mutants . such activity , if it were to occur , would have to arise via ccs - independent activation of apo - sod1 ( see below ) . human sod1 is active in ccs - null drosophila and restores their adult life span in earlier work ( 14 ) , we described the functional expression of human sod1 ( hsod1 ) in drosophila in a ccs genetic background . since then , the ccs - independent activation of hsod1 has been described ( 7 , 9 ) . here we investigated the activation of hsod1 in flies in the absence of functional dccs . as would be expected from this sod1 capable of ccs independent activation , hsod1 is quite active in ccs - null flies ( fig . moreover , hsod1 functions biologically to rescue the early mortality and reduced median life span of ccs - null flies ( fig . these results demonstrate the presence in drosophila of a ccs - independent pathway capable of robust activation of heterologous hsod1 while giving only weak activation of homologous dsod1 . ccs - independent activation of drosophila sod1 revealed in yeast expression system based on amino acid sequence alone , dsod1 is predicted to acquire copper independently of ccs . studies with yeast , human , and c. elegans sod1 have identified a pair of prolines near the c terminus that can prohibit ccs - independent activation ( 7 , 8) . yeast sod1 naturally contains these prolines and is incapable of ccs - independent activation , whereas mammalian and c. elegans sod1 with nonproline residues at these positions can be activated independently of ccs . 5a ) leading us to predict that dsod1 should not exhibit dependence on ccs for activity . and although from the phenotype of dccs - nulls we inferred the presence of a residual level of sod1 activity , the instability of dsod1 in the absence of ccs obscures any measurable ccs - independent activation of dsod1 that might occur in the drosophila model . we therefore turned to a yeast expression system to further examine drosophila ccs and sod1 . figure 2.loss of ccs affects sod1 and maconitase . a , in - gel activity assay of sod1 . h340 is the dccs parent stock from which the ccs - null mutant , ccs , was derived . extracts were treated with triton x-100 to eliminate interference from mitochondrial superoxide dismutase ( sod2 ) . note , the background activity found in 29e extracts is also present in sod1-null mutant extracts . extracts were prepared from h340 and 29e adult males , and sod1 protein was detected in western immunoblots with anti - sod1 antibody . top , immunoblot analysis ; bottom , densitometric quantitation of immunoblot where 100 = amount of dsod1 polypeptide in h340 . d , selective loss of cytosolic aconitase ( cacon ) activity in the absence of ccs . aconitase activities in extracts of 23-day - old adult males were assayed after electrophoretic separation . top , zymogram analysis ; bottom , densitometric quantitation of zymogram where 100 = level of cacon activity in h340 . loss of ccs affects sod1 and maconitase . a , in - gel activity assay of sod1 . h340 is the dccs parent stock from which the ccs - null mutant , ccs , was derived . extracts were treated with triton x-100 to eliminate interference from mitochondrial superoxide dismutase ( sod2 ) . note , the background activity found in 29e extracts is also present in sod1-null mutant extracts . extracts were prepared from h340 and 29e adult males , and sod1 protein was detected in western immunoblots with anti - sod1 antibody . top , immunoblot analysis ; bottom , densitometric quantitation of immunoblot where 100 = amount of dsod1 polypeptide in h340 . d , selective loss of cytosolic aconitase ( cacon ) activity in the absence of ccs . aconitase activities in extracts of 23-day - old adult males were assayed after electrophoretic separation . top , zymogram analysis ; bottom , densitometric quantitation of zymogram where 100 = level of cacon activity in h340 . all strains are homozygous for sod1 . to test the comparative functionality of dsod1 in the absence of ccs , the coding sequences for human , yeast , and drosophila sod1 were all placed under control of the s. cerevisiae sod1 gene promoter and analyzed for sod1 activity and protein levels in ccs1 versus ccs1 yeast . as in ccs - null flies ( fig . 5b ) , and dsod1 activity was initially difficult to discern ( fig . yet when increasing levels of cell lysate were analyzed , the dsod1 polypeptide was readily detected , and ccs - independent activation of dsod1 became apparent ( fig . 5b , lane 9 ) , similar to levels seen with human sod1 ( lane 4 ) . hence , dsod1 does have the capacity to be activated by the ccs - independent pathway , ostensibly explaining the less severe phenotypes of a ccs - null fly compared with a sod1-null mutant . role of ccs in helping to stabilize dsod1the dramatic loss of the dsod1 polypeptide in both ccs1 null yeast ( fig . 2c ) is atypical , and it is not observed with yeast and human sod1 analyzed in parallel ( fig . this loss in dsod1 can be rescued by expressing either yeast ccs or drosophila ccs in the ccs1 yeast ( fig . this apparent stabilization of dsod1 requires physical interactions between ccs and sod1 , as a k136e , g137e derivative of yeast ccs that can not dock with sod1 ( 29 ) fails to increase dsod1 levels ( fig . 6a , lane 4 ) . to test whether sod1-ccs interactions are by themselves sufficient , we employed a c229s , c231s mutant of yeast ccs . the highly conserved cysteines cys-229 and cys-231 in domain iii of ccs are needed for copper transfer and disulfide oxidation in sod1 , and a c229s , c231s mutant of ccs can dock with sod1 but can not activate the enzyme ( 29 , 30 ) . as seen in fig . 6a , lane 3 , c229s , c231s yccs failed to stabilize dsod1 , indicating that copper transfer and/or disulfide oxidation are required . to directly test the requirement for copper , yeast cells were starved for copper by treatment with the cu(i ) chelator , bathocuproine sulfonate ( 31 ) . under such copper - limiting conditions , levels of the dsod1 polypeptide were lowered even in cells expressing wild type ccs from yeast or drosophila ( fig . together , these studies indicate that apo - dsod1 is unusually unstable and that ccs affords stability to dsod1 by activating the enzyme through copper insertion and/or disulfide oxidation . unique n terminus of drosophila ccs as mentioned above , drosophila ccs lacks the mxcxxc copper - binding motif that is well conserved in ccs molecules from phylogenetically distant taxa . in fact , an inspection of ccs molecules across diverse species reveals that with the exception of drosophila and mosquito ccs , all ccs molecules identified to date harbor these cysteines ( table 1 ) . interestingly , we observed that drosophila ccs is very poor at activating yeast sod1 compared with the homologous yeast ccs expressed from the identical yeast ccs1 gene promoter ( fig . 6c , lanes 2 and 3 ) . by comparison , fly ccs was nearly as effective as yeast ccs in activating human sod1 ( fig . 6c , lanes 5 and 6 ) , and drosophila sod1 exhibited no apparent preference for ccs and showed strong activation by both ccs molecules ( lanes 8 and 9 ) . to address whether the yeast sod1 preference for yeast ccs reflected loss of the conserved mxcxxc cysteines , we tested the effects of a c17s , c20s substitution in yeast ccs . 6d , this mutant retains the ability to fully activate yeast sod1 . despite the well conserved nature of domain i cxxcin ccs , these cysteines are not essential for activating sod1 . these results also demonstrate that the apparent poor reactivity between fly ccs and yeast sod1 is not simply explained by the absence of these cxxc cysteines . in any case , sod1 molecules from phylogenetically distant organisms exhibit unique preferences in copper activation pathways . figure 3.ccs deficiency confers early adult mortality and enhanced sensitivity to the redox cycling agent , paraquat . survival of at least 125 males of each genotype on standard cornmeal food was followed at 25 c with enumeration and transfer of survivors to fresh bottles every 23 days . ccs is h340 , the ccs parent stock from which ccs was derived . sod1 is sod1/sod1 , sod1 is the wild type stock , ry . ccs is h340 , the ccs parent stock from which ccs was derived . young adult males ( at least 200 flies per genotype , 10 flies per vial ) were exposed to 2 mm paraquat . ccs deficiency confers early adult mortality and enhanced sensitivity to the redox cycling agent , paraquat . survival of at least 125 males of each genotype on standard cornmeal food was followed at 25 c with enumeration and transfer of survivors to fresh bottles every 23 days . ccs is h340 , the ccs parent stock from which ccs was derived . sod1 is sod1/sod1 , sod1 is the wild type stock , ry . ccs is h340 , the ccs parent stock from which ccs was derived . young adult males ( at least 200 flies per genotype , 10 flies per vial ) were exposed to 2 mm paraquat . figure 4.expression of hsod1 in ccs - null drosophila . a , hsod1 expressed in drosophila is enzymatically active in the absence of ccs . hsod1 was expressed in ccs and ccs flies using the gal4/uas system . genotypes ( by lane ) are as follows : lane 1 , ccs / ccs ; dsod1/dsod1 , dagal4 ; lane 2 , ccs / ccs ; dsod1/dsod1 ; lane 3 , ccs / ccs ; dsod1/dsod1 ; dagal4 ; lane 4 , ccs / ccs uas - hsod1 ; dsod1/dsod1 ; lane 5 , ccs / ccs , uas - hsod1 ; dsod1/dsod1 ; lane 6 , ccs / ccs , uas - hsod1 ; dsod1/dsod1 , dagal4 ; lane 7 , ccs / ccs , uas - hsod1 ; dsod1/dsod1 , dagal4 . the uas - hsod1 transgene is on recombinant 2nd chromosomes carrying ccs or ccs . genotypes are as follows : ccs , ccs / ccs ; dsod1/dsod1 , dagal4 . ccs , hsod1(a ) , ccs / ccs , uas - hsod1(a ) ; dsod1/dsod , dagal4 . ccs , hsod1(a ) , ccs / ccs , uas - hsod1(a ) ; dsod1/dsod1 , dagal4 . ccs , hsod1(b ) : ccs / ccs , uas - hsod1(b ) ; dsod1/dsod1 , dagal4 . ccs , hsod1(b ) : ccs / ccs , uas - hsod1(b ) ; dsod1/dsod1 , dagal4 . note that all strains are homozygous for dsod1 and hemizygous for dagal4 , the latter of which is known to impose a mild dominant reduction of adult life span ( compare with fig . hsod1(a ) , hsod1(b ) are independently selected recombinant 2nd chromosomes carrying both ccs and uas - hsod1 . expression of hsod1 in ccs - null drosophila . a , hsod1 expressed in drosophila is enzymatically active in the absence of ccs . hsod1 was expressed in ccs and ccs flies using the gal4/uas system . genotypes ( by lane ) are as follows : lane 1 , ccs / ccs ; dsod1/dsod1 , dagal4 ; lane 2 , ccs / ccs ; dsod1/dsod1 ; lane 3 , ccs / ccs ; dsod1/dsod1 ; dagal4 ; lane 4 , ccs / ccs uas - hsod1 ; dsod1/dsod1 ; lane 5 , ccs / ccs , uas - hsod1 ; dsod1/dsod1 ; lane 6 , ccs / ccs , uas - hsod1 ; dsod1/dsod1 , dagal4 ; lane 7 , ccs / ccs , uas - hsod1 ; dsod1/dsod1 , dagal4 . the uas - hsod1 transgene is on recombinant 2nd chromosomes carrying ccs or ccs . genotypes are as follows : ccs , ccs / ccs ; dsod1/dsod1 , dagal4 . ccs , hsod1(a ) , ccs / ccs , uas - hsod1(a ) ; dsod1/dsod , dagal4 . ccs , hsod1(a ) , ccs / ccs , uas - hsod1(a ) ; dsod1/dsod1 , dagal4 . ccs , hsod1(b ) : ccs / ccs , uas - hsod1(b ) ; dsod1/dsod1 , dagal4 . ccs , hsod1(b ) : ccs / ccs , uas - hsod1(b ) ; dsod1/dsod1 , dagal4 . note that all strains are homozygous for dsod1 and hemizygous for dagal4 , the latter of which is known to impose a mild dominant reduction of adult life span ( compare with fig . hsod1(a ) , hsod1(b ) are independently selected recombinant 2nd chromosomes carrying both ccs and uas - hsod1 . mutational inactivation of ccs generates a set of phenotypes that are equivalent to those from a weakly expressing sod1 hypomorphic mutation , and are consistent with the notion that ccs appears to have a single function in drosophila , which is to mediate the activation of apo - sod1 by copper . compared with the severity of phenotypes caused by sod1 deficiency ( either genomic null mutation or rna interference knockdown ) , the ccs - null phenotypes are unexpectedly mild . this likely arises from a vanishingly small amount of ccs - independent sod1 activity that is at or just below the threshold of detectability in fly extracts and could only be discerned in concentrated lysates from yeast cells expressing dsod1 . considering the rather severe phenotype of sod1-null flies with respect to adult life span and oxidative stress resistance ( 10 ) , these findings suggest that only a very small fraction of wild type sod1 is actually required for adult viability in the absence of oxidative stress , whereas the bulk of sod1 is utilized in defense against oxidative stress . this view is supported by the contrasting modest and severe effects of ccs deficiency on life span and paraquat resistance , respectively . thus , the age of onset and severity of early adult mortality in ccs - nulls is markedly less than in sod1-nulls , whereas the hypersensitivity of ccs - nulls to paraquat is essentially equivalent to that of sod1-nulls . a , an alignment of the c - terminal region of sod1 molecules from s. cerevisiae , human , and drosophila , highlighting the proline residues known to preclude ccs - independent activation of yeast sod1 ( 5 ) . b , coding sequences for s. cerevisiae , human , and drosophila sod1 were placed under control of the s. cerevisiae sod1 gene promoter and used to transform a yeast strain that was sod1 and either ccs1 ( yccs:+ ) or ccs1 ( yccs: ) as indicated . sod1 lysates were analyzed for sod1 activity ( top ) or sod1 protein levels using an antibody directed against c. elegans cu , zn - sod1 that cross - reacts well with diverse sod1 molecules ( 6 ) . amounts of lysate protein analyzed are as follows : lanes 16,5 g ; lane 7,10 g ; lane 8,25 g ; lane 9,50 g . strains analyzed include ks107 ( sod1 ) and ls101 ( sod1 ccs1 ) . a , an alignment of the c - terminal region of sod1 molecules from s. cerevisiae , human , and drosophila , highlighting the proline residues known to preclude ccs - independent activation of yeast sod1 ( 5 ) . b , coding sequences for s. cerevisiae , human , and drosophila sod1 were placed under control of the s. cerevisiae sod1 gene promoter and used to transform a yeast strain that was sod1 and either ccs1 ( yccs:+ ) or ccs1 ( yccs: ) as indicated . sod1 lysates were analyzed for sod1 activity ( top ) or sod1 protein levels using an antibody directed against c. elegans cu , zn - sod1 that cross - reacts well with diverse sod1 molecules ( 6 ) . amounts of lysate protein analyzed are as follows : lanes 16,5 g ; lane 7,10 g ; lane 8,25 g ; lane 9,50 g . strains analyzed include ks107 ( sod1 ) and ls101 ( sod1 ccs1 ) . a sod1 ccs1 yeast strain expressing the designated sod1 and ccs molecules was analyzed for sod1 activity and sod1 polypeptide levels using 10 g of cell lysate protein for each sample . a , strains expressing dsod1 and either wild type ( wt ) or the indicated mutant versions of s. cerevisiae ccs1 or empty vector were analyzed for steady state levels of the dsod1 polypeptide . top , immunoblot analysis ; bottom , quantitation of immunoblot by odyssey quantitation software ( version 1.2 ) where 100 = amount of dsod1 polypeptide accumulated in cells expressing wt ccs1 . b , cells expressing dsod1 and either s. cerevisiae ccs1 or drosophila ccs driven by the s. cerevisiae ccs1 promoter were grown in the presence of 100 m bathocuproine sulfonate where indicated ( + ) , and dsod1 polypeptide levels measured as in a where 100 = amount of dsod1 polypeptide with no addition of bathocuproine sulfonate . c , cells expressing either yeast , human , or drosophila sod1 in combination with either yeast or drosophila ccs or with vector as indicated were analyzed for sod1 activity and sod1 protein levels as fig . d , cells expressing yeast sod1 and either wild type or the indicated mutant versions of s. cerevisiae ccs1 were analyzed for sod1 activity and protein levels as in fig . 5b . a sod1 ccs1 yeast strain expressing the designated sod1 and ccs molecules was analyzed for sod1 activity and sod1 polypeptide levels using 10 g of cell lysate protein for each sample . a , strains expressing dsod1 and either wild type ( wt ) or the indicated mutant versions of s. cerevisiae ccs1 or empty vector were analyzed for steady state levels of the dsod1 polypeptide . top , immunoblot analysis ; bottom , quantitation of immunoblot by odyssey quantitation software ( version 1.2 ) where 100 = amount of dsod1 polypeptide accumulated in cells expressing wt ccs1 . b , cells expressing dsod1 and either s. cerevisiae ccs1 or drosophila ccs driven by the s. cerevisiae ccs1 promoter were grown in the presence of 100 m bathocuproine sulfonate where indicated ( + ) , and dsod1 polypeptide levels measured as in a where 100 = amount of dsod1 polypeptide with no addition of bathocuproine sulfonate . c , cells expressing either yeast , human , or drosophila sod1 in combination with either yeast or drosophila ccs or with vector as indicated were analyzed for sod1 activity and sod1 protein levels as fig . d , cells expressing yeast sod1 and either wild type or the indicated mutant versions of s. cerevisiae ccs1 were analyzed for sod1 activity and protein levels as in fig . it is also possible that the residual sod1 activity in ccs - nulls reflects the presence of enzymatically active sod1 in just one or a small number of cell types in which the ccs - independent activation pathway is particularly active . although we know of no direct evidence in support of this possibility , the ectopic expression of the ccs - independent hsod1 in motor neurons , which confers robust rescue of the sod1-null adult life span ( 14 ) , suggests that motor neurons do possess an active ccs - independent activation pathway ; consequently , motor neurons could potentially be one such cell type that could activate dsod1 in the absence of ccs . because drosophila life span is sensitive to sod1 activity in motor neurons , the functioning of the ccs - independent activation pathway in motor neurons could also help explain why ccs deficiency has such a modest effect on adult life span relative to the severe life span reduction conferred by sod1 deficiency . alternatively , and as discussed later , our results are consistent with the possibility that although most cells can survive under normal conditions with little sod1 , some cells may require more than the residual level of sod1 activity present in ccs - nulls under conditions of applied oxidative stress ( paraquat ) or increased metabolic activity ( motor neurons ) . finally , it is also plausible that cell type variation in copper availability could , in principle , underpin such cell type specificity in ccs - independent activation of sod1 . drosophila sod1 can now be added to the growing list of sod1 molecules that can obtain copper independently of ccs . currently , the only cu , zn - sod known to solely rely on ccs is the sod1 of s. cerevisiae . it is curious that yeast does not need a ccs independent pathway , yet retains the ability to activate heterologous sods independent of ccs . a likely explanation is that the factors for ccs independent activation serve another function in the cell . for example , ccs independent activation may involve metal binding or thiol - reactive molecules that participate in more general metal homeostasis and/or redox control . drosophila sod1 appears unique in that the apo - form is very unstable in the absence of the copper chaperone . the apparent instability is not because of the drosophila host , as human sod1 expressed in ccs - null flies shows no polypeptide loss , and in the heterologous yeast expression system , ccs1 mutations affect loss of dsod1 but not yeast or human sod1 examined in parallel . moreover , in ccs mouse models , the mammalian sod1 polypeptide is stable ( 32 ) . this loss of dsod1 in the absence of ccs is in fact reminiscent of what has been observed with certain amyotrophic lateral sclerosis mutants of human sod1 associated with amyotrophic lateral sclerosis ( 33 ) . because ccs both inserts copper and oxidizes the sod1 disulfide , either one or both of these post - translational modifications must be important stabilizing factors for the dsod1 molecule . prior to these studies , apo - inactive sod1 was generally thought to stably accumulate in cells , allowing for rapid enzyme activation by ccs without the need for new sod1 synthesis ( 31 , 34 ) . yet drosophila sod1 does not stably accumulate in the inactive apo - form . consequently , the high level of enzyme activity that is required to defend against oxidative challenge would require de novo sod1 synthesis . the inability of ccs - null adults , which are constitutively deficient in sod1 activity , to mount a successful defense against paraquat - induced oxidative stress is consistent with this scenario . it is also possible that available copper is limiting in drosophila , and it would be disadvantageous to produce more copper - binding sod1 than is needed . with the exception of certain insects , ccs molecules from diverse eukaryotes harbor an mxcxxc near the n terminus that is a well characterized cu(i ) site ( 35 ) . although earlier studies with human ccs indicated that the mxcxxc cysteines may be necessary for sod1 activation in vivo ( 36 ) , studies with recombinant ccs in vitro indicate that only the c - terminal cxc cysteines are needed to bind and transfer the metal ( 37 , 38 ) . we observed that the mxcxxc motif is not required for activation of yeast sod1 in vivo ; copper binding and transfer must occur exclusively via the c - terminal cxc motif of ccs and the same must be true for dccs lacking the n - terminal cysteines . it remains possible that in certain species , the n - terminal mxcxxc site becomes critical for maintaining sod1 activity when oxygen and/or copper are limiting reagents for ccs . if true , the absence of such a motif in drosophila ccs would minimize copper loading of sod1 when pools of the metal are sparse . however , the biological consequences of transient fluctuations in copper availability may be minimized by the relative stability of metalated sod1 coupled with the low cell turnover in the post - mitotic drosophila adult .

copper , zinc superoxide dismutase ( sod1 ) in mammals is activated principally via a copper chaperone ( ccs ) and to a lesser degree by a ccs - independent pathway of unknown nature . in this study , we have characterized the requirement for ccs in activating sod1 from drosophila . a ccs - null mutant ( ccsn29e ) of drosophila was created and found to phenotypically resemble drosophila sod1-null mutants in terms of reduced adult life span , hypersensitivity to oxidative stress , and loss of cytosolic aconitase activity . however , the phenotypes of ccs - null flies were less severe , consistent with some ccs - independent activation of drosophila sod1 ( dsod1 ) . yet sod1 activity was not detectable in ccsn29e flies , due largely to a striking loss of sod1 protein . in contrast , human sod1 expressed in ccs - null flies is robustly active and rescues the deficits in adult life span and sensitivity to oxidative stress . the dependence of dsod1 on ccs was also observed in a yeast expression system where the dsod1 polypeptide exhibited unusual instability in ccs - null ( ccs1 ) yeast . the residual dsod1 polypeptide in ccs1 yeast was nevertheless active , consistent with ccs - independent activation . stability of dsod1 in ccs1 cells was readily restored by expression of either yeast or drosophila ccs , and this required copper insertion into the enzyme . the yeast expression system also revealed some species specificity for ccs . yeast sod1 exhibits preference for yeast ccs over drosophila ccs , whereas dsod1 is fully activated with either ccs molecule . such variation in mechanisms of copper activation of sod1 could reflect evolutionary responses to unique oxygen and/or copper environments faced by divergent species .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION Supplementary Material

PMC3774873

the isolate of s. rolfsii causing collar rot in chickpea ( cicer arietinum ) used in the study was collected from the agricultural farm of banaras hindu university , varanasi , india . the fungus was isolated by inoculating sclerotia from the infected parts of the plant in petri dishes containing potato dextrose agar ( pda ) ( peeled potato 200 g , dextrose 20 g , agar 15 g , distilled water 1 l ) medium and incubating the plates at 252 for 7~10 days till sclerotium formation . from each plate the present experiments were conducted with fresh leaves and bulbs of two - month - old garlic ( a. sativum ) and onion ( a. cepa ) , their roots , mature garlic clove and mature red and white onion bulbs . all the plant materials were cleaned by washing in distilled water and air - dried to remove trace water from the surface . such pastes were used at different concentrations ( 1 , 3 , 5% w / v ) prepared in distilled water in conical flasks ( 100 ml ) without agar in the medium ( 30 ml in each conical flask ) . the medium was sterilized in autoclave at 121 for 10 minutes . a five millimeter ( dia ) mycelial disc from 4-day - old cultures of s. rolfsii grown on pda medium was inoculated into the liquid medium in flasks with the help of a sterilized inoculation needle . the flasks were incubated at 252 and observed for growth characters of mycelial mat after 10 and 20 days . the potato dextrose broth ( pdb ; peeled potato 200 g , dextrose 20 g , distilled water 1 l ) which was similarly inoculated and incubated served as control . thirty milliliter of broth medium supplemented with different concentrations ( 1 , 3 , 5% w / v ) of the paste prepared in distilled water was poured into conical flasks ( 100 ml ) and mycelial bits from exponentially growing culture of s. rolfsii were inoculated separately . cultures were allowed to grow for 10 and 20 days at 252 as described earlier . the mycelial mat developed in broth media 1 filter papers after 10 and 20 days and washed three times in distilled water and then placed on a pad of sterile filter paper to remove excess water . two gram of fresh mycelial mat from each treatment and each concentration of the paste was thoroughly macerated separately in the presence of ethanol : water ( 80 : 20 , v / v ) in a pestle - mortar and collected in screwcapped bottles and kept overnight . the ethyl acetate fraction was evaporated under vacuum ( buchi rotavapor re type ) to dryness and the material was dissolved in 1.0 ml methanol ( hplc grade ) for further analysis . high performance liquid chromatography ( hplc ) of the samples was performed with an hplc system ( shimadzu corporation , kyoto , japan ) equipped with two shimadzu lc-10 atvp reciprocating pumps , a variable uv - vis detector ( shimadzu spd-10 avp ) and a winchrom integrator ( winchrom ) . reverse phase chromatographic analysis was carried out in isocratic conditions using rp c-18 hplc column ( 250 4.6 mm i d , particle size 5 m , luna 5 c-18 ( 2 ) , phenomenex , usa ) at room temperature . running conditions included injection volume : 5 l , mobile phase : methanol-0.4% acetic acid ( 80 : 20 , v / v ) , flow rate : 1 ml / min , detection at 290 nm and attenuation response 0.03 aufs . samples were filtered through membrane filters ( pore size 0.20 m , millipore ) prior to injection in a sample loop . gallic , ferulic , caffeic , tannic , chlorogenic , o - coumaric and cinnamic acids were used as internal and external standards . phenolic compounds present in the samples were identified by comparing retention time ( rt ) of the standards and by the co - chromatography . contents of phenolic acids were calculated by comparing peak areas of reference compounds with those in the samples run under similar elution conditions . analyses were made three times from each sample and mean value was calculated for each compound . mean relative concentrations of phenolic compounds were statistically analyzed by anova and the normality of the results and homogeneity of the variances were tested . the isolate of s. rolfsii causing collar rot in chickpea ( cicer arietinum ) used in the study was collected from the agricultural farm of banaras hindu university , varanasi , india . the fungus was isolated by inoculating sclerotia from the infected parts of the plant in petri dishes containing potato dextrose agar ( pda ) ( peeled potato 200 g , dextrose 20 g , agar 15 g , distilled water 1 l ) medium and incubating the plates at 252 for 7~10 days till sclerotium formation . from each plate the present experiments were conducted with fresh leaves and bulbs of two - month - old garlic ( a. sativum ) and onion ( a. cepa ) , their roots , mature garlic clove and mature red and white onion bulbs . all the plant materials were cleaned by washing in distilled water and air - dried to remove trace water from the surface . such pastes were used at different concentrations ( 1 , 3 , 5% w / v ) prepared in distilled water in conical flasks ( 100 ml ) without agar in the medium ( 30 ml in each conical flask ) . the medium was sterilized in autoclave at 121 for 10 minutes . a five millimeter ( dia ) mycelial disc from 4-day - old cultures of s. rolfsii grown on pda medium was inoculated into the liquid medium in flasks with the help of a sterilized inoculation needle . the flasks were incubated at 252 and observed for growth characters of mycelial mat after 10 and 20 days . the potato dextrose broth ( pdb ; peeled potato 200 g , dextrose 20 g , distilled water 1 l ) which was similarly inoculated and incubated served as control . thirty milliliter of broth medium supplemented with different concentrations ( 1 , 3 , 5% w / v ) of the paste prepared in distilled water was poured into conical flasks ( 100 ml ) and mycelial bits from exponentially growing culture of s. rolfsii were inoculated separately . cultures were allowed to grow for 10 and 20 days at 252 as described earlier . the mycelial mat developed in broth media 1 filter papers after 10 and 20 days and washed three times in distilled water and then placed on a pad of sterile filter paper to remove excess water . two gram of fresh mycelial mat from each treatment and each concentration of the paste was thoroughly macerated separately in the presence of ethanol : water ( 80 : 20 , v / v ) in a pestle - mortar and collected in screwcapped bottles and kept overnight . the extract was fractionated with equal volume of ethyl acetate . the ethyl acetate fraction was evaporated under vacuum ( buchi rotavapor re type ) to dryness and the material was dissolved in 1.0 ml methanol ( hplc grade ) for further analysis . high performance liquid chromatography ( hplc ) of the samples was performed with an hplc system ( shimadzu corporation , kyoto , japan ) equipped with two shimadzu lc-10 atvp reciprocating pumps , a variable uv - vis detector ( shimadzu spd-10 avp ) and a winchrom integrator ( winchrom ) . reverse phase chromatographic analysis was carried out in isocratic conditions using rp c-18 hplc column ( 250 4.6 mm i d , particle size 5 m , luna 5 c-18 ( 2 ) , phenomenex , usa ) at room temperature . running conditions included injection volume : 5 l , mobile phase : methanol-0.4% acetic acid ( 80 : 20 , v / v ) , flow rate : 1 ml / min , detection at 290 nm and attenuation response 0.03 aufs . samples were filtered through membrane filters ( pore size 0.20 m , millipore ) prior to injection in a sample loop . gallic , ferulic , caffeic , tannic , chlorogenic , o - coumaric and cinnamic acids were used as internal and external standards . phenolic compounds present in the samples were identified by comparing retention time ( rt ) of the standards and by the co - chromatography . contents of phenolic acids were calculated by comparing peak areas of reference compounds with those in the samples run under similar elution conditions . analyses were made three times from each sample and mean value was calculated for each compound . mean relative concentrations of phenolic compounds were statistically analyzed by anova and the normality of the results and homogeneity of the variances were tested . the best mycelial growth was obtained in potato dextrose broth and in higher concentration ( i.e. , 5% ) of paste - added broth medium of all the treatments except mature garlic clove . mostly mycelial mat of s. rolfsii was thicker and opaque in 5% in comparison to 1 and 3% , where it was thin and transparent / translucent after 10 and 20 days of inoculation . the fungus grew in only 1% concentration of paste of mature garlic clove as thin and translucent after 10 days which became thick and opaque after 20 days of inoculation . there was no growth up to 20 days of inoculation in 3 and 5% mature garlic clove paste . similarly , there was no growth in two - month - old garlic root even at 1% concentration . formation and/or maturation of sclerotia differed in each concentration after 10 and 20 days of inoculation . however , there was no definite pattern of growth characters and sclerotium formation / maturation ( table 1 ) . basidial stage induction was observed in 3% garlic leaves after 15 days of inoculation in the center of the medium as compact , whitish , thick growth , which revealed the presence of basidial hymenia on microscopic observation and basidiospores formed singly on each of the four sterigmataof a basidium . the basidia measured was 10~12 4~5 m and basidiospores 3~5 2~4 m ( fig . hplc analysis of mycelial mat of s. rolfsii obtained from different concentrations of different treatments revealed consistently presence of 5 phenolic acids which were identified on the basis of their retention time ( rt . ) as well as by co - injection with the standard compounds . mycelial mat obtained from potato dextrose broth revealed the presence of 0.14 g / g chlorogenic ( chl.a ) , 1.01 g / g ferulic ( fa ) and 0.05 g / g o - coumaric acids ( oc.a ) after 10 days of inoculation while 2.23 g / g gallic ( ga ) , 3.20 g / g ferulic , 1.24 g / g o - coumaric and 0.13 g / g cinnamic acids ( cin.a ) after 20 days of inoculation . after treatment with 1% paste of two - month - old garlic leaves , 13.22 g / g fa was detected in 10-day - old mycelia whereas 2.38 , 0.60 , 6.27 and 0.02 g / g ga , chl.a , oc.a and cin.a after 20 days , respectively . ferulic acid increased with increasing concentrations of leaf paste in mycelial mat harvested after 10 days of inoculation . in two - month - old garlic bulbs , 21.35 g / g fa was detected in 10-day - old mycelia treated with 1% bulb paste , 2.03 g / g ga , 1.00 g / g fa and 0.58 g / g oc.a was detected after 20 days . 1.61 g / g chl.a and 3.38 g / g fa were present in 10-day - old mycelial mat treated with 3% bulb paste while 1.01 g / g ga , 0.67 g / g chl.a , 0.75 g / g oc.a and 0.01 g / g cin.a were present in 20-day - old mycelia . mycelial mat obtained from 5% bulb paste revealed 1.03 g / g fa and 0.02 g / g oc.a after 10 days . in 1% mature garlic clove treatment 1.41 g / g chl.a , 321.21 g / g fa and 19.95 g / g oc.a were detected in 10-day - old mycelial mat while 0.17 g / g ga , 0.06 g / g chl.a , 1.20 g / g oc.a and 0.07 g / g cin.a in 20-day - old mycelia . in two - month - old red onion leaves , 1.79 g / g ga , 0.25 g / g fa and 0.13 g / g oc.a in 1% concentration ; 0.23 g / g ga , 0.09 g / g chl.a , 0.06 g / g fa and 0.02 g / g oc.a in 3% ; 0.26 g / g ga , and 0.07 g / g oc.a in 5% and 0.01 g / g cin.a were detected in all the three concentrations ( 1 , 3 and 5% ) in 10-day - old mycelia whereas 4.95 g / g ga , 0.48 g / g fa , 0.96 g / g oc.a in 1% ; 0.89 g / g ga , 0.37 g / g chla , 0.18 g / g fa , 0.30 g / g oc.a in 3% ; 2.22 g / g ga , 0.62 g / g chl.a , 0.34 g / g fa , 0.15 g / g oc.a in 5% in 20-day - old mycelial mat . different amounts of phenolic acids were detected in mycelia grown in different concentrations of two - month - old red onion bulbs , mature red and white onion bulb paste ( table 2 ) . the mycelia of s. rolfsii contain some phenolic acids which seem to be needed for their growth and resistance against adverse conditions of the environment . following treatment with garlic and onion , the amount of these phenolic acids increased in comparison to control ( potato dextrose broth only ) when observed after 10 days of inoculation . in some cases they were found less than control . both the plants ( garlic and onion ) were able to increase the synthesis of phenolic acids in s. rolfsii as compared to control . the phenolic acids were found to be decreased in 20-day - old mycelial mat in some cases . interestingly , the growth of mycelial mat was thin in lower than higher concentration and control . this may be due to less amount of nutrients present in lower concentration which affect the growth . inhibition of growth of s. rolfsii in 3% and 5% of mature garlic clove paste indicates its antifungal nature at these concentrations . inhibition of growth in all concentrations of two - month - old fresh garlic root may be due to antifungal nature or unavailability of nutrients in proper amounts . results of the present investigation reveal interesting observations in relation to mycelial growth and presence of different phenolic acids in mycelia of s. rolfsii under the influence of different parts of garlic and onion . the presence of high amount of ferulic acid in mycelia does not appear to affect growth and development of s. rolfsii itself . sarma and singh ( 2003 ) reported that ferulic acid could inhibit complete mycelial growth of s. rolfsii at a concentration of 1000 g / g . since the maximum concentration of ferulic acid in mycelia obtained from 1% mature garlic clove treatment was 321.21 g / g , it becomes very difficult to interpret as to whether only ferulic acid is acting as inhibitory agent or combination of several other compounds including ferulic acid for the said purpose . considering the effects of the two herbals on mycelial growth , leaves supported better growth as compared to bulbs . the role of phenolics as secondary metabolites in living organism is to induce resistance for protection of life besides several other activities ( harborne , 1988 ) . as some of them are also functionally anti - oxidants , the senescence of the living being is expected to be prolonged . it has also been generally observed that during non - balanced growth of microbial cell due to high accumulation of precursors like acetate , malonate , pyruvate etc . cells die but the toxicity of these precursors is said to be detoxified by the secondary metabolites ( weinberg , 1970 ; woodruff , 1966 ) . the better growth of s. rolfsii on leaf paste medium may be due to the presence of high amount of simple sugars in leaves than bulbs . a detailed study is needed to confirm these observations .

high performance liquid chromatographic ( hplc ) analysis of mycelia of sclerotium rolfsii grown in broth medium supplemented with garlic ( allium sativum ) and onion ( allium cepa ) was carried out to estimate qualitative and quantitative changes in phenolic acids . several phenolic acids , such as gallic , chlorogenic , ferulic , o - coumaric and cinnamic acids were detected in varied amounts in mycelia grown on such media as compared to control . phenolic acids represents a wide range of secondary metabolite found in the cells of plants and microbes including fungi . the growth characters of s. rolfsii in various supplements also varied from thin and transparent to thick and opaque .

Materials and Methods Isolation and maintenance of the fungal culture Growth characters of mycelial mat in broth media Extraction of mycelia for phenolic acids HPLC analysis Results and Discussion

PMC1893118

cell movement or motility is a highly dynamic phenomenon that is essential to a variety of biological processes such as the development of an organism ( morphogenesis ) , wound healing , cancer metastasis and immune response . for example , during morphogenesis there is a targeted movement of dividing cells to specific sites to form tissues and organs . for wound healing to occur , cells such as neutrophils ( white blood cells ) and macrophages ( cells that ingest bacteria ) move to the wound site to kill the microorganisms that cause infection , and fibroblasts ( connective tissue cells ) move there to remodel damaged structures 1 . in all these examples , cells reach their target by crawling . there are also other kinds of motility , such as the swimming of most sperm cells , and the movement of some bacteria by the rotation of flagellar motors 1 . cell crawling , however , is the common mechanism employed by most motile eukaryotic animal cells 1 , and is the focus of this review . although cell movement was observed as early as 1675 when van leeuwenhoek saw cells crawl across his microscope slide , the molecular mechanisms behind cell movement have become a scientific focus only in the past few decades . advances in fluorescence microscopy , molecular biology and biochemistry have enabled the discovery of the processes underlying motility and the identification of the major proteins behind these processes . these experimental techniques alone , however , can not adequately explain whether these proteins are capable of generating the required forces for motility nor the physical mechanisms employed . a significant advance in this direction was made when biophysical studies helped identify regions where different force generating proteins are located , measured ( in vitro ) the exact forces generated by some of these proteins 2 - 6 , and measured ( in vivo ) the forces associated with movement 7 - 9 . theoretical studies and computational modeling have complemented the experimental work and have helped quantify how the proposed mechanisms and the forces generated at a molecular level are integrated to produce whole cell movement . this review describes the present state of knowledge gained from these experimental and theoretical studies on cell movement , with an emphasis on the physical basis of the forces that govern the crawling of single cells . it also references several websites with animations and movies , given in footnotes , to clarify the processes described . as a cell moves on a substrate ( the extracellular matrix if the cell moves inside an organism or a cover slide if it moves outside an organism ) , it experiences external forces , which include the viscous force or resistance from the surrounding medium and cell - substrate interaction forces , and internal forces that are generated by the cytoskeleton . in most animal cells , the cytoskeleton is the essential component in creating these motility - driving forces , and in coordinating the entire process of movement . the cytoskeleton is a polymer network , composed of three distinct biopolymer1 types : actin , microtubules and intermediate filaments . these biopolymers are differentiated principally by their rigidity , which can be described by the persistence length lp . the persistence length is defined as the distance over which the filament is bent by thermal forces , and increases with increasing stiffness 10 . they are ~7 nm in diameter , are built from dimer pairs of globular actin monomers , and are functionally polar in nature 1,12 . this means that they have two distinct ends : a fast and a slow growing end ( called the plus end and minus end respectively ) 1,12 . the minus end has a critical actin monomer concentration that is ~6 times higher than that at the plus end ( ~0.6 m and ~0.1 m at the minus and plus end respectively ) . when the end of an af is exposed to a concentration of monomeric actin that is above its critical concentration , the filament end binds monomers and grows by polymerization . conversely , when the concentration is below the critical concentration , monomers detach from the filament end , and the filament shrinks by depolymerization . simply by having these two different critical actin concentrations at the opposing ends of the filament , afs can grow asymmetrically , and when the actin monomer concentration lies between the two values , only the plus end grows while the minus end shrinks . this process , where the length of the filament stays roughly constant and the polymerized monomers within the af transfer momentum forward due to asymmetric plus end polymerization , is known as treadmilling ; it is a critical aspect of how polymerizing afs can generate force . microtubules ( mts ) are the stiffest of the biopolymers , with lp ranging from 100 to 5000 m depending on the filament length 13 . tubulin protein subunits assemble into protofilaments , and typically 13 of these protofilaments then align to form a hollow tube , imbuing mts with their incredible rigidity . mts exhibit similar dynamics to those of actin : they are functionally polar , treadmill , and can impart a force through polymerization 14 . intermediate filaments ( ifs ) are much more flexible than afs and mts ( lp~0.3 - 1.0 m ) . they range in diameter from 8 to12 nm , between that of afs and mts . there are different classes of ifs such as vimetin , desmin , keratin , lamin and neurofilaments , with different cell types having different ifs . unlike afs or mts , ifs are not polarized , do not treadmill , do not generally depolymerize under physiological conditions once polymerized 15 , and are therefore considered to be more static in nature than afs and mts . these three kinds of biopolymers build an internal cellular scaffold , known as the cytoskeleton an organized and coherent structure that is formed by connecting these filaments via entanglements , and also crosslinking , bundling , binding , motor and other proteins . these cytoskeletal assemblies then work together as a composite , dynamic material in cell functions such as structural integrity , shape , division , and organelle transport and cell motility . with respect to motility , although the other polymer assemblies in the cell also aid in coordinating movement and powering translocation , the actin cytoskeleton is regarded as the essential engine that drives cell protrusion 16 , 17 , the first step of movement . it is also integral to achieving the two other steps of movement : adhesion of the leading edge and deadhesion at the cell body and rear , and translocation of the bulk of the cell . the actin cytoskeleton is highly dynamic and the actin structures in the cell can be readily reorganized by the cell to adapt their behavior for movement according to the surrounding environment . the constant restructuring of the actin cytoskeleton and the transition from one actin structure to another is vital in enabling the cell to change its elastic properties rapidly , and this dynamic response is fundamental for movement . mesh - like actin networks consisting of short crosslinked afs are primarily found at the leading edge of cells 9 . the growth of these meshworks i.e. the continuous creation of new actin network at the leading edge is considered to be essential for pushing the cell forward . this network formation is carried out with the help of numerous accessory proteins 18 . activating proteins ( e.g. wasp ) enable nucleator proteins ( e.g. arp2/3 complex ) to initiate the polymerization and assembly of new actin filaments . cofilin ( also known as actin depolymerizing factor adf ) severs actin filaments and creates new plus ends for the growth of new actin filaments . actin binding proteins ( e.g. profilin , thymosine-4 ) maintain a steady actin monomer pool for polymerization , while crosslinking and bundling proteins ( e.g. filamin , -actinin , fascin ) help form connected actin networks . capping proteins ( e.g. capz ) control filament length by attaching to actin filament ends and stopping further polymerization , while severing and fragmenting proteins ( e.g. gelsolin , severin ) cut actin filaments and networks . all these proteins work together to coordinate actin network formation and bring about leading edge motility in several steps , as described previously ( such as in figure no . 2 of pollard 2000 18 , which shows the dendritic nucleation model for movement of leading edge , or actin bundles are composed of parallel arrays of individual afs that are closely packed and crosslinked by proteins such as fascin , fimbrin and scruin , and fulfill structural and sensory roles that are key to cell movement . often , these actin bundles connect distal points of adhesion , allowing tension to be propagated across the cell , and enabling the cell to apply forces on the substrate and move . they are appropriately known as stress fibers , distributing forces and positively reinforcing adhesion sites 9 . in some cells , actin bundles known as filopodia may extend out beyond the lamellar edge , and function as chemical and mechanical sensors , and aid the cell in migrating through tissue 20,21 . myosin motors consist of a head , neck , and tail region ; while some myosin motors have one head and neck , others have two . the head / neck region is responsible for attachment and force production , while the tail region is principally believed to be for connecting to cargo , such as other myosins , vesicles , or filaments . myosin motors do work on actin filaments through a general three - step process of binding , power stroke , and unbinding 1,12 . this process is continuously repeated , and leads to the generation of a contractile force ( acto - myosin contractile force ) thought to be essential in pulling the bulk of the cell forward during movement . in most cell types , the microtubules predominantly extend radially from the centrosome to the actin network at the cell periphery , with their plus ends towards the cell edge , and thus display a hub and spoke arrangement . these microtubules aid in determining the direction of cell movement 22 . intermediate filaments create a fibrous network that spans the cell interior and connects the nucleus to the cell membrane , providing structural integrity to cells . due to their more static properties , it has long been held that ifs are of little importance for cell movement 12 , since cell movement requires the cytoskeleton to be dynamic and to reorganize rapidly ; although ifs may not be critical to cell movement , recent work shows ifs to be more dynamic than previously thought 23 . a cell begins to move in response to an external signal in its surrounding environment . this can be a physical , chemical , diffusible or non - diffusible signal that is detected by receptor proteins located on the cell membrane , and transmitted by them via signaling cascades to the cell interior 1 . a cell , such as a white blood cell , yeast cell or slime mold cell , is believed to sense the signal direction by spatially recognizing external gradients ( receptor proteins become more concentrated on the side of the cell where the signal is present ) 24 . once cell movement begins , the process , which involves the constant restructuring of the actin cytoskeleton , can be divided into three stages in most cells ( figure 1 ) 25 . first , a cell propels the membrane forward by orienting and reorganizing ( growing ) the actin network at its leading edge . second , it adheres to the substrate at the leading edge and deadheres ( releases ) at the cell body and rear of the cell . finally , contractile forces , generated largely by the action of the acto - myosin network , pull the cell forward . after sensing the signal , the cell starts moving in response to the signal by polymerizing actin . if the signal is a chemoattractant , for example , actin polymerizes in the region of the cell closest to the signal , whereas if the signal is a chemorepellant , the cell moves away by polymerizing actin in the opposite side . as the extending edge moves forward , the cell constantly monitors the signal direction and tailors its direction of motion accordingly . this signal tracking is beautifully demonstrated by a cell that chases an object it attempts to engulf2 or by a cell that moves in response to a chemoattractant3 . soon after the leading edge begins to protrude , adhesion molecules gathered in the extending region help attach the leading edge to the substrate . cell - substrate attachments are created at the leading edge when actin bundles link the cytoskeleton to the substrate at certain sites via adhesion molecules ( figure 2 ) . these attachments prevent the protruding leading edge from retracting . as the cell continues to adhere at the leading edge , it deadheres at the cell body and rear of the cell , possibly by the disassembly or contraction of its attachments ( actin bundles ) . finally , the rest of the cell is pulled forward , mainly by contractile forces that are produced by myosin motors sliding on actin filaments , which are in the cell body and at the rear . all the stages or processes described above are continuously running as the cell moves on the substrate , with the actin cytoskeleton transitioning between a solid - like elastic material ( gel ) and a solution - like viscous material ( sol ) . these transitions , called gel - sol transitions , are crucial for cell movement 26 . they are likely caused by the constant net actin polymerization and network assembly at the leading edge and depolymerization and disassembly at the rear of the cell . the transition of an actin network to a more fluid - like state may also be motor driven 27 . these processes lead to local changes in the elasticity of the cell as it moves . in summary , the entire process of cell movement is coordinated spatially as well as temporally by many proteins via mechanical changes in the cytoskeletal structure and changes in the force production centers . undoubtedly , propulsion of the leading edge is a multi - step , complex process 18 - 19 , 28 - 33 , but the basic active mechanism that is believed to move the leading edge is the polymerization of actin filaments towards the cell membrane . polymerizing actin filaments alone , without any accompanying motors , can generate significant force to move a cell 's leading edge . in addition , filament - substrate adhesions are required to prevent the backward movement of polymerizing actin filaments . how does a filament elongating against a load in this case , the cell membrane and external load , if any generate such a force ? there are two main types of models which have been proposed to explain force generation by actin polymerization ( polymerization force ) : ratchet models 34,35 , and autocatalytic models 36,37 . if the membrane were fixed and immovable , an actin filament would stop polymerizing when it bumped into it and would be unable to push against it or generate force . however , the membrane does not remain stationary but undergoes constant brownian motion i.e. random thermal fluctuation due to the membrane 's small size scale and relative flexibility . in addition , an actin filament is not a stiff , immovable rod that stops growing once it reaches the membrane but is an elastic filament that can bend in response to the load . if the bending of the filament away from the membrane is adequately large ( angle > ~30 ) and the filament is sufficiently long ( > ~70 nm ) but without buckling , an actin monomer ( which is 2.7 nm in size ) can easily insert itself between the filament and membrane4 . the lengthened filament can subsequently apply an elastic force on the membrane and push it forward . each addition of a monomer ratchets the membrane , which implies that this process prohibits backward movement of the membrane and ensures that there is only a net forward motion of the cell edge . this model , proposed to explain force generation by a single polymerizing actin filament , is called the elastic brownian ratchet model 34 . an extension of the elastic brownian ratchet model is the tethered elastic brownian ratchet model which incorporates the transient attachment of actin filaments to the surface or membrane and considers two sets of filaments : working filaments , which are filaments that are not attached to the surface and can exert a force on it , and attached filaments , which can not exert a force on the surface 35 . attached filaments are converted to working filaments when they dissociate from the surface and working filaments are converted to non working ( i.e. attached ) filaments when they are capped . based on the dendritic - nucleation model and its branching mechanism , the tethered elastic brownian ratchet model importantly assumes that new actin filaments / branches are generated independent of existing branches . under this assumption , the model attempts to understand force generation from a population of filaments in an actin network . theoretical calculations , based on the above ratchet mechanism along with thermodynamic principles to find the energy of monomer addition to a polymer tip , can provide estimates for the maximum force generated by a single polymerizing actin filament . the maximum force that a filament can develop ( stall force ) is estimated by equating the work done to push a load through a distance to the energy required to add a monomer to the filament tip 38 . this is given by the thermodynamic expression fmax = ( kbt/ )ln(a*kon / koff ) , where kbt is thermal energy , is the size of an actin monomer , kon and koff are the association and dissociation constants for an actin monomer adding to or falling off from a polymer tip , and a is the monomeric actin concentration 38 . if typical values are substituted in the above equation for an actin filament and for the monomeric actin concentration in the cell , the maximum force generated by a single actin filament is 5 - 7 pn 38 . although this calculation is simplistic , it can be used to estimate the force generated at the leading edge . aggregating the predicted force of ~5 - 7 pn across the hundreds of actin filaments per micron thought to exist at the leading edge of a cell , an estimate for the polymerization force at the leading edge is several piconewtons per micron ( or is on the order of nanonewtons per micron ) 35 a force large enough to tackle the membrane load or resistance 35 ( note that membrane resistance is generated by the breaking of linkages between the actin cytoskeleton and the membrane by the growing actin network , and the membrane bending force ) . however , as noted , the force calculations with the ratchet models do not include the role of motor proteins in generating force . based on studies in the bacterial pathogen listeria and other experimental systems , recent work by dickinson and others 39 concludes that the forces predicted by the ratchet models alone are not sufficient to account for the observed polymerization forces in these systems . it shows that much larger forces can be generated by considering the action of motors at the tip of the actin filaments ( end tracking motors ) , which can efficiently convert free energy into work even in the case of persistently tethered filaments . the autocatalytic model also explores force generation by an actin filament and actin network . to understand force generation by a single filament , the model assumes that the load diffuses and that the probability of monomer addition to a polymer tip includes a boltzmann factor 36 , which is identical to the basis of the elastic brownian ratchet model . to study the problem of force generation by a network , two approaches have been used : a numerical approach that uses stochastic simulations of an actin network growing against a load , while tracking the positions and orientation of all actin filaments over time 36 , and a deterministic approach that uses an explicit rate equation for filament orientation distribution 37 . both approaches incorporate the main assumption that new actin branches are generated from existing branches , which differs from the assumption made in the tethered elastic brownian ratchet model . the transient attachment of actin filaments to the surface is not considered in either approach of the autocatalytic model . the stochastic simulations are used to study the dependence of growth velocity on capping rate , load force , branching and other parameters . the simulations show that , for a fixed actin concentration , the growth velocity has an inverse linear relation with the capping rate and equals zero when the number of branches is less than 1.5 36 . these simulations , however , could be performed only for a small parameter range that is computationally feasible . hence , the deterministic model was developed to study a larger parameter range and test the generality of the previous numerical results rigorously 37 . in addition , the deterministic equations can be easily modified to accommodate the assumption that new branches are generated independent of existing branches , as assumed in the tethered elastic brownian ratchet model . hence by using the same basic set of equations , the predictions of the autocatalytic and ratchet models are compared in carlsson 2003 37 . one main prediction of these models ( ratchet model and autocatalytic model ) that can be used to compare them and experimentally test them is the relation between the load ( force ) against which a polymerizing actin filament ( or network ) elongates and the filament 's ( or network 's ) resultant growth velocity . for a single filament , the elastic brownian ratchet model predicts an exponential force - velocity relation v = vmax exp(-f*/kbt ) - vdep , where vmax is the free polymerization velocity , f is the load force on a single filament , and vdep is the depolymerization velocity ( see 34 for derivation ) . it is not surprising that the autocatalytic model also predicts the same exponential force - velocity relation as the elastic brownian ratchet model for a single filament , given that the underlying basics of both models are the same in this case 36 . although the single filament force - velocity relation is the same , the prediction for the force - velocity relation for a network growing against a load is very different from the tethered elastic brownian ratchet model and the autocatalytic model due to the following main differences in assumptions : a ) new branches are generated independent of existing branches in the tethered elastic brownian ratchet model , while they are generated only from existing branches in the autocatalytic model , and b ) only the working filaments generate force ( opposing the load force and force from attached filaments ) in the tethered elastic brownian ratchet model , whereas all filaments generate force to oppose the load force in the autocatalytic model . the tethered elastic brownian ratchet model predicts a biphasic force - velocity relation where the velocity decreases rapidly for small forces and decreases much more slowly for high forces 35 . a qualitative explanation for this predicted behavior from the tethered elastic brownian ratchet model is that the network growth velocity depends on the ratio of working to attached filaments . in the model , the number of working filaments is independent of load , while the number of attached filaments is not independent of load due to the dependence of the dissociation rate of attached filaments on velocity . if the load is high and velocity is low , the dissociation rate decreases , leading to an increase in the number of attached filaments and a slow decrease in velocity 35 . on the other hand , the autocatalytic model predicts that the actin network growth velocity is independent of the applied force or load i.e. v= v0 ( see 36,37 for details ) . a qualitative explanation for the insensitivity of the velocity to the force in this case is that if the load increases , the number of filaments pushing the load also increases proportionately , leaving the force per filament and the growth velocity unchanged 37 . when the equations of the autocatalytic model are modified to accommodate the assumption of an independent growth of new branches , it yields a force - velocity relation that is qualitatively similar to that of the tethered elastic brownian ratchet model 37 . however , the other underlying assumptions of the models and the reasons for arriving at the same result from these two network models are different 40 . it should also be noted that when the equations of the tethered elastic brownian ratchet model are modified to accommodate branching as in the autocatalytic model , the analysis does not yield a force - velocity curve similar to that of the autocatalytic model but a biphasic result that is unchanged from that of the tethered elastic brownian ratchet model itself 35 . this is likely due to the other differences between the models ( point b ) above as well as filament orientation ( not incorporated in ratchet models ) and branching rate ( not considered explicitly in the tethered elastic brownian ratchet model modified to accommodate autocatalytic branching 40 ) . the above results emphasize that the two models are fundamentally different models for force generation by an actin network 40 . the force - velocity relations for networks from these two models have been tested experimentally in vitro . one in vitro study performed with listeria used increasing amounts of methylcellulose to slow its velocity and computed the corresponding viscous resistive force exerted on the body in each case with the stokes equation , knowing the viscosity of methylcelluose , the velocity of bacterial motion and the shape factor of the bacterium . the analysis yielded force - velocity data that are quantitatively similar to that proposed by the tethered elastic brownian ratchet model 41 . another similar study was carried out in a biomimetic system ( spherical polystyrene bead coated with wasp on which an actin gel grows in the presence of other purified proteins ) 42 ; it also used varying amounts of methylcellulose to slow the bead velocity and computed the force acting on the bead with the stokes equation . one reason for the different results of these two similar in vitro experiments may be that only the analysis of wiesner and coworkers 42 incorporates the impact of methylcellulose on the force - velocity curve other than due to its viscosity effect alone . it is possible that this and other differing assumptions between the two studies lead to analyzing different regimes of the force - velocity curve ( see 40 for further explanation ) and to supporting different models . to shed further light on these models and the polymerization force , several other in vitro motility systems have also been designed ( see table 1 ) 43 - 48 . neither model has been unambiguously favored as a result of these in vitro data . however , these studies still provide useful estimates of the polymerization forces such as the stall force of an actin filament , stall force of an actin comet / network , the maximum propulsive force generated by an actin network in the given in vitro system and the contribution of each actin filament to the total force measured . they also help us begin understanding and investigating the polymerization mechanics operating in vivo . in vivo , it is not easy experimentally to test the derived single filament or actin network force - velocity relationships of these models and thus discern between the ratchet and autocatalytic network models 37 . it is also not easy theoretically to extend these models to that for the actin network at the leading edge . membrane fluctuations and membrane load ( resistance ) crucially determine the lamellipodial protrusion velocity , and need to be considered carefully similar to the detailed analysis that has been performed for filopodial protrusion 53 . membrane resistance is , for example , an important factor that is postulated to restrain the growth ( polymerization ) of actin filaments at the leading edge and to determine the protrusion velocity 54 . experimental studies have investigated the relation between membrane resistance and protrusion velocity by changing the membrane resistance in various ways and observing the protrusion velocity . methods to change the membrane resistance include the use of detergents or fluorescent lipids to expand membrane area or an external force to stretch the membrane , all of which techniques reduce resistance , or the use of the microtubule disrupter nocodazole which contracts cells , or osmosis to swell cells , which techniques increase resistance 55,56 . the results of all these experiments clearly demonstrate an inverse relation between membrane resistance and protrusion velocity and estimate a membrane resistance force of ~30 pn/m 55 . in one modeling study , membrane resistance is incorporated in a simple manner : the single filament force - velocity relation obtained from the elastic brownian ratchet model is extended to a large number of filaments pushing the membrane ( as in the lamellipodium ) , assuming an uniform distribution of the membrane load ( resistance ) among the filaments ; an expression for the cell 's protrusion velocity is then derived 54 . the velocity is shown to be inversely proportional to membrane resistance , in agreement with the experimental results . however , such force - velocity predictions for the protruding cell still need further experimental validation in vivo . in summary , the ratchet models 34 , 35 and autocatalytic models 36,37 suggest likely molecular mechanisms of actin force generation and protrusion of the leading edge and discuss parameter regimes in which they could be applicable in vivo . since all the parameters required to test the models are not yet available for the in vivo case , neither model has been either conclusively proved or ruled out in vivo , and their applicability to cell motility is still under investigation . nevertheless , these models provide useful insights into understanding cell protrusion , since it is possible that the actual protrusion mechanism is a combination of the mechanisms advanced by both classes of models 37 . the leading edge of the cell protrudes as a result of polymerization of the actin network in concert with adhesion of the extending network to the underlying substrate via integrins and other adhesion molecules . the exact distribution of cell - substrate attachments , the force with which the protruding cell adheres to the underlying substrate in various cell types , and changes or gradients in this adhesion force within the leading edge as one moves perpendicularly away from the tip of the leading edge are still active research areas . however , both the spatial distribution pattern of attachments and the adhesive force vary from cell type to cell type and are important factors that determine the protrusion rate and rate of translocation ( rate at which the bulk of the cell/ cell body moves ) of each cell type 57,58 . the adhesions near the cell 's leading edge are crucially required to convert some of the polymerization force into protrusion ; the rest of it contributes to the flow of the actin network . to understand what actin flow means , consider the case of a neuronal growth cone 59,16 . in this case , due to polymerization at the leading edge , the actin network there is pushed forward . at the same time , polymerizing actin filaments flow away from the leading edge at a fairly constant rate , known as centripetal actin flux or actin flow 60,61 . in many migrating cells , such as neuronal growth cones , fibroblasts , and fish keratocytes , the actin flow is modulated in a phenomenon known as retrograde flow , which is an important factor that determines the cell 's translocation rate via the adhesion mechanics , as described in the section below . the term retrograde flow describes the variable movement of actin filaments rearward with respect to the substrate , and generally in a direction opposite to the movement of the cell 62 - 64 . both actin polymerization and myosin motors are crucial in driving retrograde flow . while experimental studies on fibroblasts and dictyostellium have established that actin polymerization contributes to the generation of retrograde flow by providing a constant actin source and by pushing the lamellipodial actin network backward 65,66 , the role of myosin motors in retrograde flow is still being studied as the essential myosin subtypes in many systems is unclear . myosin motors are believed to generate retrograde flow by producing contractile cytoskeletal forces ( inward pulling forces ) . observations concerning myosin motors and retrograde flow include the following : myosin ii motors have been revealed to play an essential role in generating retrograde flow in fish keratocytes 67 . mysoin iia , myosin iib , and myosin ic have been found to contribute to retrograde flow in neuronal growth cones , while myosin iib and v have been found to contribute to growth cone spreading , highlighting the varied roles of myosin in growth cone movement 68 - 70 . similarly , in fibroblasts , an important role for myosin iia in retrograde flow and cell spreading has been identified , but both events are unaffected by the deletion of myosin iib 71 . in summary , regarding movement and retrograde flow , all these studies indicate that both polymerization and molecular motors contribute critically to the generation of retrograde flow 72 , but the extent of the contribution of polymerization versus that of myosin motors to retrograde flow may be different in different cell types . the association between the cell translocation rate and retrograde flow can be explained by the existence of a molecular clutch 73,64 . the molecular clutch is thought to be composed of vinculin , talin and other adhesion complexes , and determines the extent to which the cytoskeleton and underlying substrate are linked and can interact . through this interaction , the clutch controls the transmission of the cytoskeletal contractile forces to the substrate , and the cell 's translocation rate . when the clutch is engaged , there is an increase in the rate of translocation and a decrease in the retrograde flow because this tight cytoskeleton - substrate linkage enables an effective transmission of the acto - myosin contractile forces to the substrate via the adhesion complexes . it consequently enables the cell to move forward by pushing against the substrate with traction forces . the engaged clutch also allows the actin polymerization force to drive the cell forward efficiently . in contrast , when the clutch is not engaged , the loose coupling between the cystoskeleton and substrate results in an ineffective force transmission to the substrate ; in this case , instead of the cell being moved forward efficiently , the actin meshwork is pushed backwards resulting in high retrograde flow and a low translocation rate . thus , if we consider that the wheels ( actin filament mesh ) spin at a constant velocity ( actin flow ) but sometimes stick or slip on the road ( variable traction of focal adhesions ) , we begin to see how the clutch hypothesis explains observations . the clutch hypothesis thus predicts that slow moving cells ( low translocation rate ) have a high retrograde flow and generate less traction force , while in fast moving cells , a smaller retrograde flow and a larger traction force are expected . however , although many slow moving cells have been observed to have high retrograde flow and a large number of adhesions , they are also observed to generate large traction forces , which is in contrast to the above prediction . to explain this observation and to understand the relation between cell speed , traction forces and retrograde flow more clearly , one of these studies on fish keratocytes 64 reveals that a phenomenon , called adhesion raking ( i.e. the raking inward of the cytoskeleton against the substrate ) , can also produce retrograde flow and needs to be considered . when the clutch is engaged and raking occurs , the traction forces exerted on the substrate are large , but when the clutch is not engaged and retrograde flow is high , the traction forces are small ( figure 3 ) . since it is likely that both phenomena ( adhesion raking and clutch disengagement ) occur simultaneously in the cell , their combined effect produces a non - linear ( biphasic ) relation between cell speed and adhesiveness ( the relation between cell speed and traction force is also thus non - linear ) 64 , a result in agreement with other experimental and computational studies on fibroblasts , neutrophils etc . thus , the study concludes that when the adhesion to the substrate is too strong , the contractile machinery can not function effectively to produce retraction and cell movement , whereas when it is too weak , the new adhesions that develop at the leading edge can not establish themselves firmly enough to produce cell motion 64 . hence , for rapid cell movement , the adhesion force needs to be optimum and retrograde flow minimum , while for slow movement , adhesion is below or above optimum and retrograde flow is high . note that in most steady or smooth moving cells such as fish keratocytes , the cell 's translocation rate and protrusion rate are identical . however , cells such as neuronal growth cones exhibit significant stochastic fluctuations in the protrusion velocity due to variations in actin polymerization rates that occur at a much smaller timescale than retrograde flow 16 . many of the above studies 64,75 have analyzed retrograde flow using fluorescence speckle microscopy data on the assembly and disassembly of the actin network in conjunction with computational models 79 . in the technique of fluorescent speckle microscopy ( fsm ) , a small number of proteins subunits of a macromolecule ( actin , in this case ) are labeled , and the movement of these fluorescent speckles is then recorded5 . betz and coworkers also analyzed retrograde flow rates in neuronal growth cones by tracking patterns in gfp - actin fluorescent growth cones 16 . all these studies produce accurate actin flow trajectories and velocities in vivo and have detected both protrusion of the leading edge and retrograde flow in most cell types , whose speeds and adhesion forces vary . although the adhesion force varies among cell types , estimates for the adhesion force at the leading edge can be obtained . experiments performed to determine the adhesion force the force required to break a single integrin attachment for example ( see figure 2 for integrin attachment ) yield a value of ~10 - 30 pn per attachment 80 , 52 . in a recent study 81 , the net adhesion force has been measured by applying a counter force of fluid flow ( water flow from a pipette ) to the leading edge of a moving fish keratocyte until the leading edge is locally stalled6 . when the navier - stokes equations for fluid flow are used to numerically estimate the force at the cell due to the water flow , the analysis yields the result that a force of only a few piconewtons causes the cell to lose adhesion and stop moving forward locally . additionally , the analysis reveals that the actin polymerization is not stopped by this low force from the water flow but that the nascent adhesions at the tip of the leading edge are disrupted . as already mentioned , the maximum polymerization force that can be generated by a single elongating actin filament is estimated to be 5 - 7 pn . since there are several actin filaments and integrin attachments per micron at the leading edge , the total force developed by the protruding leading edge is likely to be several nanonewtons . two recent studies have directly measured the actin polymerization protrusive force and stall force by placing an atomic force microscopy ( afm ) cantilever tip in the path of a moving fish keratocyte and blocking its movement 7,82 . brunner and coworkers measured larger main cell body motility forces ranging from 34 to 85 nn 7 . these results 7,82 combined with those of bohnet and coworkers 81 illustrate the importance of adhesion and its requirement for movement , since polymerization requires traction to produce cell movement , and must work synergistically and simultaneously with adhesion to drive motility . various other groups have measured motility forces ( traction , stall forces ) for a variety of cell types using different techniques , such as deformable elastic substrates ( e.g. silicone membranes ) , force sensor arrays and cell traction force microscopy ( see relation among force and also recent review 83 ) and some of these measurements are shown in table 2 . as the protruding leading edge adheres to the substrate , the cell also deadheres at the cell body and the rear . this is likely a biochemical process involving the disassembly of the focal adhesions 89 - 91 . such a process is mediated by several proteins including the protease calpain , as well as other signals such as src , fak and pak etc . the process of detachment is also influenced by the cytoskeletal contractile forces developed at the rear , and simple mechanics . when adhesions are no longer under the cell , they are under considerable strain , and eventually unbind . on the other hand , if the cell can not generate sufficient forces to break the adhesions , these adhesion sites become restraints , and prevent the cell from moving forward further . the exact method by which each cell type detaches , however , can differ and depends on the strength and distribution pattern of the attachments and cell speed , and thus is a continued area of research . in summary , adhesion forces are a critical ingredient of cell movement , mediating the generation of traction forces that allow the protrusion of the leading edge and the translocation of the rest of the cell . the final step of cell movement is translocation of the cell body and the rear of the cell . the retraction force required for this process is generally thought to be generated by the sliding of myosin motors , such as myosin ii or ic , on actin filaments or actin bundles in the cell body and rear . since the actin filaments and bundles are connected to the cell membrane and the substrate , the force generated can be converted to traction forces that enable the cell to move forward . this idea of contraction of the acto - myosin network as the origin of the retraction force is based on several studies that have explored the role of myosin motors in retraction 92 , 94 ( note however that contractility does not occur only at the rear and can occur elsewhere , as there is cortical tension around the cell . at the rear , for example , one experimental study has investigated the role of myosin ii motors in retraction by knocking out myosin ii in dictyostelium cells 95 . the study demonstrates that , on adhesive surfaces , the myosin minus cells do not retract as well as their normal counterparts do and move more slowly than them . another study on fish keratocytes shows that the inhibition of myosin contraction causes the cell body to halt , producing a lamellar fragment which continues forward 96 . these studies conclude that conventional myosins play an important role in the movement of the cell body and retraction of the trailing edge 95,96 . studies on the acto - myosin network also provide estimates for the acto - myosin contractile force ; it is similar in magnitude to the polymerization force at the leading edge on the order of thousands of piconewtons since there are thousands of myosin motors at the rear , each generating ~1 pn of force 38 . such a myosin generated contractile force is likely to be the primary contributor to the retraction force , but it may not be the only contributor . other theories for the retraction force include transport mechanisms , which involve the pulling of the cell , with myosin motors , on oriented actin filaments that function as tracks 97 . another mechanism by which the actin gel in some cell types can create contractile forces is by the process of solation ( decrease in gel structure , which could imply disassembly of the network by the severing of filaments or breaking of crosslinks , or unbundling of filaments ) . a solating actin gel can generate a contractile force of a few piconewtons per actin filament ( see calculations in 38,51 ) . in order to understand the generation of a contractile force by solation , and to better understand the retraction force in general , these cells offer an ideal system to study crawling , since they are simple prokaryotic cells that use major sperm protein ( msp ) to crawl , in a similar process to actin - based eukaryotic cell movement ; however , they employ msp solely to perform the job of cell movement , unlike actin which participates in several other functions of eukaryotic cells 99 . in particular , the study used vesicles derived from the leading - edge membrane of nematode sperm cells surrounded by cytoplasm including msp . the researchers observed that the disassembly or unbundling of msp bundles ( in the presence of yersinia thyrosine phosphatase ( yop ) ) attached to the vesicle caused a contraction of individual filaments and led to a contractile force 98 . thus , they found that retraction forces can be explained by considering cytoskeletal dynamics alone ( without considering motors ) , thereby suggesting an alternate mechanism of generating a contractile force at the rear of the cell . a recent model motivated by this data further elucidates how solely cytoskeletal disassembly generates the necessary force to pull the cell body forward 51 . a qualitative description of the model , which contains two steps , is the following : the first step involves the detachment of filaments from the bundle . a bundle with n filaments , each having a persistence length lp , has an effective persistence length of nlp , and is thus much stiffer than an individual filament 51 . when the filaments detach from the bundle ( caused by yop ) , its effective persistence length and rigidity ( rigidity is directly proportional to lp ) decrease , leading to its contraction due to an increase in entropy . the second step deals with the dissociation of monomers from the filaments ( depolymerization ) . thus , this kinetic model for disassembly incorporating polymer entropy is able to quantify the retraction forces due to disassembly . however , future experiments are needed to ascertain the relevance and relative contribution ( with respect to myosin generated forces , for example ) of such a mechanism in generating retraction forces in vivo . in summary , the retraction force , whose major components are believed to include cytoskeletal disassembly and acto - myosin contraction , is a vital force required to move the bulk of the cell forward and complete the migration cycle . undoubtedly , propulsion of the leading edge is a multi - step , complex process 18 - 19 , 28 - 33 , but the basic active mechanism that is believed to move the leading edge is the polymerization of actin filaments towards the cell membrane . polymerizing actin filaments alone , without any accompanying motors , can generate significant force to move a cell 's leading edge . in addition , filament - substrate adhesions are required to prevent the backward movement of polymerizing actin filaments . how does a filament elongating against a load in this case , the cell membrane and external load , if any generate such a force ? there are two main types of models which have been proposed to explain force generation by actin polymerization ( polymerization force ) : ratchet models 34,35 , and autocatalytic models 36,37 . if the membrane were fixed and immovable , an actin filament would stop polymerizing when it bumped into it and would be unable to push against it or generate force . however , the membrane does not remain stationary but undergoes constant brownian motion i.e. random thermal fluctuation due to the membrane 's small size scale and relative flexibility . in addition , an actin filament is not a stiff , immovable rod that stops growing once it reaches the membrane but is an elastic filament that can bend in response to the load . if the bending of the filament away from the membrane is adequately large ( angle > ~30 ) and the filament is sufficiently long ( > ~70 nm ) but without buckling , an actin monomer ( which is 2.7 nm in size ) can easily insert itself between the filament and membrane4 . the lengthened filament can subsequently apply an elastic force on the membrane and push it forward . each addition of a monomer ratchets the membrane , which implies that this process prohibits backward movement of the membrane and ensures that there is only a net forward motion of the cell edge . this model , proposed to explain force generation by a single polymerizing actin filament , is called the elastic brownian ratchet model 34 . an extension of the elastic brownian ratchet model is the tethered elastic brownian ratchet model which incorporates the transient attachment of actin filaments to the surface or membrane and considers two sets of filaments : working filaments , which are filaments that are not attached to the surface and can exert a force on it , and attached filaments , which can not exert a force on the surface 35 . attached filaments are converted to working filaments when they dissociate from the surface and working filaments are converted to non working ( i.e. attached ) filaments when they are capped . based on the dendritic - nucleation model and its branching mechanism , the tethered elastic brownian ratchet model importantly assumes that new actin filaments / branches are generated independent of existing branches . under this assumption , the model attempts to understand force generation from a population of filaments in an actin network . theoretical calculations , based on the above ratchet mechanism along with thermodynamic principles to find the energy of monomer addition to a polymer tip , can provide estimates for the maximum force generated by a single polymerizing actin filament . the maximum force that a filament can develop ( stall force ) is estimated by equating the work done to push a load through a distance to the energy required to add a monomer to the filament tip 38 . this is given by the thermodynamic expression fmax = ( kbt/ )ln(a*kon / koff ) , where kbt is thermal energy , is the size of an actin monomer , kon and koff are the association and dissociation constants for an actin monomer adding to or falling off from a polymer tip , and a is the monomeric actin concentration 38 . if typical values are substituted in the above equation for an actin filament and for the monomeric actin concentration in the cell , the maximum force generated by a single actin filament is 5 - 7 pn 38 . although this calculation is simplistic , it can be used to estimate the force generated at the leading edge . aggregating the predicted force of ~5 - 7 pn across the hundreds of actin filaments per micron thought to exist at the leading edge of a cell , an estimate for the polymerization force at the leading edge is several piconewtons per micron ( or is on the order of nanonewtons per micron ) 35 a force large enough to tackle the membrane load or resistance 35 ( note that membrane resistance is generated by the breaking of linkages between the actin cytoskeleton and the membrane by the growing actin network , and the membrane bending force ) . however , as noted , the force calculations with the ratchet models do not include the role of motor proteins in generating force . based on studies in the bacterial pathogen listeria and other experimental systems , recent work by dickinson and others 39 concludes that the forces predicted by the ratchet models alone are not sufficient to account for the observed polymerization forces in these systems . it shows that much larger forces can be generated by considering the action of motors at the tip of the actin filaments ( end tracking motors ) , which can efficiently convert free energy into work even in the case of persistently tethered filaments . the autocatalytic model also explores force generation by an actin filament and actin network . to understand force generation by a single filament , the model assumes that the load diffuses and that the probability of monomer addition to a polymer tip includes a boltzmann factor 36 , which is identical to the basis of the elastic brownian ratchet model . to study the problem of force generation by a network , two approaches have been used : a numerical approach that uses stochastic simulations of an actin network growing against a load , while tracking the positions and orientation of all actin filaments over time 36 , and a deterministic approach that uses an explicit rate equation for filament orientation distribution 37 . both approaches incorporate the main assumption that new actin branches are generated from existing branches , which differs from the assumption made in the tethered elastic brownian ratchet model . the transient attachment of actin filaments to the surface is not considered in either approach of the autocatalytic model . the stochastic simulations are used to study the dependence of growth velocity on capping rate , load force , branching and other parameters . the simulations show that , for a fixed actin concentration , the growth velocity has an inverse linear relation with the capping rate and equals zero when the number of branches is less than 1.5 36 . these simulations , however , could be performed only for a small parameter range that is computationally feasible . hence , the deterministic model was developed to study a larger parameter range and test the generality of the previous numerical results rigorously 37 . in addition , the deterministic equations can be easily modified to accommodate the assumption that new branches are generated independent of existing branches , as assumed in the tethered elastic brownian ratchet model . hence by using the same basic set of equations , the predictions of the autocatalytic and ratchet models are compared in carlsson 2003 37 . one main prediction of these models ( ratchet model and autocatalytic model ) that can be used to compare them and experimentally test them is the relation between the load ( force ) against which a polymerizing actin filament ( or network ) elongates and the filament 's ( or network 's ) resultant growth velocity . for a single filament , the elastic brownian ratchet model predicts an exponential force - velocity relation v = vmax exp(-f*/kbt ) - vdep , where vmax is the free polymerization velocity , f is the load force on a single filament , and vdep is the depolymerization velocity ( see 34 for derivation ) . it is not surprising that the autocatalytic model also predicts the same exponential force - velocity relation as the elastic brownian ratchet model for a single filament , given that the underlying basics of both models are the same in this case 36 . although the single filament force - velocity relation is the same , the prediction for the force - velocity relation for a network growing against a load is very different from the tethered elastic brownian ratchet model and the autocatalytic model due to the following main differences in assumptions : a ) new branches are generated independent of existing branches in the tethered elastic brownian ratchet model , while they are generated only from existing branches in the autocatalytic model , and b ) only the working filaments generate force ( opposing the load force and force from attached filaments ) in the tethered elastic brownian ratchet model , whereas all filaments generate force to oppose the load force in the autocatalytic model . the tethered elastic brownian ratchet model predicts a biphasic force - velocity relation where the velocity decreases rapidly for small forces and decreases much more slowly for high forces 35 . a qualitative explanation for this predicted behavior from the tethered elastic brownian ratchet model is that the network growth velocity depends on the ratio of working to attached filaments . in the model , the number of working filaments is independent of load , while the number of attached filaments is not independent of load due to the dependence of the dissociation rate of attached filaments on velocity . if the load is high and velocity is low , the dissociation rate decreases , leading to an increase in the number of attached filaments and a slow decrease in velocity 35 . on the other hand , the autocatalytic model predicts that the actin network growth velocity is independent of the applied force or load i.e. v= v0 ( see 36,37 for details ) . a qualitative explanation for the insensitivity of the velocity to the force in this case is that if the load increases , the number of filaments pushing the load also increases proportionately , leaving the force per filament and the growth velocity unchanged 37 . when the equations of the autocatalytic model are modified to accommodate the assumption of an independent growth of new branches , it yields a force - velocity relation that is qualitatively similar to that of the tethered elastic brownian ratchet model 37 . however , the other underlying assumptions of the models and the reasons for arriving at the same result from these two network models are different 40 . it should also be noted that when the equations of the tethered elastic brownian ratchet model are modified to accommodate branching as in the autocatalytic model , the analysis does not yield a force - velocity curve similar to that of the autocatalytic model but a biphasic result that is unchanged from that of the tethered elastic brownian ratchet model itself 35 . this is likely due to the other differences between the models ( point b ) above as well as filament orientation ( not incorporated in ratchet models ) and branching rate ( not considered explicitly in the tethered elastic brownian ratchet model modified to accommodate autocatalytic branching 40 ) . the above results emphasize that the two models are fundamentally different models for force generation by an actin network 40 . the force - velocity relations for networks from these two models have been tested experimentally in vitro . one in vitro study performed with listeria used increasing amounts of methylcellulose to slow its velocity and computed the corresponding viscous resistive force exerted on the body in each case with the stokes equation , knowing the viscosity of methylcelluose , the velocity of bacterial motion and the shape factor of the bacterium . the analysis yielded force - velocity data that are quantitatively similar to that proposed by the tethered elastic brownian ratchet model 41 . another similar study was carried out in a biomimetic system ( spherical polystyrene bead coated with wasp on which an actin gel grows in the presence of other purified proteins ) 42 ; it also used varying amounts of methylcellulose to slow the bead velocity and computed the force acting on the bead with the stokes equation . one reason for the different results of these two similar in vitro experiments may be that only the analysis of wiesner and coworkers 42 incorporates the impact of methylcellulose on the force - velocity curve other than due to its viscosity effect alone . it is possible that this and other differing assumptions between the two studies lead to analyzing different regimes of the force - velocity curve ( see 40 for further explanation ) and to supporting different models . to shed further light on these models and the polymerization force , several other in vitro motility systems have also been designed ( see table 1 ) 43 - 48 . neither model has been unambiguously favored as a result of these in vitro data . however , these studies still provide useful estimates of the polymerization forces such as the stall force of an actin filament , stall force of an actin comet / network , the maximum propulsive force generated by an actin network in the given in vitro system and the contribution of each actin filament to the total force measured . they also help us begin understanding and investigating the polymerization mechanics operating in vivo . in vivo , it is not easy experimentally to test the derived single filament or actin network force - velocity relationships of these models and thus discern between the ratchet and autocatalytic network models 37 . it is also not easy theoretically to extend these models to that for the actin network at the leading edge . membrane fluctuations and membrane load ( resistance ) crucially determine the lamellipodial protrusion velocity , and need to be considered carefully similar to the detailed analysis that has been performed for filopodial protrusion 53 . membrane resistance is , for example , an important factor that is postulated to restrain the growth ( polymerization ) of actin filaments at the leading edge and to determine the protrusion velocity 54 . experimental studies have investigated the relation between membrane resistance and protrusion velocity by changing the membrane resistance in various ways and observing the protrusion velocity . methods to change the membrane resistance include the use of detergents or fluorescent lipids to expand membrane area or an external force to stretch the membrane , all of which techniques reduce resistance , or the use of the microtubule disrupter nocodazole which contracts cells , or osmosis to swell cells , which techniques increase resistance 55,56 . the results of all these experiments clearly demonstrate an inverse relation between membrane resistance and protrusion velocity and estimate a membrane resistance force of ~30 pn/m 55 . in one modeling study , membrane resistance is incorporated in a simple manner : the single filament force - velocity relation obtained from the elastic brownian ratchet model is extended to a large number of filaments pushing the membrane ( as in the lamellipodium ) , assuming an uniform distribution of the membrane load ( resistance ) among the filaments ; an expression for the cell 's protrusion velocity is then derived 54 . the velocity is shown to be inversely proportional to membrane resistance , in agreement with the experimental results . however , such force - velocity predictions for the protruding cell still need further experimental validation in vivo . in summary , the ratchet models 34 , 35 and autocatalytic models 36,37 suggest likely molecular mechanisms of actin force generation and protrusion of the leading edge and discuss parameter regimes in which they could be applicable in vivo . since all the parameters required to test the models are not yet available for the in vivo case , neither model has been either conclusively proved or ruled out in vivo , and their applicability to cell motility is still under investigation . nevertheless , these models provide useful insights into understanding cell protrusion , since it is possible that the actual protrusion mechanism is a combination of the mechanisms advanced by both classes of models 37 . the leading edge of the cell protrudes as a result of polymerization of the actin network in concert with adhesion of the extending network to the underlying substrate via integrins and other adhesion molecules . the exact distribution of cell - substrate attachments , the force with which the protruding cell adheres to the underlying substrate in various cell types , and changes or gradients in this adhesion force within the leading edge as one moves perpendicularly away from the tip of the leading edge are still active research areas . however , both the spatial distribution pattern of attachments and the adhesive force vary from cell type to cell type and are important factors that determine the protrusion rate and rate of translocation ( rate at which the bulk of the cell/ cell body moves ) of each cell type 57,58 . the adhesions near the cell 's leading edge are crucially required to convert some of the polymerization force into protrusion ; the rest of it contributes to the flow of the actin network . to understand what actin flow means , consider the case of a neuronal growth cone 59,16 . in this case , due to polymerization at the leading edge , the actin network there is pushed forward . at the same time , polymerizing actin filaments flow away from the leading edge at a fairly constant rate , known as centripetal actin flux or actin flow 60,61 . in many migrating cells , such as neuronal growth cones , fibroblasts , and fish keratocytes , the actin flow is modulated in a phenomenon known as retrograde flow , which is an important factor that determines the cell 's translocation rate via the adhesion mechanics , as described in the section below . the term retrograde flow describes the variable movement of actin filaments rearward with respect to the substrate , and generally in a direction opposite to the movement of the cell 62 - 64 . both actin polymerization and myosin motors are crucial in driving retrograde flow . while experimental studies on fibroblasts and dictyostellium have established that actin polymerization contributes to the generation of retrograde flow by providing a constant actin source and by pushing the lamellipodial actin network backward 65,66 , the role of myosin motors in retrograde flow is still being studied as the essential myosin subtypes in many systems is unclear . myosin motors are believed to generate retrograde flow by producing contractile cytoskeletal forces ( inward pulling forces ) . observations concerning myosin motors and retrograde flow include the following : myosin ii motors have been revealed to play an essential role in generating retrograde flow in fish keratocytes 67 . mysoin iia , myosin iib , and myosin ic have been found to contribute to retrograde flow in neuronal growth cones , while myosin iib and v have been found to contribute to growth cone spreading , highlighting the varied roles of myosin in growth cone movement 68 - 70 . similarly , in fibroblasts , an important role for myosin iia in retrograde flow and cell spreading has been identified , but both events are unaffected by the deletion of myosin iib 71 . in summary , regarding movement and retrograde flow , all these studies indicate that both polymerization and molecular motors contribute critically to the generation of retrograde flow 72 , but the extent of the contribution of polymerization versus that of myosin motors to retrograde flow may be different in different cell types . the association between the cell translocation rate and retrograde flow can be explained by the existence of a molecular clutch 73,64 . the molecular clutch is thought to be composed of vinculin , talin and other adhesion complexes , and determines the extent to which the cytoskeleton and underlying substrate are linked and can interact . through this interaction , the clutch controls the transmission of the cytoskeletal contractile forces to the substrate , and the cell 's translocation rate . when the clutch is engaged , there is an increase in the rate of translocation and a decrease in the retrograde flow because this tight cytoskeleton - substrate linkage enables an effective transmission of the acto - myosin contractile forces to the substrate via the adhesion complexes . it consequently enables the cell to move forward by pushing against the substrate with traction forces . the engaged clutch also allows the actin polymerization force to drive the cell forward efficiently . in contrast , when the clutch is not engaged , the loose coupling between the cystoskeleton and substrate results in an ineffective force transmission to the substrate ; in this case , instead of the cell being moved forward efficiently , the actin meshwork is pushed backwards resulting in high retrograde flow and a low translocation rate . thus , if we consider that the wheels ( actin filament mesh ) spin at a constant velocity ( actin flow ) but sometimes stick or slip on the road ( variable traction of focal adhesions ) , we begin to see how the clutch hypothesis explains observations . the clutch hypothesis thus predicts that slow moving cells ( low translocation rate ) have a high retrograde flow and generate less traction force , while in fast moving cells , a smaller retrograde flow and a larger traction force are expected . however , although many slow moving cells have been observed to have high retrograde flow and a large number of adhesions , they are also observed to generate large traction forces , which is in contrast to the above prediction . to explain this observation and to understand the relation between cell speed , traction forces and retrograde flow more clearly , one of these studies on fish keratocytes 64 reveals that a phenomenon , called adhesion raking ( i.e. the raking inward of the cytoskeleton against the substrate ) , can also produce retrograde flow and needs to be considered . when the clutch is engaged and raking occurs , the traction forces exerted on the substrate are large , but when the clutch is not engaged and retrograde flow is high , the traction forces are small ( figure 3 ) . since it is likely that both phenomena ( adhesion raking and clutch disengagement ) occur simultaneously in the cell , their combined effect produces a non - linear ( biphasic ) relation between cell speed and adhesiveness ( the relation between cell speed and traction force is also thus non - linear ) 64 , a result in agreement with other experimental and computational studies on fibroblasts , neutrophils etc . thus , the study concludes that when the adhesion to the substrate is too strong , the contractile machinery can not function effectively to produce retraction and cell movement , whereas when it is too weak , the new adhesions that develop at the leading edge can not establish themselves firmly enough to produce cell motion 64 . hence , for rapid cell movement , the adhesion force needs to be optimum and retrograde flow minimum , while for slow movement , adhesion is below or above optimum and retrograde flow is high . note that in most steady or smooth moving cells such as fish keratocytes , the cell 's translocation rate and protrusion rate are identical . however , cells such as neuronal growth cones exhibit significant stochastic fluctuations in the protrusion velocity due to variations in actin polymerization rates that occur at a much smaller timescale than retrograde flow 16 . many of the above studies 64,75 have analyzed retrograde flow using fluorescence speckle microscopy data on the assembly and disassembly of the actin network in conjunction with computational models 79 . in the technique of fluorescent speckle microscopy ( fsm ) , a small number of proteins subunits of a macromolecule ( actin , in this case ) are labeled , and the movement of these fluorescent speckles is then recorded5 . betz and coworkers also analyzed retrograde flow rates in neuronal growth cones by tracking patterns in gfp - actin fluorescent growth cones 16 . all these studies produce accurate actin flow trajectories and velocities in vivo and have detected both protrusion of the leading edge and retrograde flow in most cell types , whose speeds and adhesion forces vary . although the adhesion force varies among cell types , estimates for the adhesion force at the leading edge can be obtained . experiments performed to determine the adhesion force the force required to break a single integrin attachment for example ( see figure 2 for integrin attachment ) yield a value of ~10 - 30 pn per attachment 80 , 52 . in a recent study 81 , the net adhesion force has been measured by applying a counter force of fluid flow ( water flow from a pipette ) to the leading edge of a moving fish keratocyte until the leading edge is locally stalled6 . when the navier - stokes equations for fluid flow are used to numerically estimate the force at the cell due to the water flow , the analysis yields the result that a force of only a few piconewtons causes the cell to lose adhesion and stop moving forward locally . additionally , the analysis reveals that the actin polymerization is not stopped by this low force from the water flow but that the nascent adhesions at the tip of the leading edge are disrupted . as already mentioned , the maximum polymerization force that can be generated by a single elongating actin filament is estimated to be 5 - 7 pn . since there are several actin filaments and integrin attachments per micron at the leading edge , the total force developed by the protruding leading edge is likely to be several nanonewtons . two recent studies have directly measured the actin polymerization protrusive force and stall force by placing an atomic force microscopy ( afm ) cantilever tip in the path of a moving fish keratocyte and blocking its movement 7,82 . brunner and coworkers measured larger main cell body motility forces ranging from 34 to 85 nn 7 . these results 7,82 combined with those of bohnet and coworkers 81 illustrate the importance of adhesion and its requirement for movement , since polymerization requires traction to produce cell movement , and must work synergistically and simultaneously with adhesion to drive motility . various other groups have measured motility forces ( traction , stall forces ) for a variety of cell types using different techniques , such as deformable elastic substrates ( e.g. silicone membranes ) , force sensor arrays and cell traction force microscopy ( see relation among force and also recent review 83 ) and some of these measurements are shown in table 2 . as the protruding leading edge adheres to the substrate , the cell also deadheres at the cell body and the rear . this is likely a biochemical process involving the disassembly of the focal adhesions 89 - 91 . such a process is mediated by several proteins including the protease calpain , as well as other signals such as src , fak and pak etc . the process of detachment is also influenced by the cytoskeletal contractile forces developed at the rear , and simple mechanics . when adhesions are no longer under the cell , they are under considerable strain , and eventually unbind . on the other hand , if the cell can not generate sufficient forces to break the adhesions , these adhesion sites become restraints , and prevent the cell from moving forward further . the exact method by which each cell type detaches , however , can differ and depends on the strength and distribution pattern of the attachments and cell speed , and thus is a continued area of research . in summary , adhesion forces are a critical ingredient of cell movement , mediating the generation of traction forces that allow the protrusion of the leading edge and the translocation of the rest of the cell . the final step of cell movement is translocation of the cell body and the rear of the cell . the retraction force required for this process is generally thought to be generated by the sliding of myosin motors , such as myosin ii or ic , on actin filaments or actin bundles in the cell body and rear . since the actin filaments and bundles are connected to the cell membrane and the substrate , the force generated can be converted to traction forces that enable the cell to move forward . this idea of contraction of the acto - myosin network as the origin of the retraction force is based on several studies that have explored the role of myosin motors in retraction 92 , 94 ( note however that contractility does not occur only at the rear and can occur elsewhere , as there is cortical tension around the cell . at the rear , for example , one experimental study has investigated the role of myosin ii motors in retraction by knocking out myosin ii in dictyostelium cells 95 . the study demonstrates that , on adhesive surfaces , the myosin minus cells do not retract as well as their normal counterparts do and move more slowly than them . another study on fish keratocytes shows that the inhibition of myosin contraction causes the cell body to halt , producing a lamellar fragment which continues forward 96 . these studies conclude that conventional myosins play an important role in the movement of the cell body and retraction of the trailing edge 95,96 . studies on the acto - myosin network also provide estimates for the acto - myosin contractile force ; it is similar in magnitude to the polymerization force at the leading edge on the order of thousands of piconewtons since there are thousands of myosin motors at the rear , each generating ~1 pn of force 38 . such a myosin generated contractile force is likely to be the primary contributor to the retraction force , but it may not be the only contributor . other theories for the retraction force include transport mechanisms , which involve the pulling of the cell , with myosin motors , on oriented actin filaments that function as tracks 97 . another mechanism by which the actin gel in some cell types can create contractile forces is by the process of solation ( decrease in gel structure , which could imply disassembly of the network by the severing of filaments or breaking of crosslinks , or unbundling of filaments ) . a solating actin gel can generate a contractile force of a few piconewtons per actin filament ( see calculations in 38,51 ) . in order to understand the generation of a contractile force by solation , and to better understand the retraction force in general , these cells offer an ideal system to study crawling , since they are simple prokaryotic cells that use major sperm protein ( msp ) to crawl , in a similar process to actin - based eukaryotic cell movement ; however , they employ msp solely to perform the job of cell movement , unlike actin which participates in several other functions of eukaryotic cells 99 . in particular , the study used vesicles derived from the leading - edge membrane of nematode sperm cells surrounded by cytoplasm including msp . the researchers observed that the disassembly or unbundling of msp bundles ( in the presence of yersinia thyrosine phosphatase ( yop ) ) attached to the vesicle caused a contraction of individual filaments and led to a contractile force 98 . thus , they found that retraction forces can be explained by considering cytoskeletal dynamics alone ( without considering motors ) , thereby suggesting an alternate mechanism of generating a contractile force at the rear of the cell . a recent model motivated by this data further elucidates how solely cytoskeletal disassembly generates the necessary force to pull the cell body forward 51 . a qualitative description of the model , which contains two steps , is the following : the first step involves the detachment of filaments from the bundle . a bundle with n filaments , each having a persistence length lp , has an effective persistence length of nlp , and is thus much stiffer than an individual filament 51 . when the filaments detach from the bundle ( caused by yop ) , its effective persistence length and rigidity ( rigidity is directly proportional to lp ) decrease , leading to its contraction due to an increase in entropy . the second step deals with the dissociation of monomers from the filaments ( depolymerization ) . thus , this kinetic model for disassembly incorporating polymer entropy is able to quantify the retraction forces due to disassembly . however , future experiments are needed to ascertain the relevance and relative contribution ( with respect to myosin generated forces , for example ) of such a mechanism in generating retraction forces in vivo . in summary , the retraction force , whose major components are believed to include cytoskeletal disassembly and acto - myosin contraction , is a vital force required to move the bulk of the cell forward and complete the migration cycle . cell motility is a complex and integrated process that is carefully and precisely orchestrated by the cell with the help of many receptor , crosslinking , bundling , binding , adhesion , motor and other proteins ; these proteins serve to determine the direction of cell movement , and to carry out the motility events in an exactly timed manner . the crawling of a cell is a cyclical process driven primarily by actin polymerization and acto - myosin contractility , and can be divided into three parts : protrusion of the leading edge , adhesion of the leading edge and deadhesion at the cell body and rear of the cell , and cytoskeletal contraction to pull the cell body forward as the cell moves . although the basic molecular mechanisms of cell movement and the associated signaling pathways are much clearer now than before , there are still a number of open questions regarding the polymerization , adhesion and retraction forces and other mechanisms . for the polymerization force , further experimental studies are required to ascertain the exact role of actin polymerization as the main ( active ) force mechanism pushing the leading edge . it is likely that in some systems it is not the sole active mechanism pushing the front , similar to the case of the nematode sperm cell , where osmotic pressure is postulated to be the active force mechanism and msp is a passive generator that fills the space 100 . hence further investigations are required into which cellular systems actin polymerization and acto - myosin contractility are essential and what their relative contributions are when other force generating mechanisms are present in the same system . if they are the core mechanisms , other questions include determining the role of motor proteins in generating the polymerization force , and testing the force - velocity relations further . as already mentioned , active research areas regarding the adhesion and retraction forces include the exact relation between the force with which the protruding cell edge adheres to the underlying substrate , the retrograde actin flow and cell speed in various cell types , and also the exact origin of the retraction force in vivo . a thorough understanding of the underlying mechanisms and forces of cell movement is essential for gaining deeper insight into several key biological processes such as morphogenesis , wound healing , neurogenesis and immune response . in addition , cell movement is critical in many diseases including tumor development and metastasis , where cells from a primary tumor move away and spread to other parts of the body . a better understanding of the effective mechanisms by which the cell generates these forces and how these forces operate at the microscopic scale may facilitate incorporating similar principles in engineering various mechanisms at microscopic scales . a schematic of the three stages of cell movement , based on 1,12 : after determining its direction of motion , the cell extends a protusion in this direction by actin polymerization at the leading edge . it then adheres its leading edge to the surface on which it is moving and de - adheres at the cell body and rear . finally , it pulls the whole cell body forward by contracile forces generated at the cell body and rear of the cell . a schematic ( based on a figure in http://www.rpi.edu/dept/bcbp/molbiochem/mbweb/mb2/part1/actin.htm ) showing how the cell adheres to the substrate . cell - substrate attachments are formed when actin bundles connect to the substrate at certain sites via adhesion molecules such as vinculin , talin and integrin . a schematic depicting two phenomena that can cause retrograde actin flow in vivo , based on 64 . retrograde flow is postulated to occur either due to release of the molecular clutch and resultant slippage , as seen in figure a ) ( where the yellow and green parts of the clutch do not fit ) , or due to adhesion raking as shown in figure b ) ( where the clutch is engaged ( yellow and green parts fit ) but there is raking of the cytoskeleton against the substrate ( blue and white parts ) ) . magnitudes of various forces measured in vitro : magnitudes of various measured forces in different cells :

cell movement is a complex phenomenon primarily driven by the actin network beneath the cell membrane , and can be divided into three general components : protrusion of the leading edge of the cell , adhesion of the leading edge and deadhesion at the cell body and rear , and cytoskeletal contraction to pull the cell forward . each of these steps is driven by physical forces generated by unique segments of the cytoskeleton . this review examines the specific physics underlying these phases of cell movement and the origins of the forces that drive locomotion .

1. Introduction 2. The Process of Cell Movement 3. The Forces of Cell Movement a) Polymerization Mechanics b) Adhesion Mechanics c) Translocation of Cell Body and Rear of the Cell or Retraction Mechanics 4. Conclusion Figures and Tables

PMC5284470

scotland has higher mortality rates than the rest of britain , higher mortality rates than all other western european countries and wider health inequalities than the rest of western europe.1 2 scotland 's mortality outcomes have not always , however , compared so badly.1 it is only from around 1950 onwards that the rate of improvement in mortality started to lag behind other populations . the rate of improvement faltered again during the 1980s with some areas experiencing an increase in mortality rates.3 around two - thirds of the higher mortality in comparison to the rest of britain could be explained by higher deprivation in 1981 , but less than half of the increasing difference was explained by deprivation by 2001.4 this excess mortality ( ie , after accounting for deprivation ) , the lagging mortality compared with the rest of europe and the greater health inequalities are due to higher mortality from alcohol - related and drug - related deaths , violence and suicide ( particularly in young adults ) and higher mortality from heart disease , stroke and cancer throughout adulthood.5 suicide rates in scotland for men and women are now ranked above those for many other european countries,68 although the rate for women has declined since 1980 and that for men is now also in decline9 ( as are mortality rates due to alcohol10 and violence11 ) . the reasons for these mortality phenomena remain unclear , although many hypotheses are under consideration.5 12 it is possible that period or cohort effects ( age effects would have been adjusted for in the age - standardised analyses ) may be partially responsible for the trends in these outcomes . it has been specifically hypothesised ( in the academic literature,5 12 13 mainstream press and autobiographical accounts1416 ) that there may have been a large section of the scottish population which was disproportionately impacted by the introduction of neoliberalism . in the uk context , this is associated with the election of the conservative government in 1979 , which dramatically changed social and economic policy during the 1980s . this new approach favoured monetarist economic policies which favoured lower taxation and the growth of the financial services industry ( particularly in south - east england ) , but resulted in increased unemployment , further deindustrialisation and a large increase in income and regional inequality.13 17 it is therefore possible that this political change may have impacted on health in the same way as in other countries,18 and particularly on the working class living in the deindustrialising regions.19 20 if this exposure was to be partially responsible for the mortality phenomena in scotland , it would be expected that the cohort of individuals most directly exposed ( ie , working class , working age adults during the period 19791990 ) would be the most profoundly affected . there are clear differences in suicide rates by deprivation.8 21 however , there is a substantial degree of ambiguity around this exposure cohort . although more men than women were likely to have been impacted directly in terms of job losses , women would have been similarly impacted by the loss of household income and the associated trauma of the changing political context . the timing of the exposure is also rather indistinct with deindustrialisation occurring earlier22 and the change in political approach being sustained thereafter ( although it is clear that the 1980s did represent a marked turning point in political outlook).13 17 there is also the potential for delayed impacts . this paper expands the existing epidemiology on suicide in scotland,69 23 by exploring age , period and cohort ( apc ) effects using novel visualisation techniques and applying the intrinsic estimator ( ie ) , a recent development in apc modelling that aims to obtain unbiased estimates of the individual components ( a , p and c ) . a particular focus is whether there is evidence of negative impacts from the political changes in the 1980s , especially for those living in the most deprived areas of scotland , which might explain the recent trends in suicide in scotland . we obtained data on the number of deaths due to probable suicide ( deaths from intentional self - harm or by events of undetermined intent ) by sex , single year of age at death and year of registration of death for scotland from 1974 to 2013 ( the age was unknown for one male death , so this was excluded from the analyses ) , and mid - year population estimates for scotland by sex and single year of age , from age 0 to 90 years and over , from 1974 to 2013 , from the national records of scotland ( nrs ) . the convention in scotland followed by nrs is to produce statistics of suicides by combining the numbers of deaths from intentional self - harm and deaths caused by events of undetermined intent because it is thought that most of the latter are likely to be suicides , see http://www.nrscotland.gov.uk/statistics-and-data/statistics/statistics-by-theme/vital-events/deaths/suicides/the-definition-of-the-statistics . this is the uk standard approach ; see the office for national statistics quality and methodology information paper http://www.ons.gov.uk/peoplepopulationandcommunity/birthsdeathsandmarriages/deaths/qmis/suicideratesintheukqmi . suicides 19741978 were coded by international classification of diseases ( icd ) 8 e950-e959 and e980-e989 , 19791999 by icd9 e950-e959 and e980-e989 , and from 2000 onward by icd10 x60 - 84 , y87.0 , y10 - 34 and y87.2 . acute intoxication with undetermined intent that would previously have been counted under mental and behavioural disorders became counted as analyses used the pre-2011 coding for all years of data with suicides post-2010 identified according to direction by nrs , see http://www.nrscotland.gov.uk/statistics-and-data/statistics/statistics-by-theme/vital-events/deaths/suicides/main-points and http://www.nrscotland.gov.uk/files/statistics/vital-events/changes-to-coding-of-causes-of-death-between-2010-2011.pdf analyses by deprivation were based on carstairs deprivation categories.24 for suicide data , carstairs decile look - up files were obtained from the information and statistics division scotland ( isd ) website and matched in on appropriate postcode sectors ( average population size in scotland of 5600 in 2011 ) . data were then assigned to an appropriate carstairs decile ; cases with no associated carstairs were removed from analyses by deprivation . population data by carstairs deciles , from age 0 to 85 years and over , were obtained from isd with carstairs deciles matched in for each census on postcode sectors and then interpolated between the censuses to obtain the populations for the in - between years . in analyses , data for the two most deprived carstairs deciles were grouped to create the most deprived quintile and the remaining deciles to create the less deprived four - fifth of the population . suicide cases are rare in those aged under 15 years or over 90 years so , unless stated otherwise , analyses were restricted to those aged 1589 years , or aged 1584 years for deprivation analysis ( the upper age limit was dictated by the age structure of the available population data ) . presentations of trends in death rates for all ages from 1974 to 2013 by sex were age - standardised to the 2013 version of the european standard population . for age effects analysis , data from 1974 to 1993 and data from 1994 to 2013 were separately combined for presentation of crude death rates by 5-year age groups ( from 04 to 85 + years ) for age at death by sex . to examine apc effects , suicide rates were plotted using data grouped by age at death ( 5-year age groups ) , year of death ( 5-year periods ) and birth cohort ( those born over 10-year periods from 1895 to 1994 ) . shaded contour plots ( scps ) were produced in r25 ( scps are levelplots ( heatmaps ) with labelled contour lines added , which link positions on the levelplot that have the same value . the same information is therefore presented in two ways in a figure ; in this paper , lines and colours show population age groups with the same suicide rates over time ) . separate plots were produced for men and women , and then separately for the most deprived fifth and the less deprived four - fifths of the population , created using rates for single age and year of death or for single age and birth year ( for more details , see online supplementary appendix 1 ) . in scps of year of death on the horizontal axis and age on the vertical axis , period effects appear as vertical patterns , age effects as horizontal patterns and cohort effects as diagonal disruptions running along diagonal lines from the bottom left to top right , with a gradient of 1 year of age per calendar year . in scps of birth year , rather than year of death on the horizontal axis cohort effects instead run vertically . scps of birth year rather than year of death were therefore used to make cohort effects easier to identify visually . for apc regression modelling , suicide and population data were grouped into 5-year age groups ( ages 1589 years for the overall population and 1584 years for the deprivation analyses ) and 5-year time periods ( from 1974 to 2013 ) stratified by sex and carstairs deprivation . the ie command ( apc_ie ) in stata v.13 ( stata corp lp , college station , texas , usa ) was used.26 as the count data were found to be overdispersed , negative binomial maximum likelihood models were used . the year for the cohorts was calculated as a function of the 5-year age and period groups , as being equal to mean period minus mean age . standard negative binomial regression models were also run to ensure that age , period or cohort alone or any combination of two factors did not fit the data better than a full apc model . we obtained data on the number of deaths due to probable suicide ( deaths from intentional self - harm or by events of undetermined intent ) by sex , single year of age at death and year of registration of death for scotland from 1974 to 2013 ( the age was unknown for one male death , so this was excluded from the analyses ) , and mid - year population estimates for scotland by sex and single year of age , from age 0 to 90 years and over , from 1974 to 2013 , from the national records of scotland ( nrs ) . the convention in scotland followed by nrs is to produce statistics of suicides by combining the numbers of deaths from intentional self - harm and deaths caused by events of undetermined intent because it is thought that most of the latter are likely to be suicides , see http://www.nrscotland.gov.uk/statistics-and-data/statistics/statistics-by-theme/vital-events/deaths/suicides/the-definition-of-the-statistics . this is the uk standard approach ; see the office for national statistics quality and methodology information paper http://www.ons.gov.uk/peoplepopulationandcommunity/birthsdeathsandmarriages/deaths/qmis/suicideratesintheukqmi . suicides 19741978 were coded by international classification of diseases ( icd ) 8 e950-e959 and e980-e989 , 19791999 by icd9 e950-e959 and e980-e989 , and from 2000 onward by icd10 x60 - 84 , y87.0 , y10 - 34 and y87.2 . acute intoxication with undetermined intent that would previously have been counted under mental and behavioural disorders became counted as analyses used the pre-2011 coding for all years of data with suicides post-2010 identified according to direction by nrs , see http://www.nrscotland.gov.uk/statistics-and-data/statistics/statistics-by-theme/vital-events/deaths/suicides/main-points and http://www.nrscotland.gov.uk/files/statistics/vital-events/changes-to-coding-of-causes-of-death-between-2010-2011.pdf analyses by deprivation were based on carstairs deprivation categories.24 for suicide data , carstairs decile look - up files were obtained from the information and statistics division scotland ( isd ) website and matched in on appropriate postcode sectors ( average population size in scotland of 5600 in 2011 ) . data were then assigned to an appropriate carstairs decile ; cases with no associated carstairs were removed from analyses by deprivation . population data by carstairs deciles , from age 0 to 85 years and over , were obtained from isd with carstairs deciles matched in for each census on postcode sectors and then interpolated between the censuses to obtain the populations for the in - between years . in analyses , data for the two most deprived carstairs deciles were grouped to create the most deprived quintile and the remaining deciles to create the less deprived four - fifth of the population . suicide cases are rare in those aged under 15 years or over 90 years so , unless stated otherwise , analyses were restricted to those aged 1589 years , or aged 1584 years for deprivation analysis ( the upper age limit was dictated by the age structure of the available population data ) . presentations of trends in death rates for all ages from 1974 to 2013 by sex were age - standardised to the 2013 version of the european standard population . for age effects analysis , data from 1974 to 1993 and data from 1994 to 2013 were separately combined for presentation of crude death rates by 5-year age groups ( from 04 to 85 + years ) for age at death by sex . to examine apc effects , suicide rates were plotted using data grouped by age at death ( 5-year age groups ) , year of death ( 5-year periods ) and birth cohort ( those born over 10-year periods from 1895 to 1994 ) . shaded contour plots ( scps ) were produced in r25 ( scps are levelplots ( heatmaps ) with labelled contour lines added , which link positions on the levelplot that have the same value . the same information is therefore presented in two ways in a figure ; in this paper , lines and colours show population age groups with the same suicide rates over time ) . separate plots were produced for men and women , and then separately for the most deprived fifth and the less deprived four - fifths of the population , created using rates for single age and year of death or for single age and birth year ( for more details , see online supplementary appendix 1 ) . in scps of year of death on the horizontal axis and age on the vertical axis , period effects appear as vertical patterns , age effects as horizontal patterns and cohort effects as diagonal disruptions running along diagonal lines from the bottom left to top right , with a gradient of 1 year of age per calendar year . in scps of birth year , rather than year of death on the horizontal axis cohort effects instead run vertically . scps of birth year rather than year of death were therefore used to make cohort effects easier to identify visually . for apc regression modelling , suicide and population data were grouped into 5-year age groups ( ages 1589 years for the overall population and 1584 years for the deprivation analyses ) and 5-year time periods ( from 1974 to 2013 ) stratified by sex and carstairs deprivation . the ie command ( apc_ie ) in stata v.13 ( stata corp lp , college station , texas , usa ) was used.26 as the count data were found to be overdispersed , negative binomial maximum likelihood models were used . the year for the cohorts was calculated as a function of the 5-year age and period groups , as being equal to mean period minus mean age . standard negative binomial regression models were also run to ensure that age , period or cohort alone or any combination of two factors did not fit the data better than a full apc model . age - standardised suicide rates ( see online supplementary figure s1 ) for men increased from the 1970s to around 2000 before subsequently declining , but decreased among women in the early 1980s before stabilising . rates for men have been consistently higher than those for women and the gap has widened . the age - standardised suicide rates were higher for those living in the most deprived areas over the time period ( see online supplementary figure s2 ) , with the decline among women only evident for the less deprived group . suicide rates differ markedly over the life course ( see online supplementary figures s3 and s4 ) and the age profiles have changed over the period and differ by sex . the peak rate for women has been older than that for men , indeed the peak age declined rapidly for men around 1990 as rates increased for young men and decreased for men aged over 50 years . subsequently , between 1990 and 2000 , the peak age of suicide began to increase for men , suggesting a cohort effect for those born between 1960 and 1980 ( figure 1 ) ; the most affected cohort being 19651974 where suicides peaked at about 4550/100 000 for 26 year olds ( around the 1972 cohort ) and also 10 years later at 45/100 000 for 35 year olds , having increased from around 25/100 000 and 35/100 000 , respectively , in the 1960 birth cohort . smoothed shaded contour plot of age - year specific crude suicide rates per 100 000 population in scotland for each single age from 15 to 49 years of age and each birth year from 1940 to 1998 stratified by sex . the colour and contour labels indicate the number of suicides per 100 000 for each single age and year . thick dashed guidelines indicate birth cohorts 1960 ( left most line ) , 1970 and 1980 ( right most line ) and thinner ones mid - decadal birth cohorts 1965 and 1975 . changes for women have been less noticeable than among men ( although the number of suicides are smaller ) , with the maximum rate declining and moving to younger ages , from 4070 to 2550 years by the 1990s , as the rates for older ages continued to fall . there is suggestion of a weak cohort effect for those born between 1960 and 1980 . there are differences in age profiles for men and women by carstairs deprivation and over time ( see online supplementary figures s5 and s6 ) . age differences are again more noticeable for men , especially for those aged 2060 years . the rise in rates for younger ages in the 1990s and the movement of this maximum rate to older ages with time , noted above , is very evident in the deprived group displaying a cohort effect for those born between 1960 and 1980 ( figure 2 ) , indicating that the cohort effect is substantially driven by men in the most deprived areas . it can similarly be proposed that the changes in women rates are also driven more by those in the deprived group . the timing of the peak of the cohort effect on suicide rates also differs by deprivation ; the effect occurs to a greater extent in those born between 1965 and 1969 in the most deprived group ( peak suicide rate of around 80/100 000 , occurring in 26 year olds ) and 5 years later in those born between 1970 and 1974 in the less deprived group ( peak suicide rate of around 40/100 000 , occurring in 26 year olds ) . smoothed shaded contour plot of age - year specific crude suicide rates per 100 000 population in scotland for each single age from 15 to 49 years , and each birth year from 1940 to 1998 stratified by sex and deprivation ( most deprived fifth and less deprived four - fifths ) . thick dashed guidelines indicate birth cohorts 1960 ( left most line ) , 1970 and 1980 ( right most line ) and thinner ones mid - decadal birth cohorts 1965 and 1975 . analysis of suicide rates for birth cohorts as they age ( see online supplementary figure s7 ) reflect the differences in age - specific suicide profiles for birth cohorts and also how these differ between the sexes . the peak age for suicide for both sexes has decreased from around middle age with successive cohorts . the rise in suicide at younger ages is marked for men , with maximum rates among young adults in the recent cohorts noticeably higher than those of older adults in older cohorts . in contrast , each subsequent birth cohort among women has a lower maximum suicide rate across the life course . figure 3 shows the major contribution of men in recent cohorts in the most deprived quintile to the substantial rise in suicide rates for younger men aged 2024 years overall . also apparent are the higher rates in women in recent cohorts in the most deprived quintile for ages 3044 years . crude suicide rate per 100 000 population per year in scotland by age ( 5-year age groups , for ages 1584 years ) , birth cohort ( 10-year cohorts 18951994 ) , sex and deprivation . the 19651974 cohort identified from shaded contour plots as that most affected by a cohort effect is identified in red and large dashes . regression analysis confirmed that no single - factor or two - factor combination of age , period or cohort provided a better fit than the full apc model ( see online supplementary appendix 2 ) . the ie coefficients ( see online supplementary appendix 2 for an explanation of how to interpret ie coefficients ) suggest ( figure 4 ) ( see online supplementary appendix 3 for the ie coefficient data with 95% cis ) that there is a strong age effect for both sexes , with a peak in middle age ( younger among men and larger for women ) ; a small period effect , again different for men and women , decreasing for women , but increasing for men to a peak in 19992003 before decreasing ; and a u - shaped birth cohort effect for both sexes , with a low around the 1944 cohort . a larger cohort effect is observed for men than women in the successive birth cohorts 19591974 ( see online supplementary figure s8a ) . intrinsic estimator coefficients for age , period and birth cohort effects for suicide in scotland stratified by sex . inclusion of deprivation ( figure 5 ) ( see online supplementary appendix 3 for the ie coefficient data with 95% cis ) showed overall similarities in the effect on the most deprived fifth and the rest of the population , although differences in the timing , and to a lesser extent magnitude , do exist . the age effect for instance , especially for women , tends to occur 5 years earlier for those in the most deprived population and the cohort effect since the 1960s ( see online supplementary figure s8b ) increased earlier in the most deprived population . intrinsic estimator coefficients for age , period and birth cohort effects for suicide in scotland stratified by sex and deprivation . age - standardised suicide rates ( see online supplementary figure s1 ) for men increased from the 1970s to around 2000 before subsequently declining , but decreased among women in the early 1980s before stabilising . rates for men have been consistently higher than those for women and the gap has widened . the age - standardised suicide rates were higher for those living in the most deprived areas over the time period ( see online supplementary figure s2 ) , with the decline among women only evident for the less deprived group . suicide rates differ markedly over the life course ( see online supplementary figures s3 and s4 ) and the age profiles have changed over the period and differ by sex . the peak rate for women has been older than that for men , indeed the peak age declined rapidly for men around 1990 as rates increased for young men and decreased for men aged over 50 years . subsequently , between 1990 and 2000 , the peak age of suicide began to increase for men , suggesting a cohort effect for those born between 1960 and 1980 ( figure 1 ) ; the most affected cohort being 19651974 where suicides peaked at about 4550/100 000 for 26 year olds ( around the 1972 cohort ) and also 10 years later at 45/100 000 for 35 year olds , having increased from around 25/100 000 and 35/100 000 , respectively , in the 1960 birth cohort . smoothed shaded contour plot of age - year specific crude suicide rates per 100 000 population in scotland for each single age from 15 to 49 years of age and each birth year from 1940 to 1998 stratified by sex . the colour and contour labels indicate the number of suicides per 100 000 for each single age and year . thick dashed guidelines indicate birth cohorts 1960 ( left most line ) , 1970 and 1980 ( right most line ) and thinner ones mid - decadal birth cohorts 1965 and 1975 . changes for women have been less noticeable than among men ( although the number of suicides are smaller ) , with the maximum rate declining and moving to younger ages , from 4070 to 2550 years by the 1990s , as the rates for older ages continued to fall . there is suggestion of a weak cohort effect for those born between 1960 and 1980 . there are differences in age profiles for men and women by carstairs deprivation and over time ( see online supplementary figures s5 and s6 ) . age differences are again more noticeable for men , especially for those aged 2060 years . the rise in rates for younger ages in the 1990s and the movement of this maximum rate to older ages with time , noted above , is very evident in the deprived group displaying a cohort effect for those born between 1960 and 1980 ( figure 2 ) , indicating that the cohort effect is substantially driven by men in the most deprived areas . it can similarly be proposed that the changes in women rates are also driven more by those in the deprived group . the timing of the peak of the cohort effect on suicide rates also differs by deprivation ; the effect occurs to a greater extent in those born between 1965 and 1969 in the most deprived group ( peak suicide rate of around 80/100 000 , occurring in 26 year olds ) and 5 years later in those born between 1970 and 1974 in the less deprived group ( peak suicide rate of around 40/100 000 , occurring in 26 year olds ) . smoothed shaded contour plot of age - year specific crude suicide rates per 100 000 population in scotland for each single age from 15 to 49 years , and each birth year from 1940 to 1998 stratified by sex and deprivation ( most deprived fifth and less deprived four - fifths ) . thick dashed guidelines indicate birth cohorts 1960 ( left most line ) , 1970 and 1980 ( right most line ) and thinner ones mid - decadal birth cohorts 1965 and 1975 . analysis of suicide rates for birth cohorts as they age ( see online supplementary figure s7 ) reflect the differences in age - specific suicide profiles for birth cohorts and also how these differ between the sexes . the peak age for suicide for both sexes has decreased from around middle age with successive cohorts . the rise in suicide at younger ages is marked for men , with maximum rates among young adults in the recent cohorts noticeably higher than those of older adults in older cohorts . in contrast , each subsequent birth cohort among women has a lower maximum suicide rate across the life course . figure 3 shows the major contribution of men in recent cohorts in the most deprived quintile to the substantial rise in suicide rates for younger men aged 2024 years overall . also apparent are the higher rates in women in recent cohorts in the most deprived quintile for ages 3044 years . crude suicide rate per 100 000 population per year in scotland by age ( 5-year age groups , for ages 1584 years ) , birth cohort ( 10-year cohorts 18951994 ) , sex and deprivation . the 19651974 cohort identified from shaded contour plots as that most affected by a cohort effect is identified in red and large dashes . regression analysis confirmed that no single - factor or two - factor combination of age , period or cohort provided a better fit than the full apc model ( see online supplementary appendix 2 ) . the ie coefficients ( see online supplementary appendix 2 for an explanation of how to interpret ie coefficients ) suggest ( figure 4 ) ( see online supplementary appendix 3 for the ie coefficient data with 95% cis ) that there is a strong age effect for both sexes , with a peak in middle age ( younger among men and larger for women ) ; a small period effect , again different for men and women , decreasing for women , but increasing for men to a peak in 19992003 before decreasing ; and a u - shaped birth cohort effect for both sexes , with a low around the 1944 cohort . a larger cohort effect is observed for men than women in the successive birth cohorts 19591974 ( see online supplementary figure s8a ) . intrinsic estimator coefficients for age , period and birth cohort effects for suicide in scotland stratified by sex . inclusion of deprivation ( figure 5 ) ( see online supplementary appendix 3 for the ie coefficient data with 95% cis ) showed overall similarities in the effect on the most deprived fifth and the rest of the population , although differences in the timing , and to a lesser extent magnitude , do exist . the age effect for instance , especially for women , tends to occur 5 years earlier for those in the most deprived population and the cohort effect since the 1960s ( see online supplementary figure s8b ) increased earlier in the most deprived population . intrinsic estimator coefficients for age , period and birth cohort effects for suicide in scotland stratified by sex and deprivation . between 1974 and 2013 , the suicide rate in scotland was consistently higher for men than women , with a decrease in the 1980s for women before stabilisation and an increase for men until around 2000 before subsequently declining . since 1990 , the peak age for suicide became noticeably lower for men due to a rise in rate among younger adults and a decline in older adults . this reflects a cohort effect for men ( and possibly , to a lesser extent , women ) starting around 1990 for those born between 1960 and 1980 . the cohort effect is more prominent for those living in the most deprived quintile for both sexes , where it also starts earlier in the 1960s , indicating that the change in suicide rate by age in recent years is largely driven by those in more deprived areas . this finding is consistent with a cohort effect for young adults ( and especially men living in deprived areas ) entering the changing , and increasingly neoliberal , labour market during the 1980s as this group have a higher risk of suicide for the following 30 years than the cohorts born before and after . deaths from suicide can be difficult to record and count accurately as it relies on being able to ascertain the intent of the individual who has died . as a result , we have used the uk standard approach to count both deaths which have been judged to have been due to intentional self - harm and deaths with an undetermined intent ( available from icd 8 after 1968 ) . the changes to the definitions of suicide over time are testament to the difficulties in recording these deaths accurately.2730 data are restricted to post-1974 when nrs introduced individual computer records containing sufficient details for our analyses . the data we used provided us with the year that the death was registered rather than the year in which the death occurred ( chosen to match the reporting convention of nrs ) . suicide is at greater risk of delayed registration than many other causes of death in scotland because of the requirement for a procurator fiscal report to be prepared . however , in scotland , only a few percentage of suicides are registered after the calendar year in which the death occurred , so the potential for this to interfere with the conclusions is minor.31 as our analyses have sought to break down the crude mortality rates by age over time , the number of deaths in each group is small , and there is therefore a substantial degree of random variation which makes it more difficult to be clear what the apc effects are . modelling apc effects simultaneously is problematic due to the three terms being mathematically dependent on one another , the identification problem. there is much debate about whether this is possible,3235 and several solutions have been proposed , all of which have limitations.36 37 the most common solutions involve imposing a constraint on the model such as assuming that the age effects in the first two age groups are equal . this is problematic as the parameter estimates are sensitive to the constraints chosen and often the constraints are difficult to justify empirically.38 the ie is a newer approach to the statistical estimation of apc accounting models and is a special form of the principal components regression estimator , which assumes that the sum of the coefficients in the vector are zero , thus avoiding the need to select a ( usually arbitrary ) constraint . while it too has limitations,26 our findings align with those from the descriptive analyses , especially clear in the scps , so adding weight to the emerging thesis on the timing and possible reasons behind the trends in suicide in scotland . the results do not directly test causal mechanisms but shed new light on the temporal trends and therefore represent an inductive exploration , rather than a deductive test , of the hypothesis . the trends in suicide over time and differences between men and women and between age groups have been previously described for scotland.7 8 23 the substantially higher rates of suicide among those living in deprived circumstances is also well recognised,21 30 and the greater increase and polarisation of suicide among young adults in the most deprived parts of scotland occurring between 19801982 and 19992001 has also been noted , although not identified as a cohort effect.39 of those dying from suicide in scotland , men are currently most likely to use a means of hanging / strangulation / suffocation and women self - poisoning as their chosen mode.30 suicides by hanging have increased since the 1960s and markedly since 1990 , mostly driven by men , and overtook those due to poisoning around the early 2000s , the rates for which has fallen since 1993.9 it is this change in method preference that is suggested as a possible cause for the increase in male suicide rates in the 1990s.23 indeed , this has contributed to the divergence in suicide rates for men between scotland and england and wales.7 trends in suicides due to hanging / strangulation / suffocation as a proportion of all suicides , however , do not indicate that this means of suicide is specific to the cohort effect identified and there is increasing gender parity in the use of that method over time ( the gender analysis involved the creation of ratios from ratios based on small numbers ; see online supplementary figures s9 and s10 ) . a substantial proportion of suicides , particularly those due to self - poisoning ( the second most common mode among men ) , are likely to be influenced by alcohol,40 and it is notable that alcohol sales and a range of alcohol - related harms have started to decline in recent years.10 scotland has also seen the introduction of restrictions in the availability of paracetamol ( a common drug used for self - poisoning ) through uk legislation,41 a trend away from the prescription of tricyclic antidepressants ( another common drug used for self - poisoning ) and the introduction of suicide barriers on sites known as suicide hotspots,42 all of which would be expected to exert downward pressure on suicide mortality rates.43 it could therefore be that the recent decline in suicide rates for men , and some of the earlier decline in women , can be attributed to good public health policy ( and in particular the national suicide strategy44 and success in related areas of policy ( alcohol ) . although only a small percentage of deaths in scotland each year are due to suicide ( 1.43% in 2013 ) , suicides disproportionately affect young adults ( thereby impacting substantially on life expectancy ) and are likely to have profound negative impacts on children and other family members left behind.45 suicides also represent an important contribution to the health inequalities and excess mortality in scotland.5 a motivation for this study was to investigate whether or not there is evidence of a cohort effect for those most likely to have been exposed to the negative consequences of deindustrialisation and neoliberalism during the 1980s . we have identified a cohort effect for suicide which appears for young men ( and to a lesser extent for women ) in the early 1990s that would seem consistent with this exposure and to have a lasting negative effect through to middle age . exposure appears to have started earlier for those in the most deprived areas , this would be consistent with deleterious exposure to neoliberal change occurring primarily when young adults became job seekers ; an earlier event for those in more deprived areas who tend to leave education and enter the changed labour market at an earlier age.46 additionally , having undertaken less formal education , it is likely that they also tended to face worse labour market conditions than those from less deprived areas , consistent with their higher suicide rates , with the job market being more adversely affected for those doing less skilled work , the sector of the labour market they would primarily enter.47 further work is required to examine the other causes of death that are responsible for the higher mortality , excess mortality and health inequalities in scotland for such cohort effects . what is already known on this subjectsuicide is one of the causes of death partly responsible for excess mortality in scotland ( ie , after accounting for deprivation).suicide , violence , alcohol - related and drug - related deaths , all increased in scotland during the 1980s and 1990s ( particularly for young adults ) , although some are now in decline.it has been hypothesised that exposure to neoliberalism from the 1980s may be part of the explanation for the mortality phenomena in scotland . what this study addsa cohort effect is evident for suicide for people born between 1960 and 1980 , the cohort 19651974 being most affected.this cohort effect was largely driven by men and those living in the most deprived areas , where the cohort at highest risk occurred earlier than for the rest of the population , 19651969 compared with 19701974.the recent rise and subsequent fall in suicide in scotland can be explained by a cohort effect that is consistent with exposure to neoliberal politics during the 1980s , which was experienced earlier and more profoundly by people living in more deprived areas . suicide is one of the causes of death partly responsible for excess mortality in scotland ( ie , after accounting for deprivation ) . suicide , violence , alcohol - related and drug - related deaths , all increased in scotland during the 1980s and 1990s ( particularly for young adults ) , although some are now in decline . it has been hypothesised that exposure to neoliberalism from the 1980s may be part of the explanation for the mortality phenomena in scotland . a cohort effect is evident for suicide for people born between 1960 and 1980 , the cohort 19651974 being most affected . this cohort effect was largely driven by men and those living in the most deprived areas , where the cohort at highest risk occurred earlier than for the rest of the population , 19651969 compared with 19701974 . the recent rise and subsequent fall in suicide in scotland can be explained by a cohort effect that is consistent with exposure to neoliberal politics during the 1980s , which was experienced earlier and more profoundly by people living in more deprived areas . between 1974 and 2013 , the suicide rate in scotland was consistently higher for men than women , with a decrease in the 1980s for women before stabilisation and an increase for men until around 2000 before subsequently declining . since 1990 , the peak age for suicide became noticeably lower for men due to a rise in rate among younger adults and a decline in older adults . this reflects a cohort effect for men ( and possibly , to a lesser extent , women ) starting around 1990 for those born between 1960 and 1980 . the cohort effect is more prominent for those living in the most deprived quintile for both sexes , where it also starts earlier in the 1960s , indicating that the change in suicide rate by age in recent years is largely driven by those in more deprived areas . this finding is consistent with a cohort effect for young adults ( and especially men living in deprived areas ) entering the changing , and increasingly neoliberal , labour market during the 1980s as this group have a higher risk of suicide for the following 30 years than the cohorts born before and after . deaths from suicide can be difficult to record and count accurately as it relies on being able to ascertain the intent of the individual who has died . as a result , we have used the uk standard approach to count both deaths which have been judged to have been due to intentional self - harm and deaths with an undetermined intent ( available from icd 8 after 1968 ) . the changes to the definitions of suicide over time are testament to the difficulties in recording these deaths accurately.2730 data are restricted to post-1974 when nrs introduced individual computer records containing sufficient details for our analyses . the data we used provided us with the year that the death was registered rather than the year in which the death occurred ( chosen to match the reporting convention of nrs ) . suicide is at greater risk of delayed registration than many other causes of death in scotland because of the requirement for a procurator fiscal report to be prepared . however , in scotland , only a few percentage of suicides are registered after the calendar year in which the death occurred , so the potential for this to interfere with the conclusions is minor.31 as our analyses have sought to break down the crude mortality rates by age over time , the number of deaths in each group is small , and there is therefore a substantial degree of random variation which makes it more difficult to be clear what the apc effects are . modelling apc effects simultaneously is problematic due to the three terms being mathematically dependent on one another , the identification problem. there is much debate about whether this is possible,3235 and several solutions have been proposed , all of which have limitations.36 37 the most common solutions involve imposing a constraint on the model such as assuming that the age effects in the first two age groups are equal . this is problematic as the parameter estimates are sensitive to the constraints chosen and often the constraints are difficult to justify empirically.38 the ie is a newer approach to the statistical estimation of apc accounting models and is a special form of the principal components regression estimator , which assumes that the sum of the coefficients in the vector are zero , thus avoiding the need to select a ( usually arbitrary ) constraint . while it too has limitations,26 our findings align with those from the descriptive analyses , especially clear in the scps , so adding weight to the emerging thesis on the timing and possible reasons behind the trends in suicide in scotland . the results do not directly test causal mechanisms but shed new light on the temporal trends and therefore represent an inductive exploration , rather than a deductive test , of the hypothesis . the trends in suicide over time and differences between men and women and between age groups have been previously described for scotland.7 8 23 the substantially higher rates of suicide among those living in deprived circumstances is also well recognised,21 30 and the greater increase and polarisation of suicide among young adults in the most deprived parts of scotland occurring between 19801982 and 19992001 has also been noted , although not identified as a cohort effect.39 of those dying from suicide in scotland , men are currently most likely to use a means of hanging / strangulation / suffocation and women self - poisoning as their chosen mode.30 suicides by hanging have increased since the 1960s and markedly since 1990 , mostly driven by men , and overtook those due to poisoning around the early 2000s , the rates for which has fallen since 1993.9 it is this change in method preference that is suggested as a possible cause for the increase in male suicide rates in the 1990s.23 indeed , this has contributed to the divergence in suicide rates for men between scotland and england and wales.7 trends in suicides due to hanging / strangulation / suffocation as a proportion of all suicides , however , do not indicate that this means of suicide is specific to the cohort effect identified and there is increasing gender parity in the use of that method over time ( the gender analysis involved the creation of ratios from ratios based on small numbers ; see online supplementary figures s9 and s10 ) . a substantial proportion of suicides , particularly those due to self - poisoning ( the second most common mode among men ) , are likely to be influenced by alcohol,40 and it is notable that alcohol sales and a range of alcohol - related harms have started to decline in recent years.10 scotland has also seen the introduction of restrictions in the availability of paracetamol ( a common drug used for self - poisoning ) through uk legislation,41 a trend away from the prescription of tricyclic antidepressants ( another common drug used for self - poisoning ) and the introduction of suicide barriers on sites known as suicide hotspots,42 all of which would be expected to exert downward pressure on suicide mortality rates.43 it could therefore be that the recent decline in suicide rates for men , and some of the earlier decline in women , can be attributed to good public health policy ( and in particular the national suicide strategy44 and success in related areas of policy ( alcohol ) . although only a small percentage of deaths in scotland each year are due to suicide ( 1.43% in 2013 ) , suicides disproportionately affect young adults ( thereby impacting substantially on life expectancy ) and are likely to have profound negative impacts on children and other family members left behind.45 suicides also represent an important contribution to the health inequalities and excess mortality in scotland.5 a motivation for this study was to investigate whether or not there is evidence of a cohort effect for those most likely to have been exposed to the negative consequences of deindustrialisation and neoliberalism during the 1980s . we have identified a cohort effect for suicide which appears for young men ( and to a lesser extent for women ) in the early 1990s that would seem consistent with this exposure and to have a lasting negative effect through to middle age . exposure appears to have started earlier for those in the most deprived areas , this would be consistent with deleterious exposure to neoliberal change occurring primarily when young adults became job seekers ; an earlier event for those in more deprived areas who tend to leave education and enter the changed labour market at an earlier age.46 additionally , having undertaken less formal education , it is likely that they also tended to face worse labour market conditions than those from less deprived areas , consistent with their higher suicide rates , with the job market being more adversely affected for those doing less skilled work , the sector of the labour market they would primarily enter.47 further work is required to examine the other causes of death that are responsible for the higher mortality , excess mortality and health inequalities in scotland for such cohort effects . what is already known on this subjectsuicide is one of the causes of death partly responsible for excess mortality in scotland ( ie , after accounting for deprivation).suicide , violence , alcohol - related and drug - related deaths , all increased in scotland during the 1980s and 1990s ( particularly for young adults ) , although some are now in decline.it has been hypothesised that exposure to neoliberalism from the 1980s may be part of the explanation for the mortality phenomena in scotland . what this study addsa cohort effect is evident for suicide for people born between 1960 and 1980 , the cohort 19651974 being most affected.this cohort effect was largely driven by men and those living in the most deprived areas , where the cohort at highest risk occurred earlier than for the rest of the population , 19651969 compared with 19701974.the recent rise and subsequent fall in suicide in scotland can be explained by a cohort effect that is consistent with exposure to neoliberal politics during the 1980s , which was experienced earlier and more profoundly by people living in more deprived areas . suicide is one of the causes of death partly responsible for excess mortality in scotland ( ie , after accounting for deprivation ) . suicide , violence , alcohol - related and drug - related deaths , all increased in scotland during the 1980s and 1990s ( particularly for young adults ) , although some are now in decline . it has been hypothesised that exposure to neoliberalism from the 1980s may be part of the explanation for the mortality phenomena in scotland . a cohort effect is evident for suicide for people born between 1960 and 1980 , the cohort 19651974 being most affected . this cohort effect was largely driven by men and those living in the most deprived areas , where the cohort at highest risk occurred earlier than for the rest of the population , 19651969 compared with 19701974 . the recent rise and subsequent fall in suicide in scotland can be explained by a cohort effect that is consistent with exposure to neoliberal politics during the 1980s , which was experienced earlier and more profoundly by people living in more deprived areas .

backgroundmortality rates are higher in scotland relative to england and wales , even after accounting for deprivation . this excess mortality is partly due to higher mortality from alcohol - related and drug - related deaths , violence and suicide ( particularly in young adults ) . this study investigated whether cohort effects from exposure to neoliberal politics from the 1980s might explain the recent trends in suicide in scotland.methodswe analysed suicide deaths data from 1974 to 2013 by sex and deprivation using shaded contour plots and intrinsic estimator regression modelling to identify and quantify relative age , period and cohort effects.resultssuicide was most common in young adults ( aged around 2540 years ) living in deprived areas , with a younger peak in men . the peak age for suicide fell around 1990 , especially for men for whom it dropped quickly from around 50 to 30 years . there was evidence of an increased risk of suicide for the cohort born between 1960 and 1980 , especially among men living in the most deprived areas ( of around 30% ) . the cohort at highest risk occurred earlier in the most deprived areas , 19651969 compared with 19701974.conclusionsthe risk of suicide increased in scotland for those born between 1960 and 1980 , especially for men living in the most deprived areas , which resulted in a rise in age - standardised rates for suicide among young adults during the 1990s . this is consistent with the hypothesis that exposure to neoliberal politics created a delayed negative health impact .

Introduction Methods Data sources Descriptive analysis Shaded contour plots IE models Results Age-sex-standardised trends over time Shaded contour plots Birth cohort analysis IE analysis Discussion Main results Strengths and weaknesses of the analysis Comparison with other studies Implications and conclusions

PMC4967475

the discovery of grid cells in the medial entorhinal cortex ( mec ) ( hafting et al . , 2005 ) has been a major advance in cortical physiology ( burgess 2014 ) . the assessment of single - unit activity in rats running in boxes has led to the discovery of a plethora of functional cell types in the mec : conjunctive ( head - directional ) grid cells ( sargolini et al . , 2006 ) , border cells ( solstad et al . , 2008 ) , boundary vector cells ( koenig et al . , 2011 ) , speed cells ( kropff et al . , 2015 ) , and cue cells ( kinkhabwala et al . grid and border cells also exist in areas neighboring the entorhinal cortex , such as the subiculum and pre- and parasubiculum ( lever et al . , 2009 , boccara et al . , computational models propose many different mechanisms to explain how grid cell discharges come about ( giocomo et al . , 2011 , zilli , 2012 ) . a better knowledge of the anatomy and spatio - temporal firing patterns of defined cell types is needed to constrain models and help prune the forest of different models . two aspects of the temporal firing patterns were highlighted in recent work : burstiness and theta cycle skipping . burstiness has been shown to be associated with grid cell firing ( newman and hasselmo , 2014 , latuske et al . , 2015 ) and might serve important functions in parahippocampal microcircuits ( welday et al . , 2011 , sheffield and dombeck , 2015 ) . burstiness has also been linked to differences in extracellular spike shape ( newman and hasselmo , 2014 , latuske et al . , 2015 ) . theta cycle skipping might be related to the computation of head - directional information and grid firing ( brandon et al . , 2013 ) . previous investigations of burstiness and theta cycle skipping have analyzed mixed extracellular recordings from both the superficial medial entorhinal cortex and the parasubiculum ( brandon et al . it has thus remained unclear whether burstiness and theta cycle skipping map onto anatomical categories or whether bursty and non - bursty neurons are simply intermingled ( latuske et al . , 2015 ) . stellate cells ( stel ) in layer 2 ( l2 ) of the medial entorhinal cortex show a tendency to fire bursts of action potentials upon membrane depolarization in vitro ( alonso and klink , 1993 , pastoll et al . , 2012 , such findings led to the hypothesis that stellate cells might display bursty firing patterns in vivo ( newman and hasselmo , 2014 , latuske et al . , 2015 ) . entorhinal grid cells phase - precess ; i.e. , they shift spike timing in a systematic way relative to the field potential during firing field transversals ( hafting et al . , 2008 , jeewajee et al . , 2013 , , it has been found that mec l2 cells phase - precess more strongly than mec layer 3 ( l3 ) cells ( hafting et al . , 2008 , this difference between mec layers 2 and 3 has not been seen at the single run level ; however , it may arise because mec l3 cells are less correlated between runs ( reifenstein et al . recently , a single run analysis of phase precession revealed differences between pyramidal and stellate neurons in mec l2 ( reifenstein et al . , 2016 ) . parasubicular neurons provide specific input to mec l2 pyramidal neurons ( pyr ) ( tang et al . , 2016 ) , but it is unknown whether parasubicular neurons phase - precess . here we analyze juxtacellular recordings from the medial entorhinal cortex ( ray et al . , 2014 , tang et al . , 2014a , tang et al . , 2015 ) and the parasubiculum ( tang et al . , 2016 ) . juxtacellular data offer two advantages ( pinault , 1996 , herfst et al . , 2012 ) . second , juxtacellular recording of the local field potential ( lfp ) and spikes has a very high temporal resolution and signal - to - noise ratio , which is crucial for investigating temporal patterns such as burstiness . does burstiness differ between parasubicular neurons , mec l2 pyramids , mec l2 stellates , and mec l3 neurons ? l2 of the mec contains patches of calbindin - positive pyramidal neurons arranged in a hexagonal grid ( ray et al . , 2014 ; figure 1a , top ) that are surrounded by calbindin - negative stellate cells ( figure 1a , top , black background ) . the parasubiculum ( pas ) is a thin elongated structure that wraps around the mec mediodorsally and has high wolframin expression ( wfs1-positive cells ; figure 1a , top ; tang et al . , 2016 ) . axons from the parasubiculum specifically target the patches of mec l2 pyramidal cells ( burgalossi et al . mec l3 neurons are not arranged in a hexagonal grid but are visible as a homogenous band of purkinje cell protein 4 ( pcp4)-positive cells below layer 2 ( l3 ; red band in figure 1a , bottom ) . figure 1b , left , shows reconstructions of example cells of the four neuron types : a parasubicular neuron ( blue ) , a mec l2 pyramidal neuron ( green ) , a mec l2 stellate cell ( black ) , and an mec l3 pyramidal neuron ( red ) , all recorded in freely moving rats . all reconstructions are from tangential sections ( i.e. , a top view of the morphology ) . in addition to the morphology , we also show juxtacellular recording traces from the reconstructed example cells ( figure 1b , right ) . two signals are visible in the recordings : the spikes of the identified cells and the prominent theta rhythm in the lfp . to determine whether a neuron was discharging in a bursty pattern , we analyzed the interspike interval ( isi ) histogram using a similar approach as latuske et al . isis below 60 ms were binned in 2-ms bins ( normalized to area = 1 to generate a probability distribution ) , which revealed that our dataset contained both non - bursty and bursty cells ( figure 2a ) . we performed a principal component analysis on a matrix of the isi probability distributions of all neurons and found that the first three principal components ( pcs ; figure 2b , bottom ) explained 69% of the variance in the data . in agreement with latuske ( 2015 ) , we found that , when the first two principal components were plotted against each other , the neurons formed a c - shaped structure , indicative of a bimodal distribution ( figure 2b , top ) . we assigned the neurons to two clusters using a k - means clustering algorithm on the first three principal components ( figure 2c , top ) . the two clusters were well separated with little overlap ( figure 2d ) . to assess the separation quality of the two clusters , we calculated the projection of the neurons onto fisher s linear discriminant . we can interpret the linear discriminant as a measure of burstiness because it is places the cells along an axis from non - bursty to bursty based on the shape of the isi histogram . we plotted all cells sorted according to burstiness , and , in agreement with latuske et al . ( 2015 ) , we found that bursty neurons were distinguished by a tendency to fire bursts at 125250 hz ( 4- to 8-ms bins ; figure 2d ) . to investigate differences in burstiness among cell types , we plotted the median isi histogram of all recorded cells , resolved by cell type . the median isi histograms of parasubicular as well as mec l2 pyramidal neurons indicated very bursty cells ( figure 3a , top ) . the median isi histograms of mec l2 stellate and mec l3 neurons were flat with no obvious burstiness ( figure 3a , bottom ) . to assess whether this difference was statistically significant , we performed two tests : one based on categorical classifications of cells as non - bursty and bursty with a guard zone ( experimental procedures ; latuske et al . , when we compared the proportions of non - bursty , guard - zoned , and bursty cells among neuron types , we found no significant difference between parasubicular neurons and mec l2 pyramids , which both contained predominantly bursty cells ( pas versus pyr , bursty / guard / non - bursty : 11/11/0 versus 15/15/1 , p we also found no difference between mec l2 stellate cells and mec l3 cells ( stel versus l3 , bursty / guard / non - bursty : 9/25/34 versus 3/5/24 , p > 0.05 , test ; figure 3b ) , which were both predominantly non - bursty . both parasubicular neurons and mec l2 pyramids contained significantly different proportions of bursty and non - bursty cells in comparison with both mec l2 stellates and mec l3 neurons ( all p < 0.001 , tests ; figure 3b ) . the width and placement of the guard zone is estimated from the bimodal fit , and thus the guard zone depends on the relative abundance of bursty and non - bursty cells , which is evidently not the same among neuron types ; i.e. , the guard zone might be either too wide or too narrow . the guard zone also discards information telling us whether a neuron is near the guard zone or closer to the extremes . these problems may inflate our estimated differences in burstiness among cell types . to make sure that no spurious results were imposed by the guard zone , we directly compared the burstiness of the neuron types and included all cells . in agreement with the estimations based on comparisons of the proportions , we found that the burstiness of parasubicular neurons and mec l2 pyramids was significantly higher than the burstiness in both mec l2 stellates and mec l3 neurons ( all p < 0.001 , mann - whitney u tests ; figure 3c ) . again , we did not find a significant difference between parasubicular neurons and mec l2 pyramids ( p > 0.05 , mann - whitney u test ; figure 3c ) , but we did find that mec l3 neurons had a significantly lower burstiness than mec l2 stellates ( p = 0.0036 , mann - whitney u test ; figure 3c ) . thus , parasubicular neurons and mec l2 pyramids are generally bursty , whereas mec l2 stellates and mec l3 neurons are generally non - bursty ( figures 3a and 3b ) . furthermore , within the non - bursty neuron types , mec l3 neurons are more strictly non - bursty than mec l2 stellates ( figure 3c ) . it should be noted , however , that even though there are large and highly significant differences in burstiness among cell types , the distributions of burstiness among cell types are overlapping . for example , a minority of l2 stellate cells and l3 neurons assume firing patterns that are otherwise classically parasubicular / pyramid - like . our dataset includes mec l2 neurons that were classified as putatively pyramidal or stellate based on theta strength and preferred theta phase ( tang et al . we therefore also checked whether there was any correlation between burstiness and theta strength because such a correlation might introduce we fitted three generalized linear models to investigate whether burstiness might be related to theta strength ( model 1 , burstinessstrength ; figure s3a , left ) , putative cell type ( model 2 , burstinesstype ; figure s3a , middle ) , or both ( model 3 , burstinesstype + strength ; figure s3a , right ) . both comparisons of the akaike information criterion ( aic ; akaike , 1974 ) and likelihood ratio tests of nested models indicated that model 2 is superior to the other models ( figure s3b ) ; i.e. , the burstiness depends only on putative cell type ( model 2 , ptype = 0.0000076 ; figure s3c , middle ) and not on theta strength ( model 2 versus model 3 , p = 0.54 , likelihood ratio test ; figure s3b ) . second , we plotted the burstiness among cell types twice : once where we include the classified mec l2 cells ( figure s3d , left ) and once where we only include identified mec l2 cells ( figure s3d , right ) . the pattern of burstiness among cell types remained the same when we only included the identified cells ( figure s3d ) . we thus conclude that cell type - specific differences in burstiness are not an artifact of our classification approach . in tetrode recordings of parasubicular and mec l2/3 neurons , differences in spike shape have been linked to burstiness ( latuske et al . , 2015 ) and theta phase preference of grid cells ( newman and hasselmo , 2014 ) . we therefore investigated whether there was a difference in spike shape among our four anatomical categories of neurons . first we removed a subset of cells for which the signal - to - noise ratio of spike waveforms was insufficient to reliably assess the spike shape . second , we removed spikes that happened within 100 ms of the previous spike to disregard potential effects of spike shape adaptation during bursts ( experimental procedures ) . in figure 4a , we plot the remaining spike shapes ( normalized for display ; experimental procedures ) for all four neuron types . we did not find any differences among neuron types in spike amplitude , peak - to - trough ratio , or spike half - width ( all p > 0.05 , kruskal - wallis test ) . this was expected for two reasons : overall spike amplitude depends strongly on the particular recording pipette and relation to the soma ( gold et al . , 2009 ) , and narrow spikes and a small peak - to - trough ratio are indicative of interneurons ( mountcastle et al . we noticed , however , a large variability in the repolarization phase of the cell type : parasubicular neurons and mec l2 pyramids contained many cells that quickly reached the trough and repolarized , whereas mec l2 stellates and mec l3 neurons reached the trough more slowly ( figure 4a ) . this tendency was also evident in the mean spike shape of the four neuron types ( figure 4b ) . when we compared the peak - to - trough time of the cell types , we found significant differences ( p = 0.0014 , kruskal - wallis test ) . parasubicular neurons and mec l2 pyramids had significantly shorter peak - to - trough times than both mec l2 stellates and mec l3 neurons ( all p < 0.05 , mann - whitney u test ; figure 4c ) . because parasubicular neurons and mec l2 pyramids have faster peak - to - trough times and are also the most bursty cell types , we wondered whether , as has been suggested ( latuske et al . , 2015 ) , the burstiness of the cell predicts the spike shape . alternatively , the spike shape might simply be different among neuron types , or it might depend on neuron types as well as burstiness . to figure this out , we decided to employ a generalized linear regression approach . because peak - to - trough time can not assume negative values , we modeled peak - to - trough time as a gamma - distributed variable ( experimental procedures ) . we selected the appropriate model using the following approach : we first modeled peak - to - trough time as a function of only burstiness ( glm1 , peak - to - trough 1 + burstiness ) and found a significant dependence ( anova , pburstiness = 0.0087 ; figure s4a , dashed gray line ) . ( 2015 ) . also , when we modeled peak - to - trough time as a function of only neuron type ( glm2 , peak - to - trough 1 + type ) , we also found a significant dependence on neuron type ( anova , ptype = 0.0015 ; figure s4a , solid lines ) . however , when we modeled peak - to - trough time as a function of both burstiness and neuron type ( glm3 , peak - to - trough 1 + burstiness + type ) , we found that the dependency on type but not the dependency on burstiness remained significant ( anova , pburstiness = 0.22 , ptype = 0.017 ; figure s4c ) . we also fitted a model where we allowed for interactions between burstiness and type ( glm4 , peak - to - trough 1 + burstiness + type + burstinesstype ) , where all effects became non - significant ( anova , all p > 0.05 ; figure s4c ) . to determine which model best explains the data , we calculated the aic of all models and found that , despite the four fitted parameters , glm2 had the lowest aic , indicating that the peak - to - trough time depends on neuron type , but not on burstiness ( figure s4b ) . similarly , when comparing nested models , we found that glm3 better explains the data than glm1 ( p = 0.0023 , likelihood ratio test ; figure s4b ) ; i.e. , including neuron type as a predictor makes the model better . we did not find that glm3 explains the data better than glm2 ( p = 0.32 , likelihood ratio test ; figure s4b ) ; i.e. , it is unnecessary to include burstiness as a predictor in addition to neuron type . we thus infer that the differences in spike shape primarily reflect the anatomical type and not the burstiness of the neuron . to determine whether a neuron was theta cycle - skipping , we used a maximum likelihood estimation ( mle ) of a parametric model of the isi histogram ( climer et al . , 2015 ; experimental procedures ) . our dataset contained neurons that showed no theta modulation and also neurons that had strong rhythmic components ( figure 5a ) . for every cell , we fitted three models to the isi distribution : a flat model with no rhythmic components ( figure 5a , left ) , a rhythmic , non - skipping model with a theta - rhythmic modulation of the isi histogram ( figure 5a , middle ) , and a rhythmic , cycle - skipping model with a theta - rhythmic modulation of the isi histogram and a second parameter introducing theta cycle skipping ( i.e. , a higher amplitude of every other peak in the isi histogram ; figure 5a , right ) . the three fitted models were compared using the appropriate statistic ( calculated from the maximum log likelihood of the models ) to generate two p values : prhythmic ( comparing the flat and the rhythmic , non - skipping models ) and pskipping ( comparing the rhythmic , non - skipping and the rhythmic , cycle - skipping models ) . the cells were classified using a two - level classification ( figure 5b ) : first , we determined whether a cell was rhythmic ( prhythmic < 0.05 ) or non - rhythmic ( prhythmic > 0.05 ) . then we classified the rhythmic cells as either rhythmic , cycle - skipping ( pskipping < 0.05 ) or rhythmic , non - skipping ( pskipping > 0.05 ) . using the mle approach , we found that parasubicular neurons and mec l2 pyramids were overwhelmingly rhythmic ( 93% ; pas , 20/22 ; pyr , 29/31 ; figure 5c , left ) . mec l2 stellates and mec l3 neurons were rarely rhythmic ( 26% ; stel , 16/68 ; l3 , 9/32 ) , both significantly less rhythmic than both parasubicular neurons and mec l2 pyramids ( all p < 0.001 , tests ; figure 5c , left ) . this is in agreement with previous observations in which evaluated spike train rhythmicity of cell types using a theta index was used ( ray et al . , 2014 , tang et al . we found that the generally rhythmic cell types were also significantly more likely to also be theta cycle - skipping than the generally non - rhythmic cell types ( p = 0.018 , mixed - effects logistic regression ; figure 5c , right ; experimental procedures ) : approximately 49% of the rhythmic parasubicular neurons and rhythmic mec l2 pyramids were also theta cycle - skipping ( pas , 9/20 ; pyr , 15/29 ; figure 5c , right ) . of the mec l2 stellates and mec l3 neurons , which were classified as rhythmic using the mle approach , only 20% were also theta cycle - skipping ( stel , 4/16 ; l3 , 1/9 ; figure 5c , right ) . our dataset includes mec l2 neurons that were classified as putatively pyramidal or stellate based on theta strength and preferred theta phase ( tang et al . obviously , we expect a correlation between the theta rhythmicity ( which is calculated from the isi distribution ) and the theta strength ( locking to the lfp theta rhythm ) . however , the mle approach of climer et al . ( 2015 ) returns a p value of the rhythmicity per cell and is sensitive to very low amounts of rhythmicity , which could potentially have been present in , e.g. , putative stellates with a low locking strength and locking to the peak of the lfp theta rhythm ( figure s1 ; climer et al . , 2015 , tang et al . , 2014a ) . more importantly , our classification procedure considers simply strength of locking to the local lfp , and there is no way of distinguishing a simply theta - rhythmic cell from a rhythmic and cycle - skipping cell based on theta strength because they might show equally strong locking . to be sure that the cell type differences were not an artifact of including the classified cells , we plotted the burstiness among cell types twice : once where we included the classified mec l2 cells ( figure s5a , left ) and once where we only included identified mec l2 cells ( figure s5a , right ) . the proportions among cell types remained the same when restricting the analysis to identified cells only ( figure s5a ) . to compare the magnitude of phase precession among cell types at the single - run level , we first selected single runs of high firing based on the firing rate ( figures 6a , top , and 6b ; experimental procedures ) . from these single runs , we determined the slope and range of phase precession by a circular - linear fit of time and theta phase angle of the spikes in each run ( figure 6a , bottom ; experimental procedures ) . figure 6c shows example single runs from example cells of the four neuron types . the example mec l2 stellate and l3 neurons have steep phase precession slopes and cover larger ranges of theta phase angles during a single run . in contrast , the example parasubicular neuron and mec l2 pyramid only weakly phase - precess . across the population , we found the same result : first , identified and putative mec l2 stellate and l3 neurons had approximately 3-fold steeper phase precession slopes than parasubicular neurons and identified and putative mec l2 pyramids ( figure 6d ; median slopes : pas / pyr / stel / l3 = 16.7/25.9/76.7/64.8 degrees / s ; p(pas versus stel ) = 0.0001 ; p(pas versus l3 ) = 0.003 ; p(pyr versus stel ) = 0.0001 ; p(pyr versus l3 ) = 0.01 ; mann - whitney u tests ) . second , identified and putative mec l2 stellate and l3 neurons covered a much larger range of theta phase angles per run than parasubicular neurons and identified and putative mec l2 pyramids ( approximately 2-fold ; figure 6e ; median ranges : pas / pyr / stel / l3 = 63.2/48.7/127.5/114.2 degrees ; p(pas versus stel ) = 0.00008 ; p(pas versus l3 ) = 0.0007 ; p(pyr versus stel ) = 0.0000002 ; p(pyr versus l3 ) = 0.00005 ; mann - whitney u tests ) . we did not find any differences in the circular - linear correlation coefficient among the cell types ( p = 0.38 , kruskal - wallis test ) . we used advanced statistical techniques to tease apart how differences in burstiness , spike shape , theta modulation ( rhythmicity , locking , skipping ) , and phase precession map onto regular spiking layer 3 medial entorhinal neurons , layer 2 medial entorhinal pyramidal neurons , layer 2 medial entorhinal stellate neurons , and parasubicular regular spiking cells . we found significant differences in spike shape , burstiness , theta modulation ( rhythmicity , locking , cycle skipping , phase precession ) , and theta phase precession between the four groups of cells investigated . thus , our data suggest that cell type is a major determinant of discharge patterns in the rat parasubiculum and superficial medial entorhinal cortex . although our data emphasize the significance of cell types , the discharge patterns we observed do not directly match what is expected based on the analysis of intrinsic properties of these neurons in vitro . in vitro recordings of parasubicular neurons have suggested an intrinsic disposition for theta rhythmicity ( glasgow and chapman , 2008 ) . it is known that in vitro measurements of l2 mec cell properties are very sensitive to recording conditions ( alonso and klink , 1993 , pastoll et al . , 2012 ) . however , mec l2 stellates often display some intrinsic burstiness in vitro ( alonso and klink , 1993 , pastoll et al . , 2012 , alessi et al . , 2016 , fuchs et al . , 2016 ) , but they are generally not very bursty in vivo ( ray et al . , 2014 ; figure 2 ) . thus , it is probably incorrect to assume that bursty cells recorded extracellularly in the superficial mec and the parasubiculum are mec l2 stellates ( newman and hasselmo , 2014 , latuske et al . , 2015 ) because we show that bursty cells are more likely to be mec l2 pyramids or parasubicular neurons . although phase precession is arguably the most intensely studied example of temporal coding in the brain , its underlying mechanism is still a matter of debate . parasubicular neurons , which show only weak phase precession , project to pyramidal cells in mec l2 ( tang et al . , 2016 ) . also , these mec l2 pyramidal cells express only a low degree of phase precession . conversely , stellate cells in mec l2 and pyramidal cells in mec l3 phase precess with steep slopes . the latter finding is somewhat surprising because it challenges the long - held belief that cells in mec l3 do not phase - precess ( hafting et al . , 2008 , in contrast to that , we analyzed phase precession in single runs ( schmidt et al . , 2009 ) . we argue that the single - run approach is more appropriate because the animal needs to process information online and does not have the opportunity to pool over trials . our finding of substantial mec l3 phase precession is in line with a previous single - run account ( reifenstein et al . , 2014 ) . mec l2 stellate cells project to the dentate gyrus , whereas mec l2 pyramidal cells send output to ca1 ( varga et al . , 2010 , kitamura et al . , 2014 , ray et al . , 2014 ) . because mec l2 pyramidal cells show only weak phase precession , it seems unlikely that they substantially contribute to ca1 phase precession . therefore , ca1 either generates phase precession de novo or inherits phase - precessing inputs via the strongly precessing stellate cells in mec l3 ( jaramillo et al . , 2014 ) . it is presently unknown how the functional categories ( grid cells , border cells , speed cells , cue cells , etc . ) for example , it is unknown whether mec l2 grid cells are predominantly pyramidal cells ( tang et al . , 2014a ) or stellate cells ( domnisoru et al . , 2013 ) or whether they show no preference for either cell type ( sun et al . , 2015 ) similarly , some authors have reported that about a third to half of mec l3 neurons are grid cells ( sargolini et al . , 2006 , boccara et al . , 2010 ) , whereas others have estimated that if l3 grid cells exists , then they must be rare ( 1% ; tang et al . parasubicular neurons and mec l2 pyramids are more bursty , have narrower spikes , and are more likely to be theta - rhythmic , theta - locked , and theta cycle - skipping than mec l2 stellates and mec l3 neurons . these differences remain even when we statistically control for interactions between spike shape , burstiness , and rhythmicity . some studies have tried to elucidate the grid cell generation mechanism by characterizing the firing properties of the entorhinal network . from these studies we know that grid cells are bursty whereas border cells are not ( newman and hasselmo , 2014 , latuske et al . , it has also been shown that theta cycle skipping is somehow necessary for maintaining grid cell firing ( brandon et al . , 2013 ) . in agreement with tang et al . , 2014a , tang et al . , 2016 , we conclude that , based on burstiness and theta cycle skipping , parasubicular neurons and mec l2 pyramids are likely to play a key role in generating grid cell activity in the parasubiculum and superficial medial entorhinal cortex . in line with the differences in temporal discharge patterns , we observed that parasubicular and mec l2 pyramidal cells had shorter spike durations than mec l2 stellates and mec l3 neurons . several previous studies have noticed significant differences between mec l2 pyramidal and mec l2 stellate cells , most notably , that stellate cells have larger depolarizing afterpotentials ( alonso and klink , 1993 , alessi et al . in in vivo recordings , it was generally observed that stellate cells had a shorter spike duration than pyramidal cells ( alonso and klink , 1993 ) . interestingly , however , it was also found that the spike duration of both pyramidal and stellate cells varied depending on the depolarizing current pulse ( alonso and klink , 1993 ) . thus , the juxtacellularly observed differences in spike shape are probably not primarily a reflection of differences in intrinsic cell properties . however , the distributions of spike durations are largely overlapping ( figure 4c ) , probably precluding a classification of extracellularly recorded mec l2 regular spiking neurons into pyramidal and stellate cells based purely on spike shape . we observed a strong similarity between spike shapes and firing patterns of parasubicular neurons and mec l2 pyramids . these two neuron groups were different in spike shapes and firing patterns from layer 3 cells and layer 2 stellate cells , which were similar to each other , however . parasubicular axons specifically target patches of mec l2 pyramidal cells ( tang et al . , 2016 ) , which might be a pathway for head - directional information from the medial septum to reach the grid cell system ( winter et al . , 2015 , unal et al . , 2015 , tang et al . , l3 cells and layer 2 stellate cells provide a massive direct ( l3 ) and indirect input to the hippocampus , whereas projections from both layer 2 pyramids and the parasubiculum are minor or absent ( varga et al . , 2010 , thus , analysis of spike shapes and firing patterns , direct connectivity , and projection targets supports the distinction of layer 3 cells and layer 2 stellate cells on one hand and parasubiculum and layer 2 pyramids on the other hand as two distinct processing systems . they both express wolframin ( ray and brecht , 2016 ) , and , in the early development stages , they also express calbindin ( ray and brecht , 2016 ) . likewise , layer 3 neurons and layer 2 stellate cells also have an anatomical likeliness in their protein expression profile , with both expressing reelin in adult rats ( ray and brecht , 2016 ) . this might allude to the electrophysiological and functional characteristics of these two groups being perhaps somewhat genetically determined , with the protein expression profiles of these respective cell groups shaping their inputs and outputs . our results will constrain future modeling of network activity in the hippocampus and para - hippocampal cortices . because different anatomical cell types have different projection patterns , burstiness , and theta rhythmicity / skipping might be passed on differentially to hippocampal subfields like the dentate gyrus , which receives massive mec l2 stellate input ( varga et al . , 2010 ) , and ca1 , which receives some mec l2 pyramidal input ( kitamura et al . , 2014 ) . some grid cell models suggest that grid cells are generated by network mechanisms where a large number of similar ( stellate ) cells self - organize to generate symmetrical firing patterns either via continuous attractors or via oscillatory interference ( for reviews , see giocomo et al . , 2011 , others have suggested mechanisms based on anatomical microcircuits ( brecht et al . , 2013 ) . our results do not resolve this question , but we add to the picture that the network mechanism distributes firing patterns differentially according to cell type . we conclude that the anatomical identity of the neuron is a strong determinant of the firing pattern . analysis of burstiness , theta cycle skipping , and phase precession jointly suggest similarities between layer 3 cells and layer 2 stellate cells on one hand and layer 2 pyramidal cells and parasubicular cells on the other hand . all experimental procedures were performed according to the german guidelines on animal welfare under the supervision of local ethics committees . in this paper , we analyzed a dataset of juxtacellular recordings from the superficial medial entorhinal cortex and the parasubiculum that we have published previously ( ray et al . 2014a , tang et al . , 2015 , tang et al . , detailed descriptions of recording procedures ( pinault , 1996 , lee et al . , 2006 , herfst et al . , 2012 , tang et al . , 2014b ) , quality control ( joshua et al . , 2007 ) , tissue preparation , immunohistochemistry , and image acquisition ( naumann et al . , 2016 , ray and brecht , 2016 ) in addition to labeled cells , we included a number of unlabeled , regularly spiking cells from mec l2 in our analysis . these cells were assigned as either putatively calbindin - positive ( pcb+ ) pyramidal cells or putatively calbindin - negative ( pcb ) stellate cells based on their theta strength and preferred theta phase angle using the classification approach of tang et al . ( 2014a ) ; i.e. , based on the theta strength and preferred theta phase angle of spiking activity . as in tang et al . ( 2014a ) , we used a 0.1 guard zone and found that the cells were well separated with no cells in the guard zone ( figure s1 ) . in the manuscript , we refer to the pooled groups of identified and putative calbindin - positive pyramidal cells simply as when we show example cells of the four cell types ( figures 1a and 1b and 6a6c ) , we show only identified cb+/ cells . in figures s3 and s5 , we show analysis of a dataset where we only included identified cells . to determine whether a neuron was discharging in a bursty pattern , we analyzed the isi histogram using a similar approach as latuske et al . isis below 60 ms were binned in 2-ms bins and normalized to area = 1 to generate a probability distribution ( figure 2a ) . a principle component analysis ( pca ) was done on a matrix of the isi probability distribution of all neurons ( pca in matlab , mathworks ) . for plotting , the density of cells in this space was estimated with a 2d gaussian kernel density estimator ( kde2d ; botev et al . , 2010 ) . the neurons were assigned to two clusters using a k - means clustering algorithm on the first three principal components ( kmeans ; matlab ; figure 2c , top ) . to assess the separation quality of the two clusters , we calculated the projection of the neurons onto fisher s linear discriminant ( the burstiness , using lda from scikit - learn in python ) and found that the two clusters ( non - bursty and bursty ) were well separated with little overlap ( figure 2c , top ) . to check whether the distribution of burstiness was bimodal , thus reflecting two distinct classes of isi histograms , we fitted probability density functions for gaussian mixture models with between one and three underlying gaussians and compared the models using the akaike information criterion ( akaike 1974 ; aic from gmdistribution.fit in matlab ) . a bimodal distribution best explained the data ( aicunimodal = 622.7 , aicbimodal = 609.6 , aictrimodal = 614.7 ) . based on the mean and variance of the two gaussian distributions underlying the observed distribution of burstiness ( figure 2c , bottom , dashed red lines ) , we estimated that excluding cells where 0.4 < burstiness < 1.5 would yield > 95% correct labeling of non - bursty and bursty neurons in the non - bursty and bursty categories and used this as a guard zone ( latuske et al . , 2015 ) . during recording , the juxtacellular traces were digitized at 20 khz . to analyze the spike shapes , we first zero - phased high pass - filtered the raw signal at 100 hz with a finite impulse response filter of order 2 ( fir1 in matlab ) . the spike times were detected by thresholding the filtered signal and saving each threshold crossing 2.5 ms . spike sorting based on the first principal components was performed on these 5-ms snippets to remove any threshold crossings because of artifacts in the signal ( tang et al . , 2014a ) . to align the spike shapes optimally after spike sorting , the 5-ms snippets were oversampled at five times their original sampling rate using a spline interpolation ( interp1 in matlab ) and were then aligned to the peak sample . to ensure that we were only analyzing shapes free of distortions because of drift of the pipette and that the spikes were well above the noise floor , we only analyzed spikes for which the spike amplitude was in the top 60 - 90 percentile and where the z score of the spike amplitude was > 17 . the noise floor was defined as the mean of the first and last 0.5 ms of each 5-ms spike snippet . we also removed any spikes where there was another spike in the preceding 100 ms . in the four cell groups , there were only a few cells where the spikes did not have sufficient quality to analyze the spike shape , and we could analyze 19/22 parasubicular cells , 24/31 mec l2 pyramidal cells , 58/68 mec l2 stellate cells , and 27/32 mec l3 cells . we calculated the mean spike shape of every cell and determined the spike features from these traces . for plotting the comparison between cells and for illustrating the differences in peak - to - trough time ( figures 4a and 4b ) , we normalized the spike shape by subtracting the noise floor , dividing the mean spike by the peak - to - trough height , and setting the peak height to 1 . to determine whether a neuron was rhythmic and theta cycle - skipping , we used an mle of a parametric model of the isi histogram ( mle_rhythmicity ; climer et al . , 2015 ) for every cell , we fitted three models to the isi distribution : a flat model with no rhythmic components , a rhythmic , non - skipping model with a rhythmic modulation of the isi histogram , and a rhythmic , cycle - skipping model with a rhythmic modulation of the isi histogram and a second parameter introducing theta cycle skipping ( i.e. , a higher amplitude of every other peak in the isi histogram ) . when fitting the models , we searched for a rhythmic component with a theta frequency between 5 and 13 hz and for cycle skippings > 0.01 . the three fitted models were compared using the appropriate statistic ( calculated from the maximum log likelihood of the models ) to generate two p values : prhythmic ( comparing the flat and the rhythmic , non - skipping models ) and pskipping ( comparing the rhythmic , non - skipping and the rhythmic , cycle - skipping models ) . the cells were classified using a two - level classification ( figure 5b ) . first we determined whether a cell was rhythmic ( prhythmic < 0.05 ) or non - rhythmic ( prhythmic > 0.05 ) . then we classified the rhythmic cells as either rhythmic , cycle - skipping ( pskipping < 0.05 ) or rhythmic , non - skipping ( pskipping > 0.05 ) . to statistically assess whether theta cycle skipping cells were rarer among rhythmic cells in the generally non - rhythmic cell types ( mec l2 stellates and mec l3 neurons ) than in the generally rhythmic cell types ( parasubicular neurons and mec l2 pyramids ) , we fitted a mixed - effects logistic regression . we constructed a vector , isgenrhytm ( which takes the value 1 for parasubicular neurons and mec l2 pyramids and the value 0 for mec l2 stellates and mec l3 neurons ) . we also constructed a vector type that simply dummy - coded the four neuron types from 1 , 2 , 3 , and 4 . we dummy - coded when the neuron was theta cycle - skipping in the vector isskipping . we then modeled the probability of being rhythmic as a function of being generally rhythmic while controlling for the different number of cells in the four categories of neurons : isskippingisgenrhytm + ( 1|type ) using fitglme in matlab ( aarts et al . , in addition to the mle approach , we also calculated the theta strength and preferred theta phase of every cell . the local field potential was bandpass - filtered in the theta range ( 412 hz ) , and a hilbert transform was used to determine the instantaneous phase of the theta wave for every spike . the theta locking strength and the preferred phase angle were calculated as the modulus and argument of the rayleigh average vector of the theta phase at all spike times . statistical modeling ( generalized linear models ) was done in matlab using the glmefit function . we modeled burstiness as a function of theta strength as a normally distributed variable ( figures s3a s3c ) . we modeled the peak - to - trough time as a gamma - distributed variable with a reciprocal link function in matlab because it can only assume positive values ( figures s4a s4c ) . to compare models , we either calculated and compared the aic ( akaike , 1974 ) or , in the case of nested models , calculated the p value from likelihood ratio tests . in the manuscript , we describe all statistical models using standard wilkinson notation ( wilkinson and rogers , 1973 ) . to identify coherent periods of elevated firing ( single runs ) , we follow a previously applied strategy based on the temporal structure of the recorded spike trains ( reifenstein et al . , 2016 ) . briefly , we convolved the spike train with a gaussian kernel to estimate the instantaneous firing rate . we then used a firing rate threshold to locate periods of elevated firing ( figure 6a , top ) . for each of the single runs , we quantified phase precession by calculating the slope , phase range , and circular - linear correlation coefficient of the circular - linear regression line ( figure 6a , bottom ; kempter et al . , 2012 , c.l.e . analyzed burstiness , spike shape , theta rhythmicity , and cycle skipping . e.t.r . , r.k . , and m.b . provided expertise and feedback on the analysis and supervised the project .

summarythe medial entorhinal cortex ( mec ) and the adjacent parasubiculum are known for their elaborate spatial discharges ( grid cells , border cells , etc . ) and the precessing of spikes relative to the local field potential . we know little , however , about how spatio - temporal firing patterns map onto cell types . we find that cell type is a major determinant of spatio - temporal discharge properties . parasubicular neurons and mec layer 2 ( l2 ) pyramids have shorter spikes , discharge spikes in bursts , and are theta - modulated ( rhythmic , locking , skipping ) , but spikes phase - precess only weakly . mec l2 stellates and layer 3 ( l3 ) neurons have longer spikes , do not discharge in bursts , and are weakly theta - modulated ( non - rhythmic , weakly locking , rarely skipping ) , but spikes steeply phase - precess . the similarities between mec l3 neurons and mec l2 stellates on one hand and parasubicular neurons and mec l2 pyramids on the other hand suggest two distinct streams of temporal coding in the parahippocampal cortex .

Introduction Results Discussion Experimental Procedures Author Contributions

PMC3502318

let us imagine a population with n individuals where we recruit 1 control per case into a cc study and follow up the coca and the coco . the exposure ( e ) and outcome ( o ) are distributed as shown in figure 1 . figure 1.venn diagram showing the distribution of 2400 cases and 2400 controls in relation to an exposure and an outcome in a population of 100 000 individuals . venn diagram showing the distribution of 2400 cases and 2400 controls in relation to an exposure and an outcome in a population of 100 000 individuals . our incident cases are recruited into this ccf study within a short time window after the onset of a case - defining event ( d ) ; those who do not develop d within that time window are noncases ( nc ) . the population was generated using functions ( given in the supplementary appendix ) that describe how e influences d , and how e and d separately and in combination , influence o ( figure 2 ) . figure 2.schematic presentation of associations between an exposure ( e ) , a case disease ( d ) , and an outcome ( o ) in a population , where arrows indicate the direction of causality . schematic presentation of associations between an exposure ( e ) , a case disease ( d ) , and an outcome ( o ) in a population , where arrows indicate the direction of causality . because our study recruits an equal number of cases and controls , n , the number of nc in the population is because n of all nc are recruited into the coco , the sampling fraction is calculated as n/(n - n ) , and the sampling weight as ( n - n)/n . in our example , where n = 2400 and n = 100 000 , the sampling fraction is 2400/(100 0002400 ) = 0.02459 , the corresponding sampling weight ( 100 0002400)/2400 = 40.67 . the relative risk ( rr ) of experiencing o given e for the whole population , in the coca , among the nc , and in the coco is shown in table 1 , rows a d . the slight difference in rr between nc ( table 1 , row c ) and the coco ( table 1 , row d ) is an artifact of rounding . table 1.two-by-two tables showing distributions of exposure ( e ) and outcome ( o ) or disease defining case status ( d ) as a basis for the conceptual framework of the reconstructed population methodo + totalriskrr95% ciasource populatione+70493050000.0142.650894 49295 0000.005bcocae+254755000.0502.243185719000.023cnce+45445545000.0102.046592 63593 1000.005dcocoe+11101110.0091.911227822890.005d + ( odds)(or)ecc studye+5001114.5055.4190022890.83o + fcocae+254755000.0502.21.43.643185719000.023gcocoe+11101110.0091.9.2414.411227822890.005hcoca + cocoe+265856110.0433.32.15.254413541890.013abbreviations : cc , case / control ; ci , confidence interval ; coca , cohort of cases ; coco , cohort of controls ; d , case - defining illness ; nc , noncases ; or , odds ratio ; rr , relative risk . two - by - two tables showing distributions of exposure ( e ) and outcome ( o ) or disease defining case status ( d ) as a basis for the conceptual framework of the reconstructed population method abbreviations : cc , case / control ; ci , confidence interval ; coca , cohort of cases ; coco , cohort of controls ; d , case - defining illness ; nc , noncases ; or , odds ratio ; rr , relative risk . the odds ratio ( or ) describing the association between e and getting d ( ie , becoming a case ) can be calculated from table 1 , row e ( derived from table 1 , rows b and d ) , which distributes e among the cases ( d ) and the controls ( d ) . we now make a shift to the real world of epidemiology where only the ccf study represented by the coca ( table 1 , row b ) and the coco ( table 1 , row d ) is known . when analyzing the ccf study with the rpm , the exposure - outcome distributions in the coca and the coco should be identical but because they represent samples of our population , estimated associations should be provided with confidence intervals ( cis ) ( table 1 , rows f and g ) . as an estimate for the association between e and o in the source population , it may seem tempting to ignore the cc sampling scheme and simply calculate rr on the combined data of the 4800 individuals in coca and coco ( table 1 , row h ) . this corresponds to what jiang et al lists as the first ad hoc approach to secondary analysis of cc data . however , this approach assumes that d is conditionally independent of o given e , ie , when none of the effect of e on o is mediated by d. when getting d , on the other hand , does change the risk of o , this approach yields an unbiased estimate of the association between e and o only when the ratio of cases to nc in the source population is 1:1 , ie , when d risk is 50% . in many situations , including in the global enteric multicenter study ( gems ) [ 9 , 10 ] , not only may e increase the incidence risk of d , this risk is usually much lower than 50% , and such an approach would accordingly overestimate the strength of the association between e and o ( table 1 , row h ) . another approach , which is suggested by nagelkerke et al , is to base the estimates only on the 2400 coco individuals ( table 1 , row g ) . jiang et al argues that this , in what they call the second ad hoc approach , may be approximately valid when d is rare , but emphasizes , just as do reilly et al , that it is inefficient because it discards the case data . if there is an interaction between e and d on o , ie , when the association between e and o differs between coca and coco individuals , the bias may be substantial and even more unpredictable . a third approach is to calculate rrs for the coca and for the coco , and , if there is no interaction between e and d on o , report the average of the 2 rrs using mantel - haenszel stratified analysis . this corresponds to jiang et al 's third ad hoc approach where the combined analysis of coca and coco individuals is adjusted for d . this approach , which gives an rr estimate of 2.2 ( 95% ci , 1.43.5 ) in our example , not only disregards the fact that cases are oversampled ( see table 1 , row h and the first ad hoc approach ) but also de facto removes the effect of e on o that operates through , ie , is mediated by , d. to use ccf data to estimate the association between e and o in a given population , we need to perform the analysis on the population reconstructed from the coca plus the nc . the sampling fraction needed to estimate nc can not be calculated directly , but must be derived from an independent source of d incidence risk . thus , if r is the incidence risk of d in the time window during which cases are recruited , and because we assume equal numbers of cases and controls , the sampling fraction of controls is proportional to the corresponding incidence odds , ie , r/(1 r ) . if we assign a weight of 1 to the cases , the sampling weight of the controls is its reciprocal , ( 1 r)/r . in our example , let us assume that the d incidence risk , derived from a perfectly representative survey in the population , is 0.024 . to reconstruct the population 's nc , we multiply the number of individuals in the coco with the reciprocal of its corresponding incidence odds , the sampling weight , ie , 40.67 , to obtain the reconstructed number of exposed and unexposed noncases ( rnc ) ( table 2 , row a ) . we can then estimate the association between e and o in our reconstructed population consisting of the coca plus the rnc ( table 2 , row b ) . table 2.reconstructing the populationo + totalriskrrarnce+1 40.67 = 40.67110 40.67 = 4473.3345140.0131.911 40.67 = 447.332278 40.67 = 92 639.6793 0860.005breconstructed populatione+40.67 + 25 = 65.674473.33 + 475 = 4948.3350140.0132.5447.33 + 43 = 490.3392639.67 + 1857 = 94 496.6794 9860.005two - by - two tables showing distributions of exposure , outcome , and disease that defines case status in the reconstructed noncases and in the reconstructed source population.abbreviations : d , case - defining illness ; e , exposure ; o , outcome ; rnc , reconstructed noncases ; rr , relative risk . rnc + cohort of cases . reconstructing the population two - by - two tables showing distributions of exposure , outcome , and disease that defines case status in the reconstructed noncases and in the reconstructed source population . abbreviations : d , case - defining illness ; e , exposure ; o , outcome ; rnc , reconstructed noncases ; rr , relative risk . the difference in cell numbers between the imagined ( table 1 , row a ) and this reconstructed population is an artifact of the rounding we undertook to generate the coco ( table 1 , row d ) . we do not include a 95% ci for this rr estimate because the sampling error should be derived from the coca and coco , not from the reconstructed population . to explain the difference between the rr in the combined coca and the coco ( table 1 , row h ) and that in the reconstructed population ( table 2 , row b ) , and to provide a transition into regression analysis of such data , figure 3 illustrates how the weighting of the data influences the estimated effect of e on o. figure 3.regression lines reflecting the relative risk for an outcome during follow - up for ( a ) the cohort of cases ( coca ) + the cohort of controls ( coco ) and ( b ) the reconstructed population ( coca + noncases that have been reconstructed from the coco sampling weight [ rnc ] ) . the data underlying each line corresponds to the 2 2 tables in table 1 and table 2 , so that ( t1b ) is the 2 2 in row b of table 1 , and ( t2a ) is the 2 2 table in row a of table 2 . notice that the change in weights , or individuals , between ( a ) and ( b ) alters the end - point positions , and thus the slope of the middle line . ( a ) depicts the ill - advised approach to analyze the combined coco and coca data ( table 1 , row h ) . the area of each circle is proportional to the number of exposed ( exposure = 1 ) and unexposed ( exposure = 0 ) individuals in the coca and the coco . ( b ) depicts the reconstructed population method ( table 2 , row b ) . the area of each circle is proportional to the number of exposed and unexposed individuals in the coca and the rnc . abbreviations : coca , cohort of cases ; coco , cohort of controls ; rnc , noncases that have been reconstructed from the coco sampling weight ; rr , relative risk . regression lines reflecting the relative risk for an outcome during follow - up for ( a ) the cohort of cases ( coca ) + the cohort of controls ( coco ) and ( b ) the reconstructed population ( coca + noncases that have been reconstructed from the coco sampling weight [ rnc ] ) . the data underlying each line corresponds to the 2 2 tables in table 1 and table 2 , so that ( t1b ) is the 2 2 in row b of table 1 , and ( t2a ) is the 2 2 table in row a of table 2 . notice that the change in weights , or individuals , between ( a ) and ( b ) alters the end - point positions , and thus the slope of the middle line . ( a ) depicts the ill - advised approach to analyze the combined coco and coca data ( table 1 , row h ) . the area of each circle is proportional to the number of exposed ( exposure = 1 ) and unexposed ( exposure = 0 ) individuals in the coca and the coco . ( b ) depicts the reconstructed population method ( table 2 , row b ) . the area of each circle is proportional to the number of exposed and unexposed individuals in the coca and the rnc . abbreviations : coca , cohort of cases ; coco , cohort of controls ; rnc , noncases that have been reconstructed from the coco sampling weight ; rr , relative risk . as a more versatile analytic approach than that depicted in the previous section , we will now describe a weighted generalized linear model ( glm ) , illustrated graphically in figure 3b ) . it is based on a dataset containing individual records for the 4800 individuals in the ccf , with variables indicating e and o status , as well as the above - mentioned sampling weight . our supplementary appendix contains instructions for the use of r - functions and a spreadsheet to generate the data we have used in this paper and data with other underlying associations between e , d , and o. we further address how weighted glm can be used to depict interactions between e and d on o , and show how to estimate the extent to which getting d mediates an effect that e has on o. we have chosen to use stata version 12.1 ( stata corp ) to illustrate the analyses , but other statistical software , such as r ( the r foundation for statistical computing ; www.r-project.org ) can also be used for the weighted regression analysis , notably using survey weights [ 11 , 12 ] . the 3 above - mentioned ad hoc approaches disregard the fact that in cc studies where d incidence risk is < 50% , the cases are oversampled . several of the cited papers advise weighting the cases and the controls according to their relative probability of being sampled into the study [ 4 , 6 , 8 ] . for the analysis of ccf studies where 1 control is included per case , and the sampling weight for each individual in the coca , ie , for each case , is set to 1 , the weight for the controls is then ( 1 r)/r , as described above . if there are n cases and m controls , the weights are 1 for cases and ( n / m)(1 r)/r for controls . specifying sampling weights ( called pweight in stata , and hereafter given the variable name pw ) in the regression model de facto reconstructs the source population while basing the estimation of the corresponding standard error of the association between e and o on the actual observations in ( table 1 , rows b and d ) , rather than on the reconstructed population in ( table 2 , row b ) , the latter being an approach that would underestimate the standard error and thereby overestimate the precision of the rr . to estimate the rr of outcome o given exposure e using a glm of the binomial family with a log link and with sampling weight = pw requires the following command in stata : eform directs stata to yield rr instead of ln(rr ) , which is the default . in our example this rr corresponds to the rr point estimate derived from the reconstructed population ( table 2 , row b ) . failing to account for the fact that the cases are oversampled , by omitting the sampling weights , as in depicted graphically in figure 3a , yields an rr identical to that derived from the exposure - outcome distribution in the combination of the coca and the coco ( table 1 , row h ) , ie , a substantial overestimation . regression analyses carry several other benefits , including the ease of adjusting estimates of associations between e and o for both categorical and continuous confounders . by including an interaction term , they enable us to effectively identify and estimate the size and statistical precision of any effect measure modification between e and d on o. an interaction means that rrcoca and rrcoco are different ; ie , that getting d changes the risk of getting o given e. simply adjusting for d would under such circumstances not only violate regression model assumptions , but also iron out any differential effect of e on o between those getting d and the nc . when estimating the effect of e on o in the underlying cohort , one should refrain from adjusting for d , so that the resulting rr incorporates any effect of e on o mediated through d as well as any interaction between e and d on o. there are , however , situations where adjustment for d is warranted . for instance , to advise public health action , it may be important to break down the effect of e on o by the extent to which it is mediated through d. the size of such mediation can be measured as the relative change in the rr associated with e when estimated from models including and excluding , respectively , d as a covariate . the change in rr of o given e observed by including d as a covariate , ie , corresponding to jiang et al 's third ad hoc approach but now with an appropriate balance between cases and nc , would describe the relative change in o risk given e above and beyond that mediated by d. in our example , this adjusted rr is 1.97 ( 95% ci , .498.0 ) . if the 2 models were run on independent data sets , the estimated log rr values for e could be compared using their asymptotic standard errors and their independence . in the present case , however , the 2 models are run on the same data and the 2 estimates of log rr are thus dependent . the dependence may be accounted for with either of 2 different approaches . in stata , the postestimation command suest stores individual score values from the weighted maximum ( pseudo)likelihood estimation . the score values are then utilized to compute a robust standard error for the difference of the log rr values in the 2 models . the syntax for a log - binomial regression is the suest command requires the sampling weights to be used as importance weights ( iweight ) rather than probability weights ( pweight ) . this yields the ( same ) point estimate of 0.78 for the mediation and provides us with its 95% ci , which spans .64 to .93 . summarizing , one could say that of the rr = 2.5 that describes the effect of e on o , d contributes with 22% ( 95% ci , 7%36% ) . alternatively , a bootstrap approach can be followed . for each bootstrap sample from the observed data , the usual bootstrap standard errors and cis can then be computed for the difference , and the ci can be converted to a ci for the ratio of the 2 rrs [ 13 , 14 ] . if a glm with a log link for the binomial family does not converge , as may be the case when o is common , or we for other reasons wish to describe the association between e and o with an or using logistic regression , we can replace the log link with a logit link . glm of the binomial family with an identity link estimates the absolute risk difference rather than the rr . using this link enables us to model interactions on an additive scale , which may well be more relevant than doing so on a multiplicative scale in studies such as gems , which addresses exposures against which public health interventions , such as vaccination , may be warranted [ 2 , 3 , 15 ] . we have so far considered binary e and o variables , but the rpm is also valid for continuous outcomes . thus , we can model symmetrically distributed continuous variables , such as infant development scores and growth using an identity link combined with a gaussian distribution : which is equivalent to the simpler linear regression command : the effect estimate describes the change in o associated with e. the rpm approach can also be used to model the effects of e on a count , such as that captured in an incidence rate or incidence density , using poisson regression analysis : or , when there is overdispersion , using a negative binomial distribution : the effect estimate describes the incidence rate ratio for o where the exposure is e. finally , switching from glm to time - to - event analysis , the cox proportional hazards model is well adapted to weighted analysis . time - to - event analysis requires 2 outcome variables , t is the time from recruitment into the ccf to censoring or to the occurrence of o , which here has the value 1 when the event ( eg , death ) occurs , or 0 if the individual is censored . in stata , the sampling weights are included when the data is declared to be time - to - event data : the hazard ratios for the event where e is the exposure is returned by as in cc studies , having served as a control in a ccf study does not preclude an individual from later serving as a case or again being recruited as a control for another case [ 2 , 3 ] . similarly , having been enrolled as a case should not bar an individual from again being included as a case , nor from later being included as a control . the weight is calculated from the incidence risk , which we can not obtain from the ccf study . in gems , the risk of d is estimated using healthcare utilization and attitude surveys ( huas ) , which are undertaken every 46 months throughout the study [ 9 , 18 ] . these estimates carry sampling errors , which need to be taken into account when ultimately estimating the effect of e on o in the underlying cohort . in the supplementary appendix rpmparametersandtablesaug2012.xlsx , which generates joint probabilities and 2 2 tables describing an imagined source population based on chosen parameters explained in the sheet we used it to generate the 2 2 tables presented in the current manuscript . this population ( ie , the underlying cohort ) has an exposure ( e ) , a case disease ( d ) , and a dichotomous outcome ( o ) , the latter recorded during follow - up . data enables the user to change the underlying probabilities and associations . in cell c30 , it produces an r command highlighted in yellow which , using our r function rpmbootstrap.r , also provided in the supplementary appendix , estimates the composite measures of association , ie , the rr describing the effect of e on o in the reconstructed population ( unadjusted rr ) , the effect of e on o above and beyond that mediated by d ( adjusted rr ) , and the proportion of the unadjusted rr which is mediated by d ( mediation rr ) . these estimates incorporate not only the sampling error of the ccf study but also that of the d incidence risk estimate obtained from an independent survey . this sampling weight is calculated based on the number of individuals who developed d ( huas.d ) and the number of individuals who did not ( huas.nod ) . for example , the effect of e on o might be modeled in terms of an or in logistic regression ; as a dichotomous outcome on an additive scale , using an identity link to measure risk difference ( rd ) ; as the numerator of incidence density or rate in poisson or negative binomial regression ; as a continuous variable in linear regression ; or as a hazard ratio in cox regression . rpmbootstrap.r also generates a stata ( test.dta ) and a comma - separated values ( test.csv ) data set , which contain data from the imagined ccf study and which can be used in a weighted glm regression of the binomial family to estimate the rr , or , and rd describing the effect of e on a dichotomous o. this approach , described in some detail in this paper , does not , however , incorporate the sampling error of the sampling weight estimate , and should accordingly be used only when this value is known , as when analyzing data from a ccf study nested in a defined cohort , or when surveys used to estimate d incidence risk are of a size that the derived sampling weights can be considered known values . to illustrate how a change in parameters that define critical associations in the underlying population influences the observed effect and to guide the reader on how to use the material in the supplementary appendix , let us consider the alterations that occur if we change the association between e and d so that rr changes from 5 to 3 . the reader will in cell q84 find that the association described by the rr in the reconstructed population between e and o is reduced from 2.5 to 2.3 . moreover , because we in this example keep the exposure prevalence in the population unchanged at 0.05 , the incidence of d is reduced accordingly , in this example from 0.0240 to 0.0220 . such an incidence can be obtained in a survey of 273 individuals that identifies 6 new cases of d. by running the command returned in cell c30 using the function rpmbootstrap.r in r and then the command glm o e [ pweight = pw ] , family(binomial 1 ) link(log ) eform on the generated dataset test.dta , stata will return not only the rr of 2.3 but also its 95% ci of .4811.1 . this assumes that the incidence risk of 0.0220 is a fixed number , an assumption which is questionable unless the survey has a very large sample size . encompassing the sampling error of the sampling weight , our r bootstrap run yielded an rr of 2.4 ( 95% ci , .266.9 ) . adjusting for d reduced the rr to 2.0 ( 95% ci , .246.3 ) and quantified the mediation to be 0.86 ( 95% ci , .69.96 ) , ie , d contributing with 14% ( 95% ci , 4%31% ) of the effect of e on o. if , on the other hand we change the association between e and d so that the rr changes from 5 to 10 , the incidence increases to 0.0299 , which can be obtained by a survey of 276 individuals of which 8 develop d. under this scenario , cell q84 in the sheet data returns an rr of 3.1 ; stata also yields its 95% ci of 1.27.9 . taking the sampling error of the survey - derived incidence estimate into account using rpmbootstrap , r yielded an rr of 3.4 ( 95% ci , .958.1 ) , which was reduced to 1.9 ( 95% ci , .585.3 ) after adjustment for d ; the mediation was 0.57 ( 95% ci , .42.82 ) , so according to this analysis , d contributed with 43% ( 95% ci , 18%58% ) of the effect of e on o. the supplementary data can also be used to illustrate jiang et al 's argument that , if d changes the risk of o , the first ad hoc approach is valid only if incidence risk is 50% or 0.5 . an incidence risk of 50% can be achieved by for example changing the population incidence of d for individuals not exposed to e , ie , p.0.d , to 0.41667 . it can be seen that in this unrealistic scenario , jiang 's first ad hoc approach ( cell q54 ) yields an estimate identical to that obtained with the rpm ( cell q84 ) . if cases and controls are sampled independently of the exposures and a reliable measure of case disease occurrence can be obtained , such studies can with high efficiency estimate the association between the exposure recorded when the individuals are recruited into the ccf study and outcomes captured during follow - up thereafter . ccf studies exploit the condensation of individuals who develop the case disease into the coca , and are thereby more efficient than the corresponding cohort studies . previous reports have explored the reuse of cc data to estimate the association between exposures and alternative outcomes [ 6 , 8 , 19 , 20 ] . while the suggested approaches range from inverse probability weighting to semiparametric marginal and full likelihood models , the key issue of obtaining appropriate sampling weights is hidden from view . moreover , there is no suggestion of how to incorporate the standard error of the sampling weight into the composite effect measure generated by the proposed analyses . in general , the rarer the case disease and the smaller the surveys , the more extensive is the contribution from the sampling weight estimates to this joint sampling error . a well - designed ccf study should be planned with the intent of estimating the association between antecedent exposures and outcomes during follow - up of the 2 cohorts . to enable the necessary weighting , such studies will ensure that appropriate estimates of case disease incidence , and thereby sampling weight , is captured . this poses particular challenges for ccf studies of infectious diseases , of which the gems to our knowledge is a conspicuous first . because the incidence of infectious diseases , such as diarrhea , varies over time and often between relatively closely situated locales , this risk in gems is estimated using huas rounds undertaken periodically during the study [ 9 , 18 ] , and not as a one - time snapshot . the huas - based sampling weights are thereby likely to approach a real - time representation of the exposures , case disease , and outcomes , thus increasing the validity of the weighted regression analyses . an estimate of incidence risk derived from such survey data pooled over the duration of the study might be used , if estimates for individual survey rounds seem sufficiently similar . when incidence risk estimates from sequential surveys differ substantially and pooling over time accordingly is questionable , our advice is to first estimate the composite estimates , which describe the effect of e on o for each survey round . this may be of particular relevance for studies that describe microbial agents ' contribution to infectious disease , where microepidemics can cause substantial monthly , seasonal , and year - to - year variations [ 22 , 23 ] . when relevant and appropriate , one can thereafter pool the composite effect sizes , thereby ensuring transparency and epidemiological clarity . this paper deals with single - population ccf studies that do not recruit controls matched to their corresponding cases , when sampling weights may need to be estimated differently ( manuscript in preparation ) . further , in a pooled analysis across populations ( strata ) , the weights should be based on the relative stratum sizes and the incidence of d within each stratum . in most ccf studies relatively few children will be enrolled more than once . even in a cohort study in guinea - bissau , where children were followed with weekly stool specimen examination to identify infections with enteropathogens from birth up to 2 years of age , generalized estimating equations or frailty correction to account for between - child differences did not substantially alter point estimates or precision [ 2426 ] . we suggest that if such correction yields no or little effect on point estimates and their precision , it need not be incorporated in the bootstrapping approaches that capture the sampling error of the sampling weight estimates . it is beyond the scope of this paper to describe in further detail how to take into account between - individual differences in the occurrence of exposures and/or outcomes [ 27 , 28 ] . in this paper , we describe how a ccf study can be analyzed using weighted regression analysis and , using a bootstrap approach , incorporate the sampling error not only of the ccf component but also of the sampling weight derived from concurrently undertaken survey . using the spreadsheet and an r function supplied in the supplementary appendix , we also show how changes in population parameters , exemplified by a change in the association between e and d , will change the association between e and o in the reconstructed population . it is our contention that if reliable data on disease incidence are captured , thereby allowing sampling weights to be estimated , weighted regression analysis of ccf data can provide a useful , flexible , and effective analytic tool . we hope that by presenting the conceptual framework for ccf study design and guidance for rpm analysis using weighted regression , we will foster collaboration among infectious disease specialists , epidemiologists , and biostatisticians . such collaboration in conceptualizing , designing , undertaking , analyzing , and interpreting ccf studies will improve the studies and make it more likely that the analyses and results will address issues of relevance to clinical infectious diseases and communicable disease epidemiology . with constraints on financial and human resources to address critical questions of relationships between specific infections and outcomes ( such as clinical sequelae , nutritional impact of infection as well as illness- and infection - associated mortality ) , ccf studies , because of their efficiency , become particularly attractive . the rpm described in this paper provides a basis for estimating relationships in the population between infection with a pathogen ( eg , a diarrheal pathogen as detected in gems ) and consequences of infection over the period of follow - up . with respect to death possibly related to infection with a diarrheal pathogen , for example , ccf and rpm provide a way to go beyond describing case fatality among enrolled cases who are infected with the pathogen of interest to assessment of the association between the pathogen and mortality in the source population . one would anticipate that this addition to the toolbox of analytic epidemiology might also be useful in estimating the impact of interventions that decrease the frequency of a particular infection on specific outcomes ( eg , stunting or death ) . this can help set priorities for choosing among potential interventions aimed at control of infectious diseases encountered by clinicians on the frontline of clinical care . supplementary materials consist of data provided by the author that are published to benefit the reader .

if individuals in a case / control study are subsequently observed as a cohort of cases and a cohort of controls , weighted regression analyses can be used to estimate the association between the exposures initially recorded and events occurring during the follow - up of the 2 cohorts . such analyses can be conceptualized as being undertaken on a reconstructed source population from which cases and controls stem . to simulate this population , the cohort of cases is added to the cohort of controls expanded with the reciprocal of the case disease incidence odds ( the sampling weight ) to include all individuals in the source population who did not develop the case disease . we use a simulated dataset to illustrate how weighted generalized linear model regression can be used to estimate the association between an exposure captured during the case / control study component and an outcome that occurs during follow - up .

DECOMPOSING THE POPULATION AND THEN RECONSTRUCTING THE UNDERLYING COHORT FROM THE CASES AND THE CONTROLS ANALYSIS OF CCF DATA USING WEIGHTED REGRESSION ANALYSIS EFFECTS OF CHANGING THE POPULATION PARAMETERS DISCUSSION Supplementary Data

PMC4417551

although the polymerase chain reaction ( pcr ; [ 1 , 2 ] ) is the most widely used method , newer more sensitive techniques are favored for some applications particularly diagnostic testing . mediated isothermal amplification ( lamp ; ) is perhaps the most promising of these new methods . lamp can quickly generate large quantities of amplicon from low abundance template without temperature cycling thereby lowering the cost and complexity of necessary laboratory equipment . in addition , lamp amplicons , unlike pcr amplicons , can be directly detected via a colorimetric chemical reaction [ 5 , 6 ] . unlike pcr which requires one pair of primers , lamp requires a minimum of two nested primer pairs . generally , primers should be 1525 bases long with 4060% gc content ( annealing 5565c ) . the amplicon defined by the outer primers should be 280 base pairs and the spacing between inner and outer primers including the outer primers 40 base pairs [ 5 , 7 ] . primers are synthesized such that the reverse complements of the inner primers are connected to the outer primers by a linker ( usually five thymine residues ) . optionally , additional sets of primers can be used to increase amplification efficiency [ 3 , 79 ] . the quality of lamp primer / template match ( i.e. , the percent mismatched bases and the distribution of mismatches ) necessary for efficient amplification has not been studied , but there are studies of pcr primer / template match quality that suggest an exact 3 match of 2 - 3(4 ) bases is required for taq polymerase to extend the primer along the template [ 1014 ] and mismatches outside of the 3 end of the primer have little effect on amplification efficiency [ 10 , 14 ] . although pcr and lamp have many similarities , different polymerases and extension temperatures are typically used . it is not known to what degree the conclusions from these studies on pcr can be applied to lamp . although lamp is increasingly used for diagnostic testing ( e.g. , [ 1519 ] ) , electronic tools for lamp are limited to primer design [ 20 , 21 ] . one of the available tools , lava , can design lamp primers from aligned sequences and thus could be used to design either universal or selectively discriminatory primers . unfortunately lava can not be used to predict the activity of existing primer sets useful when one wishes to determine if a newly discovered sequence variant can be amplified with existing primers . electronic testing of primers allows researchers to quickly and inexpensively determine if newly designed primers will work with known sequence variants . this is particularly useful when meaningful consensus sequences can not be used in primer design ( e.g. , difficult to align regions ) . because two , or more , nested primer sets are used , lamp can not be directly modeled with conventional pcr simulation software ( e.g. , re - pcr ) . here we present elamp , a perl script that simulates lamp and outputs an estimate of amplification success given target sequence(s ) and primers . by default , input template(s ) are evaluated only in the given orientation , but a user option allows evaluation of both orientations ( resulting in roughly double the analysis time ) . input primers are checked for compliance with standard lamp design parameters this can be overridden by the user . the matching algorithm is the same for all primer pairs.each pair is divided into forward ( left ) and reverse ( right ) primers.the reverse primer is converted to its reverse-complement.exact matching . if there are exact matches between the forward primer and the template , the reverse primer is used for additional exact matching . otherwise approximate matching is initiated ( step 4).for each forward exact match , the reverse primer is checked against the template within an expected range ( by default 151 bp for inner , 81280 for outer primers , and 25 bp between adjacent inner and outer primers , other values may be specified by the user ; [ 3 , 5 , 7 ] ) . if a match is found , both primer - binding positions are stored . if no exact match is found for the reverse primer , approximate matching is initiated ( step 4).approximate matching . this procedure is triggered only if there are no exact matches , for both primers , and approximate matching parameters have been set . the user specifies the number of exact 3 matches ( 13 ) and the percent of matching bases for the remainder of the primer.approximate matches between template and the forward primer are stored.if there are any forward matches , reverse primer approximate matches are identified and stored . pairs of forward and reverse positions that would produce an amplicon within an expected range ( see step 3a ) are saved . each pair is divided into forward ( left ) and reverse ( right ) primers . if there are exact matches between the forward primer and the template , the reverse primer is used for additional exact matching . otherwise approximate matching is initiated ( step 4 ) . for each forward exact match , the reverse primer is checked against the template within an expected range ( by default 151 bp for inner , 81280 for outer primers , and 25 bp between adjacent inner and outer primers , other values may be specified by the user ; [ 3 , 5 , 7 ] ) . if a match is found , both primer - binding positions are stored . if no exact match is found for the reverse primer , approximate matching is initiated ( step 4 ) . for each forward exact match , the reverse primer is checked against the template within an expected range ( by default 151 bp for inner , 81280 for outer primers , and 25 bp between adjacent inner and outer primers , other values may be specified by the user ; [ 3 , 5 , 7 ] ) . if a match is found , both primer - binding positions are stored . if no exact match is found for the reverse primer , approximate matching is initiated ( step 4 ) . this procedure is triggered only if there are no exact matches , for both primers , and approximate matching parameters have been set . the user specifies the number of exact 3 matches ( 13 ) and the percent of matching bases for the remainder of the primer . approximate matches between template and the forward primer are stored.if there are any forward matches , reverse primer approximate matches are identified and stored . pairs of forward and reverse positions that would produce an amplicon within an expected range ( see step 3a ) are saved . approximate matches between template and the forward primer are stored . if there are any forward matches , reverse primer approximate matches are identified and stored . pairs of forward and reverse positions that would produce an amplicon within an expected range ( see step 3a ) are saved . for each template and primer set , if the predicted annealing positions of all constituent primer pairs are nested and appropriately spaced ( see step 3a ) , elamp predicts successful amplification . the algorithm has been implemented as a gnu public license ( gpl ) perl script ( http://www.nybg.org/files/scientists/dlittle/elamp.html ) . a graphic user interface for mac os x , linux , and windows is provided by perl / tk ( figure 1 ) . users of elamp specify a .fasta file with template sequence(s ) and a comma - separated value ( .csv ) file of primers . each cell of the .csv file corresponds to a primer and each line to a primer set . primer pairs are ordered from the innermost to the outermost . within each primer pair , the forward ( left ) primer should be followed by the reverse ( right ) . the alternative fib / bip primer format can be used , but linkers must be delimited by hyphens ( e.g. , ttttt ) . output is a .csv file : the first row indicates the primer set(s ) and the first column the template(s ) . electronic amplification of the diagnostic glta / gltb region was attempted from all 40 complete staphylococcus genomes deposited in genbank ( table 1 ; median = 2,813,126 base pairs , iqr = 180,199 base pairs ) . two similar and overlapping sets of primers designed to only amplify from s. aureus were used (; lava f1 : 3-gga ata gtt tgt aag aca cct gc ca-5 , lava r1 : 3-caa aaa caa agc gaa ctg cca at-5 , lava f2 : 3-acc aac acc aaa aat cgg t-5 , lava r2 : 3-tgg cat tat tac ttg cca tca-5 , lava f3 : 3-gct aca att gca ggc gtt t-5 , lava r3 : 3-ttg atg tcg aaa aca ctg gaa-5 ; primerexplorer f1 : 3-tgt tgg aat agt ttg taa gac acc t-5 , primerexplorer r1 : 3-caa aaa caa agc gaa ctg cca ata-5 , primerexplorer f2 : 3-tta cca aca cca aaa atc gg-5 , primerexplorer r2 : 3-gca tta tta ctt gcc atc att g-5 , primerexplorer f3 : 3-gct aca att gca ggc gtt-5 , primerexplorer r3 : 3-tgt cga aaa cac tgg aac at-5 ) . reported times are the median of five sequential single - threaded executions on an intel pentium d 950 ( 3.4 ghz ) with 4 gb of 533 mhz ram running 64-bit ubuntu 12.04.1 lts . all files were placed on a 256 mb ram disk ( a tmpfs volume ) prior to analysis . elamp was instructed to evaluate the genome sequence in both possible orientations , to perform exact primer matching , and to perform approximate matching ( an exact match of 3 ultimate , penultimate , and antepenultimate bases and 75% similarity for the remaining bases ) . the two primer sets behaved similarly ( table 1 ) : the exact matching procedure predicted , depending on the primer set , a lamp amplicon for 25 or 26 of the 31 s. aureus genomes . approximate matching resulted in a predicted lamp amplicon for 29 of the 31 s. aureus genomes . although the total predicted by approximate matching was the same for both primer sets , predictions for cp003166.1 and cp003194.1 varied by primer set . no matter the primer set or the matching procedure used , none of the nine remaining genomes , representing six other staphylococcus species , were predicted to produce an amplicon . the s. aureus genomes not predicted to produce an amplicon have various mismatches : a single mismatch within the f1 primers of both primer sets ( cp001996.1 , fr821779.1 ) ; a single mismatch within the f1 primers of both primer sets , different from the above mismatch ( he681097.1 ) ; a mismatch at the penultimate 3 base of the lava f1 primer ( cp003166.1 ) ; a single mismatch within the f2 primers of both primer sets ( including the antepenultimate 3 base of the primerexplorer primer ) as well as a single mismatch within the lava r2 primer ( cp003194.1 ) ; and single mismatches within the f3 and r1 primers of both primer sets as well as multiple mismatches within both primer sets for the f1 ( including the ultimate 3 base of the primerexplorer primer ) , r2 ( including the penultimate 3 base of the primerexplorer primer ) , and r3 ( including the ultimate 3 bases of both primer sets ; fr821777.2 ) . given the large number of mismatches and their distribution , elamp does not predict amplification of the s. aureus sequence fr821777.2 with either exact or approximate matching . this sequence is from a highly divergent strain of s. aureus that could , arguably , be classified as a different species s . the sequences of 14 s. aureus genomes were considered when the lava and primerexplorer primer sets were designed (; aj938182.1 , ap009324.1 , ap009351.1 , ba000017.4 , ba000018.3 , ba000033.2 , bx571856.1 , bx571857.1 , cp000046.1 , cp000253.1 , cp000255.1 , cp000703.1 , cp000730.1 , and cp000736.1 ) . these genomes were predicted to produce a lamp amplicon for both primer sets under both matching procedures . the s. aureus genomes that were , under various circumstances , predicted to not produce any amplification were not consulted when primers were designed ( cp001996.1 , cp003166.1 , cp003194.1 , fr821777.2 , fr821779.1 , and he681097.1 ) . given the assumptions of match quality , neither primer set is entirely diagnostic of s. aureus either the primers must be used in combination or , ideally , new primers that use less variable sites should be designed . elamp with exact primer matching required 10.556 seconds and approximate matching required 9 minutes 18.608 seconds . the graphical interface is simple allowing easy use by nonspecialists while the command - line interface is suitable for use in pipelines . results , presented either in a single .csv file or in a gui panel , are straightforward to interpret .

we present elamp , a perl script , with tk graphical interface , that electronically simulates loop - mediated amplification ( lamp ) allowing users to efficiently test putative lamp primers on a set of target sequences . elamp can match primers to templates using either exact ( via builtin perl regular expressions ) or approximate matching ( via the tre - agrep library ) . performance was tested on 40 whole genome sequences of staphylococcus . elamp correctly predicted that the two tested primer sets would amplify from s. aureus genomes and not amplify from other staphylococcus species . open source ( gnu public license ) perl scripts are available for download from the new york botanical garden 's website .

1. Introduction 2. Algorithm 3. Implementation 4. Empirical Example 5. Conclusions

PMC3794536

lactobacillus strains are extensively used as probiotics in the food industry , and certain lactobacillus species are also of importance in general health , providing a beneficial microflora in the oral cavity , intestinal tract [ 2 , 3 ] , and vagina . the genus lactobacillus contains a diverse assemble of gram - positive , catalase negative , and nonsporulating , rod - shaped organisms and includes more than 140 species [ 5 , 6 ] . among those , lactobacillus casei group , especially l. casei , l. paracasei , and l. rhamnosus , has attracted a lot of attention over the last 20 years . this is linked to the considerable economic importance of the l. ( para)casei species , which is used in many food and feed applications such as dairy products and has a proven record in human and animal health . however , the taxonomy among the species in lactobacillus casei group is still vague . the uncertain identification leads to confusion in phenotypic characters . in recent years , the identification of lactobacilli evolving along with molecular methods based upon the 16s rrna gene has been widely used . many attempts to discriminate these species yielded inaccurate results , and only limited success could be achieved , largely due to the failure of differentiation between l. paracasei and l. casei strains [ 7 -10 ] . for example , most of the strains described as l. casei much more differ from the species type strain of atcc 393 than from l. zeae strains [ 11 , 12 ] . the technique of pcr - denaturing gradient gel electrophoresis ( dgge ) has been introduced recently . this technique provides the information of variations in target genes within bacterial population which allows differentiating among the species that the dna sequences differs in at least one base pair . in this study , it revealed that the dgge method with the designed primers of the v1 and region of v2-v3 region of 16s rrna genes enabled to distinguish among the species of l. casei , l. paracasei , l. rhamnosus , and l. zeae . in addition , the phenotypic characters of acid production and inhibitory effect on oral pathogen were analyzed . four reference strains of lactobacillus casei atcc 393 , l. paracasei ccug 32212 , lactobacillus rhamnosus atcc 7469 , and l. zeae ccug 35515 and 46 clinical strains , 10 l. casei isolates , 21 l. paracasei isolates and 15 l. rhamnosus isolates , were included in this study . all clinical strains were previously identified according to 16s - rrna gene profiles by restriction fragment length polymorphism analysis ( pcr - rflp ) and protein profiles by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) . two set of primers were used in this study , the first set of primers was hda-1-gc ( 5-cgcccggggcgcgccccgggcggggcgggggcacggggggactcctacgggaggcagcagt-3 ) and hda-2 ( 5-gtattaccgcggctgctggcac-3 ) according to walter et al . , which was designed targeting 200 bp of the v2-v3 region of 16s - rrna gene . the second set of primers was carp-1-gc ( 5-cgcccggggcgcgccccgggcggggcgggggcacggggggggcgtgcctaatacatgcaa-3 ) and carp-2 ( 5-ggcaggttacccacgtgtt-3 ) , which was designed in this study targeting 112 bp of the v1 region . all dna samples were extracted using a genomic dna extraction kit ( rbc bioscience , taipei , taiwan ) , following the manufacturer 's protocol for gram - positive bacteria . the condition of pcr was that each 50 l pcr reaction contained 10 mm tris - hcl ph 8.3 , 50 mm kcl ( geneamp pcr buffer ii from applied biosystems , foster city , ca , usa ) , 1 mm mgcl2 ( applied biosystems ) , 0.1 mm each dntp , 0.4 m both primers , 5 g / ml template , and 0.5 u ampli - taq dna polymerase ( applied biosystems ) . the pcr reactions were run using a geneamp pcr system 2400 ( applied biosystems , foster , ca , usa ) . for primers carp-1-gc and carp-2 , reactions were run with an initial touchdown step in which the annealing temperature was lowered from 61 to 57c in intervals of 2c every 3 cycles , and 20 additional cycles were done with annealing at 55c . denaturation was performed at 95c for 1 min , and extension was performed at 72c for 1 min and 30 s. for primers hda-1-gc and hda-2 , reactions were run for 35 cycles of denaturation at 95c for 60 s , annealing at 56c for 45 s , and extension at 72c for 60 s. for both amplification cycles , an initial denaturation at 94c for 5 min and a final extension at 72c for 7 min were carried out . the dcode universal mutation detection system ( biorad , hercules , ca , usa ) was used for the sequence - specific separation of the pcr products . electrophoresis was performed in a 8% polyacrylamide gel with gradient of 35 to 60% urea - formamide denaturant , and electrophoresis running time was adjusted to 6 h at 120 v. after electrophoresis , they were stained for 10 min in a sybr green solution ( molecular probes , or , usa ) and analyzed under uv illumination . the inhibitory effect of the lactobacillus strains against streptococcus mutans atcc 25175 was assessed by an agar overlay method . in brief , lactobacillus strains ( producer strains ) were inoculated on the surface of the brain heart infusion agar and incubated anaerobically ( 80% n2 , 10% h2 , and 10% co2 ) for 2448 h at 37c to develop visible macrocolonies . the indicator strain was precultivated in the brain heart infusion broth ( bhi ) , and the suspension of cells was adjusted to an optical density ( od ) 0.25 at 600 nm . thereafter , 5 ml of bhi soft agar ( 7 g / l agar ) was seeded with 100 ul of an overnight culture of the indicator strain and immediately poured over the macrocolonies of lactobacillus . the plates were incubated anaerobically at 37c for 24 h to generate an inhibitory zone . the acid production of the lactobacillus strains was assessed according to piwat et al . . lactobacillus strains were initially grown anaerobically ( 80% n2 , 10% h2 , and 10% co2 ) to exponential phase in filter sterilized ( pore size 0.22 m , nalgene , ny , usa ) de man , rogosa and sharpe ( mrs ) broth ( lab m , bury , uk ) at 37c for 1618 h. cells were harvested by centrifugation at 3000 rpm for 5 min at 4c , washed twice in phosphate buffered saline ( pbs ; oxoid , basingstoke , uk ) , and inoculated into 50 ml fresh , prewarmed mrs broth containing 2% ( w / v ) glucose , ph 7.0 , to give an optical density of 1.0 at 650 nm ( corresponding to 10 cells / ml ) . the cultures were then incubated in an anaerobic chamber ( minimacs anaerobic workstation , don whitley scientific ltd , uk ) at 37c . two milliliters of each sample was collected and analyzed for the growth and acid production at the start ( 0 ) and after 1.5 , 3 , 5 , 7 , and 24 h. bacterial growth was determined by measuring the final od reached at 650 nm , and the change in od was calculated . in addition , the number of viable cells was also counted as cfu / ml on mrs agar following anaerobic incubation for 48 h. acid production was studied by ph measurements using a ph electrode and ph - meter ( hanna ph 211 , hanna instrument , uk ) . the amount of hydrogen ion [ h ] was obtained from ph values according to the formula : [ h ] = ( 10 ) . the rate of acidification by each strain ( acid production rate ) was determined from the change in h ( h ) divided by the average number of bacterial cells per hour in the logarithmic growth phase , as shown in the following equation : ( 1)acid production rate=(h+)[((n2n1)/2)t2t1 ] , where n1 and n2 are number of bacterial cells at time point 1 ( t1 ) and time point 2 ( t2 ) , respectively . the data presented are means of triplicate measurements , and all experiments were performed twice . the dgge patterns of v1 region of 16s rrna sequences are shown in figure 1 , and each type species strain and identified clinical isolate showed its own unique dgge pattern . l. rhamnosus atcc 7469 and identified clinical isolates revealed a different band from l. zeae ccug 35515 , l. casei atcc 393 , and l. paracasei ccug 32212 . it was noted that those of clinical strains identified as l. casei gave the different dgge patterns from the type strain l. casei atcc 393 . all clinical strains identified as l. casei had a major band with the same distance as l. paracasei strains ; however , l. paracasei ccug 32212 and most of clinical isolates identified l. paracasei showed the extraminor bands . when hda1 and hda2 primers were used to produce the dgge patterns of v2-v3 region of 16s rrna sequences , l. casei atcc 393 showed the same dgge pattern as all clinical strains identified as l. casei . l. paracasei ccug 32212 and all clinical isolates revealed the same multiple bands which clearly differed from all l. casei strains ( figure 2 ) . the ability for growth inhibition of l. casei group against s. mutans atcc 25175 is shown in figure 3 . a statistically significant difference among the species was found ( kruskal - wallis test , p < 0.05 ) after l. paracasei strains were identified separately . had a significantly higher inhibitory effect than either l. casei or l. rhamnosus ( mann - whitney u test , p < 0.01 ) . acid - production rate of clinical strains of l. casei group in the exponential growth phase was calculated from the 1.5 to 5 h incubation period . also , a statistically significant difference among the species was found ( kruskal - wallis test , p < 0.05 ) after l. paracasei strains were identified separately . l. paracasei strains had a significantly lower acid - production rate than either l. casei or l. rhamnosus ( mann - whitney u test , p < 0.05 ) ( figure 4 ) . the lactobacillus casei group is one of special relevance for dairy food ( cheese , yoghurt , and other fermented milk products ) and pharmaceutical industry due to its important role in promoting human health . based on their ability to inhibit the growth of various pathogens , they have been used as probiotics in the gut for decades . previously , a single species with five subspecies has been reclassified into three species including l. casei , l. paracasei , and l. rhamnosus in 1989 . however , this classification initiated a controversy , generally due to the failure of differentiation between l. paracasei and former l. casei strains even by molecular technique [ 19 -21 ] including our previous study . research has been focused on the application of molecular biology techniques for accurate differentiation among the strains . we developed a dgge method with the designed primers of the v1 in combination with v2-v3 region of 16s rrna genes , which enabled to distinguish the close related strains in the l. casei group . it was shown that carp1-gc and carp2 primers could clearly distinguish the type strain l. rhamnosus atcc 7469 , l. zeae ccug 35515 , l. casei atcc 393 , and l. paracasei ccug 32212 from each other , and each individual revealed its own unique pattern ( figure 1 ) . it was surprising that those of clinical strains identified as l. casei gave different dgge patterns from the type strain l. casei atcc 393 . the results indicated that there were different sequences in v1 region of all the type strains , which confirmed the information obtained by comparing the published 16s rrna gene sequences of the type strain l. rhamnosus atcc 7469 , l. zeae ccug 35515 , l. casei atcc 393 , and l. paracasei ccug 32212 . hda1-gc and hda2 primers could distinguish the type strain and identified clinical isolates of l. casei from the type strain and identified clinical isolates of l. paracasei ( figure 2 ) . by showing the multiple bands of all strains of l. paracasei , it indicated that l. paracasei strains contained more than one copy of 16s rrna gene . it is likely that the taxonomy of this group will undergo further changes with more extensive studies providing more evidence in the coming years . thus , such method may assist in future taxonomic considerations of this group and could be a beneficial implementation for future research . in oral cavity , lactobacilli have frequently been isolated from carious lesions , and thus , they are thought to be associated with the development of dental caries . the reason was based on their acid production and aciduric characteristics [ 16 , 23 , 24 ] . as many lactobacillus species have similar nutritional and growth requirements , it is often difficult to use classical microbiological methods to identify close related species . consequently , in most of the dental literature , they are specified and are referred to only as lactobacilli . however , it is important to understand the certain role of various lactobacilli , whether they are harmful , beneficial , or neutral for the development of dental caries . the inhibitory activities of oral lactobacillus against oral pathogens , for example , cariogenic bacteria , periodontopathogens , and candida , have been reported [ 15 , 25 , 26 ] . however , the cariogenic characteristics of lactobacillus , especially acidogenic activity , have always been concerned . in most clinical studies , l. casei and l. paracasei they have been reported for their association with dental caries in several studies [ 28 -30 ] . however , less is known of how acidogenicity and inhibitory activity differ among these two species identified with current taxonomic methods . in this study , the heterogeneity of acid production and antimicrobial effect among the species of l. casei / l . it was found that l. paracasei strains were stronger in growth inhibition against oral pathogens and were weaker in acid production . the results indicated that l. paracasei may be of benefit as probiotics for the prevention of oral diseases than the others due to its low acidogenic and high inhibition effect . in conclusion , the technique has been proven to be able to differentiate between closely related species in l. casei group and thus provide reliable information of their phenotypic appearances .

this study aimed to develop a 16s rrna pcr - denaturing gradient gel electrophoresis ( dgge ) to identify the species level of lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect . pcr - dgge has been developed based on the 16s rrna gene , and a set of hda-1-gc and hda-2 , designed at v2-v3 region , and another set of carp-1-gc and carp-2 , designed at v1 region , have been used . the bacterial strains included l. casei atcc 393 , l. paracasei ccug 32212 , l. rhamnosus atcc 7469 , l. zeae ccug 35515 , and 46 clinical strains of l. casei / paracasei / rhamnosus . inhibitory effect against streptococcus mutans and acid production were examined . results revealed that each type species strain and identified clinical isolate showed its own unique dgge pattern using carp1-gc and carp2 primers . hda1-gc and hda2 primers could distinguish the strains of l. paracasei from l. casei . it was found that inhibitory effect of l. paracasei was stronger than l. casei and l. rhamnosus . the acid production of l. paracasei was lower than l. casei and l. rhamnosus . in conclusion , the technique has been proven to be able to differentiate between closely related species in l. casei group and thus provide reliable information of their phenotypic appearances .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

PMC5067112

. feedback control may actually increase rather than decrease instability due to the inherent time - delay in the neural close - loop control . we focused on measuring the mechanical impedance of the ankle during walking because the ankle is critical for propulsion , shock absorption , and balance during standing and walking . the ankle plantarflexors contribute as much as 50% of positive mechanical work in a single stride to enable forward propulsion . in pre - swing , ankle plantarflexors also act to advance the leg into swing phase while promoting knee flexion at toe - off . additionally , the ankle helps maintain body - weight support during gait and balance , . finally , the ankle dorsiflexor musculature helps absorb impact forces during foot strike to enable controlled landing and foot - floor swing clearance at toe off . extensive previous work estimating ankle impedance has provided a rich characterization of these properties during stationary , non - moving conditions . when investigated in the sagittal plane , ankle joint impedance has been shown to vary with many factors , including mean ankle torque , ankle position , perturbation amplitude , and muscle fatigue . the research showed that modulation of these factors can vary the stiffness component of impedance by up to three orders of magnitude . few studies have focused on characterizing ankle impedance during non - stationary conditions , which are more relevant to locomotion . these studies use changes in ankle torque or position in combination with time - varying system identification techniques to estimate ankle impedance . rather than using a perturbation , researchers have instead focused on measuring the instantaneous slope of the ankle s torque - angle profile during locomotion , known as the quasi - stiffness , . these studies has provided important insight into how torque and angle co - vary at a joint during locomotion . however , because the agonist and antagonist muscles that cross human joints are capable of producing net - positive mechanical work , joint torque and angle can be varied independently of joint stiffness . thus , there is no causal relationship between quasi - stiffness and joint impedance , and these concepts are decoupled . ultimately , a perturbation is required to assess the joint impedance that characterizes the underlying system dynamics . the mechanical impedance of the human ankle plays a critical role in locomotion , and has broad applications in assistive technologies , such as robotic prostheses and exoskeletons , as well as humanoid robotics . for the first time , the previous studies have been merged to provide a comprehensive representation of how joint impedance varies throughout the entire gait cycle . specifically , we have examined the work of lee and hogan and rouse et al . to characterize ankle impedance from pre - swing to terminal stance . the purpose of this study is to show how ankle impedance varies throughout the gait cycle , and discuss the implications of the results in the context of the whole gait cycle . this study motivates many changes in the development of future assistive technologies , as well as a new understanding of neuromuscular pathologies , and the neural mechanisms that underlie these disorders . two different approaches were used to construct a trajectory of ankle impedance modulation during walking . a mechatronic platform and a wearable ankle robot were utilized to quantify ankle impedance from late - loading response to terminal stance phase and from pre - swing phase to early - loading response , respectively ( fig . a : from late - loading response to terminal stance phase , i.e , when the foot was flat to the ground , ankle impedance was estimated by a mechatronic platform , recessed into a walkway ( b ) . from pre - swing phase through the entire swing phase to early - loading response , i.e , when the toe and/or heel were off the ground , ankle impedance was estimated by a wearable ankle robot ( c ) . b : the perturberator robot was comprised of a force platform coupled to a gear - motor that was controlled by a servodrive and microcontroller . c : the anklebot was mounted onto the knee brace and its end - effectors were connected to a rigid u - shaped bracket attached to the bottom of a shoe ( left ) . the robot was properly attached to the subjects right leg , and subjects were instructed to walk on a treadmill with the robot ( right ) . a : from late - loading response to terminal stance phase , i.e , when the foot was flat to the ground , ankle impedance was estimated by a mechatronic platform , recessed into a walkway ( b ) . from pre - swing phase through the entire swing phase to early - loading response , i.e , when the toe and/or heel were off the ground , ankle impedance was estimated by a wearable ankle robot ( c ) . b : the perturberator robot was comprised of a force platform coupled to a gear - motor that was controlled by a servodrive and microcontroller . c : the anklebot was mounted onto the knee brace and its end - effectors were connected to a rigid u - shaped bracket attached to the bottom of a shoe ( left ) . the robot was properly attached to the subjects right leg , and subjects were instructed to walk on a treadmill with the robot ( right ) . joint impedance was estimated using perturbations and data recorded from a mechatronic platform , termed the perturberator robot ( fig . the mechatronic platform was comprised of a multi - axis force platform coupled to a gear - motor that received position commands from a servodrive and microcontroller . the mechatronic platform was recessed into a walkway and the platform section of the robot was flush with the walkway surface . the total length of the walkway was 5.25 m. ten healthy , able - bodied subjects participated in this experimental protocol . subjects walked at a self - selected pace that ranged from 85 90 steps per minute across the walkway that included the recessed mechatronic platform . when subjects stepped on the mechatronic platform , a 2.0 ramp perturbation was randomly applied with a probability of 50% in either the dorsiflexion or plantarflexion directions . one hundred perturbation trials were recorded at each timing point , and approximately 400 trials were recorded where no perturbation occurred , totaling approximately 800 walking trials . perturbations were applied at four timing points , ranging from 2070% of the stance phase of walking , chosen randomly during each trial . the mid - stance region of stance phase was chosen because it is a critical part of the gait cycle when the body is supported by a single leg . additionally , during mid - stance , the perturbation caused changes in the ankle s angle , rather than deformations of the mid - foot , improving the accuracy of our estimation . data were low - pass filtered at 20 hz and segmented to include the 100 ms window beginning with the perturbation onset . to obtain ankle torque , ground reaction force data were resolved to the equivalent force - torque couple at the ankle s center of rotation . the ankle s center of rotation was identified from high - definition video obtained during the experiment . a second - order model consisting of stiffness , damping , and the stiffness , damping , and inertia describe the position , velocity , and acceleration dependent components of response torque , respectively . prior to estimation , a bootstrapping procedure was used to provide a more reliable estimate of the parameter variability . the bootstrapping procedure involved included the following steps : a random selection of 60% of the torque and angle profiles from perturbed trials were chosen and averaged . 2a black lines ) was then subtracted from the mean of the perturbed torque and angle profiles ( fig . the joint impedance was then estimated from these resultant profiles using the optimal least squares estimate , with a constraint that the estimated parameters be greater than zero . this process was repeated one hundred times for each perturbation direction and time point . in summary , the purpose of the bootstrapping procedure was to remove the torque and angle changes that occurred simply as a result of walking , while preserving the variability of the parameter estimates that were subsequently obtained . figure 2.samples of inputs and outputs for the estimation of ankle impedance . a : data during the analysis window ( 100 ms ) from the perturberator robot . random torque inputs ( top ) and the corresponding angles ( black curve - blue curve ; bottom ) . the blue curve is the nominal angle trajectory . only the data in the shaded region ( from pre - swing phase to early - loading response ) a : data during the analysis window ( 100 ms ) from the perturberator robot . random torque inputs ( top ) and the corresponding angles ( black curve - blue curve ; bottom ) . only the data in the shaded region ( from pre - swing phase to early - loading response ) were used . the aforementioned methods that were used to estimate ankle impedance during locomotion were previously validated and shown to provide highly accurate estimates of ankle stiffness . the stiffness of a passive prosthetic foot was estimated during locomotion at four time points and compared to values obtained independently using a materials testing machine . the stiffness component of impedance was shown to be accurate within 5% on average during walking . a correction has been applied to the data from , which updated the inertia estimates . the stiffness and damping values did not change significantly , however , the data presented here reflect the updated values . a wearable ankle robot , anklebot , was used to estimate ankle impedance from pre - swing phase before toe - off to early - loading response right after heel - strike . the robot was designed to have very low intrinsic mechanical impedance at the interaction port , i.e , the ankle . this made possible highly back - drivable operations , allowing subjects to have movements with minimal resistance . the robot was mounted onto the knee brace and its end - effectors were connected to a rigid u - shaped bracket attached to the bottom of a shoe . the robot was properly attached to the subjects dominant leg , and subjects were instructed to walk on a treadmill to familiarize themselves with the experimental setup and select their preferred walking speed . in a main experiment , subjects walked with the anklebot on a treadmill for 13 minutes while the robot continuously applied mild random torque perturbations ( band - limited white noise with stop frequency 100 hz ) to the ankle . the magnitude of perturbations was determined low enough not to disturb normal walking but strong enough to perturb the ankle . the resulting ankle displacements due to the mild perturbations were 1.6 ( root - mean - square value ) and 4.1 3.9 ( peak - to - peak value ; negative and positive denote plantarflexion and dorsiflexion , respectively ) when averaged across subjects . ankle impedance was estimated based on ensemble - based linear time - varying system identification . this is an effective and robust system identification technique when repetitive and periodic data are available . in this study , more than 500 realizations for ensemble sets were generated from human walking , where each realization was defined by a gait cycle that begins with heel - strike of one foot and ends with another heel - strike of the same foot . 2b bottom ) , respectively . by applying correlation - based system identification approach to the ensemble sets , measured dynamics were estimated along the time axis ( at 2 ms intervals ) in the form of finite impulse response functions ( irf ) . the irf estimates were further approximated by second order models consisting of inertia , damping , and stiffness . finally , ankle parameters ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i_{ankle } $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ b_{ankle } $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k_{ankle } $ \end{document } ) were obtained by compensating actuator dynamics . as the ankle and the robot share the same displacement , ankle impedance was calculated by subtracting impedance of the robot ( also approximated as a second order model consisting of inertia , damping , and stiffness ) from the measured impedance . while impedance was estimated every 2 ms , representative ankle parameters were calculated at five sub - gait phases , ranging from 010% ( early - loading response : elr ) , 5060% ( pre - swing phase : psw ) , 6073% ( initial swing phase : isw ) , 7486% ( mid - swing phase : msw ) , and 87100% ( terminal swing phase : tsw ) of the gait cycle by averaging estimated parameters within each sub - gait phase into single values . based on the aforementioned two methods , ankle impedance was estimated for the whole gait cycle . time - varying ankle stiffness and damping were summarized at nine timing points ( fig . 3 ) . first , ankle stiffness linearly increased from loading response to terminal stance ; the correlation coefficient ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ r)$ \end{document } between the gait phase and the corresponding ankle stiffness was 0.998 . ankle stiffness substantially decreased at the end of the stance phase , remained relatively constant through the entire swing phase , and increased again around heel - strike ( fig . stiffness in the swing phase was comparable with results from a previous steady - state study of matched muscle activation . the change in damping during the gait cycle is much less than the changes observed in stiffness ( fig . inertia estimates by the anklebot were rather invariant throughout the gait phase around 0.008 kgm , consistent with previous findings , . although also generally invariant throughout the gait cycle , inertia estimates by the perturberator robot were approximately 0.027 kgm , differing by a factor of three . this is likely a result of residual inertia of the mechanism structure and other coupled body segments . figure 3.time-varying ankle mechanical impedance during walking.ankle stiffness and damping were summarized at nine timing points . the stance and swing phases account for approximately 60% and 40% of the gait cycle . time - varying ankle mechanical impedance during walking.ankle stiffness and damping were summarized at nine timing points . the stance and swing phases account for approximately 60% and 40% of the gait cycle . red and blue colors denote results from the perturberator robot and anklebot , respectively . dashed black lines indicate the regions where no results were available . the quality of estimation by second order models was demonstrated by high percentage variance accounted for ( % vaf ) between measurements and estimates by the models . when averaged across subjects and timing points from late - loading response to terminal stance phase , the % vaf between torque measurements and estimates by second order models was 926.3% ( meanstandard deviation ) . for timing points from pre - swing phase to early - loading response , the % vaf between angle measurements and predicted outputs by input torques and estimated second order models was 85.74.5% . for the first time , we were able to provide an estimate of the ankle impedance in the sagittal plane during the complete gait cycle . especially , stiffness increases from heel strike to terminal stance , remaining low during the swing phase . this modulation of ankle mechanical impedance parallels functional need to prevent foot slap following heel strike , increase stability demands during stance and metabolic savings during the swing phase . it goes beyond coarse correlations between anthropometric measures with intrinsic ankle or prior studies showing that humans adjust leg stiffness to accommodate surface changes . we speculate that proper modulation of ankle impedance is part of the learning process of human biped walking and it is an important aspect when assessing neurological deficits , aging , and determining proper therapeutic interventions as well as designing prosthetics . this study highlights the need to incorporate joint impedance in the design of future wearable robotic technologies , including prostheses and exoskeletons . historically , the development of wearable robotic technologies has only used the torques and angles of lower limb joints to define the design criteria , and accompanying metrics of success . however , the present study demonstrates that joint impedance is also regulated during gait , and must be included in the future design and control of wearable robotic systems that can truly mimic the system dynamics of the healthy neuromuscular system . the impedance control law proposed in rouse et al . has been implemented in a robotic prosthetic leg , which led to more natural locomotor behavior in amputees . additionally , a quasi - passive prosthetic ankle has been developed that can provide the natural joint impedance relationship during gait , which will be soon be tested in amputees . because impedance is at the interface between the neural controller and the world , one implication of our working model is that restoring appropriate impedance occurs concurrently with recovery . in a preliminary study krebs and colleagues found that the ankle quasi - stiffness of neurologically impaired subjects was significantly different from that of age - matched healthy subjects , . they also found that seated ( open - chain ) robot - aided therapy successfully resolved this abnormality and most important this therapy resulted in a 20% improvement of over - ground walking speed . the identification techniques described here will allow rehabilitation therapy to be targeted toward the specific deficits in the mechanical impedance and its modulation that each individual exhibits . we will alter the adaptive algorithm developed in 2003 to compensate also for impedance abnormalities . it will include time - varying impedance that depends on arm reaching or gait phase and aims to normalize the patient s impedance with respect to unimpaired impedance ( suitably scaled ) . while the stiffness and quasi - stiffness are distinct entities in general , linearly increasing stiffness from loading response to terminal stance in this study are remarkably similar to those from the slope of the ankle s torque - angle profile , which was not previously known . however , despite their similar magnitudes , the joint stiffness and quasi - stiffness have different meanings and , therefore also different interpretations . moreover , we do not yet know if these properties are similar for other joints or regions of the gait cycle . most notably , the identification procedures assume a linear , time - invariant behavior of the ankle within a short period of time ; the procedure with the perturberator robot used data within the 100 ms window beginning with the perturbation onset , and the procedure with the anklebot used data within the 40 ms window for irf estimation . furthermore , the system structure was assumed to be second order , consisting of stiffness , damping and inertia . the rationale for this assumption was based on previous findings that the human ankle was accurately described as a second order system over a wide range of ankle positions and muscle activation , . even with these assumptions , the methods were valid under the given experimental conditions , supported by the high % vaf between measurements and predicted outputs across the gait cycle . this study used two distinct approaches to complete a trajectory of ankle impedance modulation throughout the gait cycle . to estimate ankle impedance , we should apply external energy to the ankle and analyze the corresponding responses . in the entire swing phase and when the toe or heel were off the ground , a wearable device is proper to provide accurately controlled perturbations to the ankle . however , when weight bearing is substantial during stance phase , i.e. , when the foot was flat to the ground , a wearable device is less desirable . due to high weight bearing , mild perturbations are not strong enough for the purpose of identification . strong perturbations from the wearable device are not desirable either ; they will significantly change the normal gait or may cause slippage at the interface of the lower - limb and the device . on the other hand , a robotic platform recessed into a walkway can provide strong perturbations to the ankle without disturbing the normal gait . however , this approach can be used only when solid contact exists between the foot and the device . thus , we would argue that the current best practice to estimate ankle impedance across the full gait - cycle could be achieved through the combination of two distinct experimental setups : a wearable ankle device and an instrumented walkway . however it is important to note that the robot is not mounted at the ankle , but mounted proximally to the leg and anterior to the shank to minimize perception of loading . we further examined the effects of asymmetric or unilateral loading of the limb during task - oriented gait in adults both healthy and stroke ( 9 chronic stroke patients ) . specifically , we sought to assess the effects of the added inertia and friction of unpowered ankle robot on gait parameters , interlimb symmetry , and lower extremity joint kinematics . in summary , our results demonstrated that the added inertia anterior to the shank ( above knee ) had no statistically significant effect on spatiotemporal parameters of gait , including paretic and nonparetic step time and stance percentage , in both overground and treadmill conditions . noteworthy , interlimb symmetry as characterized by relative stance duration was greater on the treadmill than overground regardless of loading conditions . the current study utilized a treadmill in estimating ankle impedance from pre - swing phase to early - loading response . the primary reason for this is to minimize the duration of the experiment in a limited space . one recent study has demonstrated that the anklebot could be well used for overground walking . since dynamics of treadmill walking may be significantly different from those of overground walking , it is important to compare the modulation of ankle impedance in these two conditions . as a limitation of this study , the analysis methods presented do not attempt to separate intrinsic and reflex components of joint impedance . however , it is presumed that impedance measurements by both systems are dominated by the intrinsic components of the ankle joint , supported by the time course of short latency reflexes that occur approximately 40 ms following the onset of an imposed movement , and an additional 60 ms delay in subsequent force production , . it was also demonstrated that the influence of random perturbations by the anklebot on muscle activity is small ; the change of muscle activation due to perturbations was less than 0.5% mvc of each of the major ankle muscles . together , it is presumed that reflex contribution to ankle impedance measurements is minimal in this study . the current results are limited to the characterization of ankle impedance in the dorsi - plantarflexion direction . we plan to expand this work and measure healthy young subjects ankle mechanical impedance in all 2-d directions during walking , including dorsi - plantarflexion and inversion - eversion directions . we will employ the anklebot described above and the mit - skywalker or a new multi - axis robotic platform during stance to implement in 2-d what the perturberator robot did in 1-d . in addition , it is possible to extend the current study to characterize ankle impedance in different walking conditions , such as walking in different speeds and slopes .

the human ankle joint plays a critical role during walking and understanding the biomechanical factors that govern ankle behavior and provides fundamental insight into normal and pathologically altered gait . previous researchers have comprehensively studied ankle joint kinetics and kinematics during many biomechanical tasks , including locomotion ; however , only recently have researchers been able to quantify how the mechanical impedance of the ankle varies during walking . the mechanical impedance describes the dynamic relationship between the joint position and the joint torque during perturbation , and is often represented in terms of stiffness , damping , and inertia . the purpose of this short communication is to unify the results of the first two studies measuring ankle mechanical impedance in the sagittal plane during walking , where each study investigated differing regions of the gait cycle . rouse et al . measured ankle impedance from late loading response to terminal stance , where lee et al . quantified ankle impedance from pre - swing to early loading response . while stiffness component of impedance increases significantly as the stance phase of walking progressed , the change in damping during the gait cycle is much less than the changes observed in stiffness . in addition , both stiffness and damping remained low during the swing phase of walking . future work will focus on quantifying impedance during the push off region of stance phase , as well as measurement of these properties in the coronal plane .

Introduction Methods Results Discussion

PMC4338493

requisition storage and retrieval are an integral part of any routine or specialized outpatient clinical laboratory . patients receive a paper requisition from their physician indicating the required tests , and the patient brings it to a satellite bleed - station or a hospital for specimen collection . the process of specimen collection involves a number of individuals : the ordering physician or their surrogate , data entry clerk , phlebotomist , and laboratory assistant or technologist . as such , there is ample room for errors at the level of order - entry . these are either brought to the hospital bleed - station or , for referred - in specimens , come from another laboratory along with the collected specimen . if the patient has no record in our laboratory information system ( lis ) , a record is created , the specimens are then electronically recorded as arrived , a barcode label is created for each tube and then the specimen is appropriately routed for testing in high volume and/or specialized areas as needed . in the past , when there was a question about the possibility of order - entry error ( misidentification of ordering physician , demographic information in question , unusual test - request or test combination , question from referring laboratory , possible specimen mix - up ) , we would manually retrieve the paper requisition from filing cabinets located in the accessioning area by date . these were stored in the order they were received . after a period of about 6 weeks , these requisitions are placed into a long - term storage facility for a period required by the accreditation body . as we receive up to 1000/day , the task of finding a requisition is laborious . however , since a one - dimensional barcode label with the patient 's unique provincial health number ( phn ) is automatically generated along with the barcodes produced for each of the collected samples , it seemed natural to apply this label to each requisition and build a solution to electronically store the requisitions for more rapid retrieval . while a number of commercial products for requisition storage and retrieval exist , our budgetary constraints did not permit us to pursue these options . given the simplicity of our needs , namely , to be able to find a requisition for confirmation of demographic , diagnostic and test - request information , it seemed reasonable to use a number of available open - source tools to create a program capable of scanning a patient requisition and storing the it by according to the content of any barcode(s ) present . the goal of this project was to produce a simple , entirely open - source tool solution to this problem . the software we developed , dubbed reqscan , requires an automatic document feed ( adf ) scanner connected to a computer running gnu / linux . it is written in python , a cross - platform scripting language , which functions as a python and all other required software tools for this project ( so - called dependencies ) , are free and open - source . reqscan interfaces with scanners using scanner access now easy ( sane ) , a public domain application programming interface which supports thousands of devices . the program scans all pages in the adf source ( which can be specifically selected if there is more than one ; for example , a front and rear loader ) , and the files are saved to a temporary directory as tiff files according to the scanning resolution chosen . they are then processed using zbar , a barcode scanning library , and saved in portable document format ( pdf ) in a folder named after the current date , with file names consisting of the barcode data . if more than one barcode is found on a given page , the requisition is saved as multiple separate pdfs each having a unique barcode name corresponding to the barcode content . if the same barcode is found on different pages , the files are saved as ( barcode_content).pdf , ( barcode_content)_2.pdf , ( barcode_content)_3.pdf etc . , where ( barcode_content ) represents the encoded information . errors ( such as records with missing barcodes ) are handled robustly , such that jobs with hundreds of records will not terminate due to an error with individual files . the scans of the failed files are saved as individual pdf files named failed_1.pdf , failed_2.pdf etc . , these can easily be concatenated for rapid review if desired as we discuss below . both a command - line and a graphical interface [ figure 1 ] are available . reqscan window displayed on ubuntu linux 14.04 reqscan supports all the barcode symbologies supported by zbar ( ean-13 , ean-8 , upca , upce , isbn-13 , isbn-10 , i25 , code 39 , code 128 , and qr ) . we have also undertaken a preliminary implementation of data matrix barcodes using libdmtx but , unlike the other symbologies implemented through zbar , currently this is not searching for multiple data matrix barcodes per page . retrieval of previously scanned requisitions is a simple matter of searching the file system ( or an appropriately dated folder ) for a phn using gnu / linux built - in file searching commands ( e.g. find or locate ) or search options in gui file managers . reqscan is available for download for free at https://github.com/eviatarbach/reqscan under the gnu general public license version 3 . paul 's hospital in vancouver , bc , canada since january of 2013 , scanning all incoming outpatient requisition forms daily . between early january and mid - march 2013 , the second author invested approximately 10 h implementing customizations specific to the work - flow of his laboratory setting . specifically , barcode data containing undesirable characters , e.g. , : % , | , and , $ , # , and ^ were removed by renaming using linux command - line shell scripts . additionally , a short shell script using the open source tool , pdf toolkit ( pdftk ) , to concatenate pages named failed_1.pdf , failed_2.pdf failed_n.pdf into a single file was undertaken . requisitions from the in - hospital outpatient phlebotomy station were generally hand - written original from the ordering physician . these are barcoded in our laboratory accessioning with a label containing the patient phn in code 128 symbology . those from in - hospital clinics are also the original but may contain one or more barcodes associated with the work - flow of the clinic . requisitions accompanying referred - in specimens were provided as : ( 1 ) a photocopy of the original ( 2 ) a single - page printed request for a single patient ( without a photocopy of the original requisition ) stating the patient 's identifying information , off - site sample collection information ( e.g. ordering and cc physicans , collection time , receive - time , shipment date , accession number , test requests ) ( 3 ) as in situation ( 2 ) but with a stapled appended photocopy of the original requisition ( 4 ) a single page containing printed requests for multiple patients and the accompanying off - site sample / demographic information but without copies of the original requisitions . our label - printing process for accessioning specimens using sunquest v 6.4 ( tucson , az ) is currently set to print container i d barcode labels for all collected specimens from a given patient , but only one barcode referring to the patient 's phn . for this reason , single - page requisitions referring to a single patient ( original , photocopy or printed ) are barcoded with a single phn label . multipage requisitions for a single patient are barcoded with the phn on their first page only . single - page requisitions referring to multiple patients are barcoded with multiple labels for the phns of all patients to which the requisition refers . in our situation , we set reqscan to search for code 39 and code 128 only , as these are the symbologies used by our laboratory and some onsite clinics . numerous other barcode symbologies may appear on requisitions sent to us from other hospitals , clinics or private laboratories , but these are set to be ignored since the barcode content may refer to patient identifiers or sample accession numbers of other hospitals or their lis . a 1-week period from sunday october 5 to saturday october 11 , 2014 was selected to examine barcode retrieval success rates . in that week , 4,653 individual sheets were scanned at 300 dpi . reqscan found 4,528 ( code 39 and code 128 ) barcodes and corresponding pdf files were created . these files were visually inspected for barcode labels that should have been identified under ideal circumstances . thirty - five of the 1,025 pages did contain barcode information that reqscan should have picked up . this meant that a total of 4,528 of 4,563 available barcodes were successfully identified over 4,653 pages making a success rate of 97.3% for conversion to a searchable pdf document . notably ; however , only about 1/3 of missed barcodes were those applied in - house . barcodes that were not successfully found were typically rotated to angles between about 35 and 55. we have not observed examples of a barcode being decoded incorrectly , nor would we expect to since barcodes generally contain internal checksums . though we do not currently employ two - dimensional barcodes for patient identification labels , a decision outside the authors control , we did want to assess the success of reqscan on qr codes at various sizes and orientations . using the linux command - line utilities qrencode and the open - source utility latex , qr codes were generated as 1,200 dpi png images and incorporated into a pdf document at a base size of 20 mm 20 mm and rotated to angles of 0340 at increments of 20 ( for 18 equally sized barcodes at various rotations ) . each barcode was encoded with the phrase the quick brown fox at x degrees , where x was the extent of rotation . duplicate images were produced but scaled down in size to 60% ( 12 mm 12 mm ) , 40% ( 8 mm 8 mm ) and 30% ( 6 mm 6 mm ) relative to their original size . this four - page document containing 72 barcodes and various size and rotation combinations was printed at 600 dpi and run through reqscan in triplicate . barcode identification success was 100% at 600 dpi scan resolution , 97.6% at 500 dpi , 94.9% at 400 dpi and 78.2% at 300 dpi . not surprisingly , unrecognized barcodes were uniformly among the smallest ( 6 mm 6 mm at 500 and 400 dpi scanning resolution and both 6 mm 6 mm and 8 mm 8 mm at 300 dpi scanning resolution ) . the setup of this software requires some knowledge of installing and updating a linux system . it also requires the user to be able to use the linux software repository of their linux distribution ( there are many ) , and use the appropriate command - line tools to install the necessary additional open - source tools ( dependencies ) required . these tools are : python 2.7 , tkinter , the python - zbar bindings , python imaging library , scanimage , dmtx - utils , and imagemagick . it is natural for the user to want to customize the output to suit their needs . for example , they may wish to delete files of a certain name - format , shorten the names to the first n characters , suppress undesirable symbols arising in barcodes from outside sources , or concatenate the one - page pdf files of pages on which no barcode information was retrieved . these kinds of file manipulations are part of basic knowledge of linux shell scripting and could be undertaken by any determined individual with introductory programming experience such as that afforded by an introductory undergraduate course in computer science . another natural file - manipulation a user might wish to undertake is to concatenate a barcoded page that is followed by a series of unbarcoded pages that all refer to the same patient . of course , this approach assumes that pages remain in the order they were received . although we considered doing this , we found that it too often led to the concatenation of documents from two separate patients . however , this procedure can be performed programmatically by sorting the current directory 's file list by the pdf 's timestamp ( i.e. time of creation ) and concatenating a page successfully named by its barcode content with the successive n files named failed_1.pdf , the user could also use the shell scripts and pdftk to concatenate all pdfs for which identical barcode content was found , as these are named according to the barcode information followed by an integer that is incremented as many times as necessary . finally , it would be a trivial task to make the day 's pdfs write - protected by scheduling a so - called cron - job wherein the administrator ( root ) could schedule a command to alter the write - privileges of new folders at a fixed time daily . because there are so many possibilities of customizations that could be undertaken with shell - scripts , we have not implemented them , realizing that sites should decide on their needs and undertake these simple shell scripting tasks themselves . however , if this seems daunting , the easiest way to make reqscan function out of the box is to make sure a clear , near - horizontal barcode is applied to each page of all single - page and multi - page requisitions . if care is taken in application and scanning resolution is high enough , near 100% scanning success rate is expected , and the small number of failures could be manually renamed . it has been our observation that one - dimensional barcodes can be consistently found at 300 dpi scanning resolution but that two - dimensional barcodes require 600 dpi . we elect to scan images at 300 dpi resolution rescale images to 25% of their original length and width which results in scanned pages which are between 50 and 150 kb and have dimensions of 636 and 825 pts and 75 dpi . this has not impeded our interpretation of a scanned requisition , but we could certainly re - evaluate this and store at a higher resolution if needed . it is important to note that we use this software to take care of a clerical problem and not an accreditation - mandated document storage requirement . at present , reqscan is not compliant with the provincial requirements for requisition storage . the provincial requirement is tiff only storage , 400 dpi resolution and storage on write - only media . notwithstanding , for the simple purpose it was designed , we have been satisfied with reqscan as the process of retrieval is essentially instantaneous . in terms of sunk - cost in setup , development and implementation - related customization , reqscan was written on a budget of $ 1500 by the first author . it was tested by a premedical student volunteer daily for 30 days and shell - script customizations requiring about 10 h of invested time were undertaken by the second author . systematic review of the causes of laboratory errors demonstrates that most can be traced to the preanalytical phase which includes errors in patient identification , the order - entry process , the specimen type , handling , processing , and storage . with respect to errors related to the interpretation of a paper requisition , in a study of 660 medical institutions in multiple countries , it was demonstrated that approximately 5% of outpatient laboratory requisitions were associated with at least one order - entry error and that 10% of institutions had errors with at least 18% of requisitions . after those related to the physician name , not surprisingly , these errors were traced to the laboratory ordering a test that was not on the requisition or not ordering a test that was . error rates for send - out tests are substantially lower ( about 2% ) but as high as 5% in some laboratories . while software of the type we provide here is not designed to prevent preanalytic errors per se , it does facilitate rapid requisition retrieval for confirmation and correction of any errors or omissions at the order - entry level in a post - hoc manner . one context in which the corresponding author has found reqscan particularly useful relates to samples collected for tumor localization . paul 's hospital performs aldosterone analysis for a province - wide primary aldosteronism screening program . part of this service involves tumor localization procedures in which multiple specimens are collection from the left and right adrenal veins and the inferior vena cava . errors in specimen identification could lead to the surgical removal of the incorrect adrenal gland . because 12 specimens from three anatomical locations , collected in close temporal proximity arrive for accessioning and order - entry at our laboratory all at once , there is always a risk for specimen mix - up . it is now very easy to electronically retrieve the scanned requisition from radiology and confirm that labeling is correct . additionally , those performing billing procedures for the laboratory have also found reqscan useful for correcting or confirming billings to the insurer that have been questioned or rejected . we recognize that there are advantages and disadvantages to the development of in - house software . the ways that we have managed to keep costs down on this project were : ( 1 ) to limit the scope of the project to a specific task that would be done well ( 2 ) to avoid developing features that would require professional consultants ( 3 ) to build the project around the work - flow rather than the converse ( 3 ) and to undertake modest customizations using the programming expertise available on site . keeping the project simple and dedicated as made reqscan a good solution for the one problem it addresses . the benefits of dedicated commercial scanning technology for rapid retrieval paper work associated with internal and referred - in anatomical pathology consult cases has been described , and although financial benefits were modest , uptake and utility was high . the availability of scanned requisitions is considered a standard part of the laboratory automation solution and in ideal circumstances would be integrated within or seamlessly link to the lis . from the perspective of avoidance of order - entry error , however , electronic physician order - entry has been found to be superior . obviously , commercial solutions would offer the manner of convenience and refinement of lis integration that is out of scope for this small - scale project . nevertheless , user satisfaction has been observed to be high and document retrieval is essentially instantaneous . the weekday human time - commitment for scanning is approximately 45 min for the 700 requisitions scanned each day . approximately half of this time is consumed with removing staples from requisitions sent from other hospitals and institutions . since implementation , we have had no downtime due to scanner breakdown and maintenance of the scanner is simply cleaning of the reflective surfaces read by the scanner optics . the rate at which zbar successfully identifies the barcode is not 100% but in real - world environments , barcodes are often haphazardly applied , partially obscure or accidentally marred with marker or pen . in the absence of these problems , we have not observed situations where the barcode could not be identified . two - dimensional qr barcodes , by nature of their built - in error correction , are much less vulnerable to information loss from distortion and marring and would be a good choice for an application like this where the documentation may have suffered some abuse before arrival . the setup of this software requires some knowledge of installing and updating a linux system . it also requires the user to be able to use the linux software repository of their linux distribution ( there are many ) , and use the appropriate command - line tools to install the necessary additional open - source tools ( dependencies ) required . these tools are : python 2.7 , tkinter , the python - zbar bindings , python imaging library , scanimage , dmtx - utils , and imagemagick . it is natural for the user to want to customize the output to suit their needs . for example , they may wish to delete files of a certain name - format , shorten the names to the first n characters , suppress undesirable symbols arising in barcodes from outside sources , or concatenate the one - page pdf files of pages on which no barcode information was retrieved . these kinds of file manipulations are part of basic knowledge of linux shell scripting and could be undertaken by any determined individual with introductory programming experience such as that afforded by an introductory undergraduate course in computer science . another natural file - manipulation a user might wish to undertake is to concatenate a barcoded page that is followed by a series of unbarcoded pages that all refer to the same patient . of course , this approach assumes that pages remain in the order they were received . although we considered doing this , we found that it too often led to the concatenation of documents from two separate patients . however , this procedure can be performed programmatically by sorting the current directory 's file list by the pdf 's timestamp ( i.e. time of creation ) and concatenating a page successfully named by its barcode content with the successive n files named failed_1.pdf , the user could also use the shell scripts and pdftk to concatenate all pdfs for which identical barcode content was found , as these are named according to the barcode information followed by an integer that is incremented as many times as necessary . finally , it would be a trivial task to make the day 's pdfs write - protected by scheduling a so - called cron - job wherein the administrator ( root ) could schedule a command to alter the write - privileges of new folders at a fixed time daily . because there are so many possibilities of customizations that could be undertaken with shell - scripts , we have not implemented them , realizing that sites should decide on their needs and undertake these simple shell scripting tasks themselves . however , if this seems daunting , the easiest way to make reqscan function out of the box is to make sure a clear , near - horizontal barcode is applied to each page of all single - page and multi - page requisitions . if care is taken in application and scanning resolution is high enough , near 100% scanning success rate is expected , and the small number of failures could be manually renamed . it has been our observation that one - dimensional barcodes can be consistently found at 300 dpi scanning resolution but that two - dimensional barcodes require 600 dpi . we elect to scan images at 300 dpi resolution rescale images to 25% of their original length and width which results in scanned pages which are between 50 and 150 kb and have dimensions of 636 and 825 pts and 75 dpi . this has not impeded our interpretation of a scanned requisition , but we could certainly re - evaluate this and store at a higher resolution if needed . it is important to note that we use this software to take care of a clerical problem and not an accreditation - mandated document storage requirement . at present , reqscan is not compliant with the provincial requirements for requisition storage . the provincial requirement is tiff only storage , 400 dpi resolution and storage on write - only media . notwithstanding , for the simple purpose it was designed , we have been satisfied with reqscan as the process of retrieval is essentially instantaneous . in terms of sunk - cost in setup , development and implementation - related customization , reqscan was written on a budget of $ 1500 by the first author . it was tested by a premedical student volunteer daily for 30 days and shell - script customizations requiring about 10 h of invested time were undertaken by the second author . systematic review of the causes of laboratory errors demonstrates that most can be traced to the preanalytical phase which includes errors in patient identification , the order - entry process , the specimen type , handling , processing , and storage . with respect to errors related to the interpretation of a paper requisition , in a study of 660 medical institutions in multiple countries , it was demonstrated that approximately 5% of outpatient laboratory requisitions were associated with at least one order - entry error and that 10% of institutions had errors with at least 18% of requisitions . after those related to the physician name , not surprisingly , these errors were traced to the laboratory ordering a test that was not on the requisition or not ordering a test that was . error rates for send - out tests are substantially lower ( about 2% ) but as high as 5% in some laboratories . while software of the type we provide here is not designed to prevent preanalytic errors per se , it does facilitate rapid requisition retrieval for confirmation and correction of any errors or omissions at the order - entry level in a post - hoc manner . one context in which the corresponding author has found reqscan particularly useful relates to samples collected for tumor localization . paul 's hospital performs aldosterone analysis for a province - wide primary aldosteronism screening program . part of this service involves tumor localization procedures in which multiple specimens are collection from the left and right adrenal veins and the inferior vena cava . errors in specimen identification could lead to the surgical removal of the incorrect adrenal gland . because 12 specimens from three anatomical locations , collected in close temporal proximity arrive for accessioning and order - entry at our laboratory all at once it is now very easy to electronically retrieve the scanned requisition from radiology and confirm that labeling is correct . additionally , those performing billing procedures for the laboratory have also found reqscan useful for correcting or confirming billings to the insurer that have been questioned or rejected . we recognize that there are advantages and disadvantages to the development of in - house software . the ways that we have managed to keep costs down on this project were : ( 1 ) to limit the scope of the project to a specific task that would be done well ( 2 ) to avoid developing features that would require professional consultants ( 3 ) to build the project around the work - flow rather than the converse ( 3 ) and to undertake modest customizations using the programming expertise available on site . keeping the project simple and dedicated as made reqscan a good solution for the one problem it addresses . the benefits of dedicated commercial scanning technology for rapid retrieval paper work associated with internal and referred - in anatomical pathology consult cases has been described , and although financial benefits were modest , uptake and utility was high . the availability of scanned requisitions is considered a standard part of the laboratory automation solution and in ideal circumstances would be integrated within or seamlessly link to the lis . from the perspective of avoidance of order - entry error , however , electronic physician order - entry has been found to be superior . obviously , commercial solutions would offer the manner of convenience and refinement of lis integration that is out of scope for this small - scale project . nevertheless , user satisfaction has been observed to be high and document retrieval is essentially instantaneous . the weekday human time - commitment for scanning is approximately 45 min for the 700 requisitions scanned each day . approximately half of this time is consumed with removing staples from requisitions sent from other hospitals and institutions . since implementation , we have had no downtime due to scanner breakdown and maintenance of the scanner is simply cleaning of the reflective surfaces read by the scanner optics . the rate at which zbar successfully identifies the barcode is not 100% but in real - world environments , barcodes are often haphazardly applied , partially obscure or accidentally marred with marker or pen . in the absence of these problems , we have not observed situations where the barcode could not be identified . two - dimensional qr barcodes , by nature of their built - in error correction , are much less vulnerable to information loss from distortion and marring and would be a good choice for an application like this where the documentation may have suffered some abuse before arrival . we present an open source solution for requisition scanning that finds the barcode(s ) on a document and saves the document as a pdf in a folder specified by the date and named according to the barcodes identified .

requisition storage and retrieval are an integral part of the outpatient laboratory testing process . it is frequently necessary to review an original requisition to confirm the ordering physician , patient demographics , diagnostic information , and requested tests . manual retrieval of a paper requisition is time - consuming and tedious . although commercial solutions exist for the scanning and archiving of barcoded paper requisitions , the tools to accomplish this are freely available from the open source software community . we present a simple dedicated piece of software , reqscan , for scanning patient laboratory requisitions , finding all barcode information , and saving the requisition as a portable document format named according the barcode(s ) found . this python application offers a simple solution to patient requisition digitization . reqscan has been successfully tested and implemented into routine practice for storage and retrieval of outpatient requisitions at st . paul 's hospital , department of pathology and laboratory medicine in vancouver , british columbia , canada .

INTRODUCTION APPROACH RESULTS DISCUSSION Set Up and User Modifications General Comments CONCLUSION

PMC4220389

laparoscopic pyeloplasty ( lp ) is a minimally invasive alternative to open pyeloplasty for the treatment of ureteropelvic junction obstruction . it has been shown to provide lower patient morbidity , shorter hospitalization and faster convalescence . however , there is an inherent risk of surgical ( blind trocar insertion , colonic injury , hemorrhage , ileus , urinoma formation ) and anesthetic complications ( gas embolism , extraperitoneal insufflation and surgical emphysema , pneumothorax and pneumomediastinum ) . an 18-year - old girl was diagnosed to have left pelviureteral junction obstruction during evaluation of left flank pain . three trocars , including a 10 mm umbilical trocar , a 10 mm trocar midway between the umbilicus and the symphysis , and a 5 mm trocar midway between the umbilicus and xiphoid were used . the entire procedure was completed in 90 minutes , and a drain was placed after confirming hemostasis . however , at the end of the procedure , before evacuation of pneumoperitoneum , the patient developed hypotension . this was initially responsive to fluid administration , but quickly deteriorated after weaning , resulting in severe hypovolemic shock with worsening of respiratory parameters ( tidal volumes , peak pressures and blood gases ) . a pneumothorax was suspected , and a needle was inserted into the left fifth intercostal space . however , it drained blood and a chest x - ray revealed a hazy left hemithorax . screening ultrasound and contrast enhanced chest computed tomography ( ct ) scan showed a massive hemothorax with no hemoperitoneum ( ultrasound and ct installations are within the operation theatre complex ) . a 28 french intercostal tube was inserted in the left fifth intercostal space , and 2 l of blood was drained . the patient continued to remain hypotensive despite resuscitation and blood transfusion and more than 500 ml blood drained through the intercostal tube during the subsequent 2 h. an emergency surgical exploration was planned . in any case of hemothorax following an abdominal procedure , the first suspicion is towards an abdominal cause and a laparotomy was performed but it did not reveal any blood in the peritoneal cavity . a subsequent left mini - thoracotomy revealed fresh and clotted blood in the pleural cavity , which was evacuated . an active bleeding vessel became evident in the thoracic aspect of the left diaphragm near the anterior costophrenic angle secondary to a small diaphragmatic cautery burn . hemostasis was achieved by under - running the bleeding vessel , and an intercostal drain was placed . the post - operative course was uneventful , and complete lung re - expansion was achieved . thoracic bleeding is a rare complication of elective abdominal surgery , mainly resulting from unrecognized , iatrogenic diaphragmatic tears . cristian et al . , have reported a case of massive right hemothorax causing hemorrhagic shock after laparoscopic cholecystectomy . the bleed was from a laceration of the parieto - diaphragmatic adherence , which was attributed to change in diaphragmatic position during pneumoperitoneum , as it has been demonstrated in animal models . have reported severe hemothorax after laparoscopic surgery for endometriosis , which was attributed to pulsatile active bleeding from scattered small endometriotic lesions present on the pleural surface of the right diaphragm . abreu et al . have reported that thoracic surgical complications occurred in only 0.5% of the patients . of the 1,129 patients operated laparoscopically , only one patient demonstrated a hemothorax ( 0.08% ) , which was attributed to accidental supracostal placement of the posterior port . most complications occur during creation of pneumoperitoneum and blind insertion of the first trocar , not because of the laparoscopic surgery . in this case , the hemothorax was probably caused due to injury to the diaphragm by an unrecognized cautery burn , following which the bleeding vessel retracted into the thorax . the colon is routinely mobilized during lp . the cautery burn probably occurred during the mobilization of the splenic flexure with the monopolar cautery . hypotension due to blood loss was evident only after evacuation of the intra - abdominal gas and the natural suspicion was of an intra - abdominal source of bleeding . there needs to be a high index of suspicion of possible thoracic complications during laparoscopic urological procedures .

laparoscopic pyeloplasty is viable standard minimally invasive alternative to open pyeloplasty for the treatment of ureteropelvic junction obstruction . intrathoracic bleeding is an extremely rare complication after laparoscopic urological surgery , but it should be suspected and promptly diagnosed in case of worsening hemodynamic status and respiratory parameters during the intra or post - operative course . we report a case of hemothorax complicating an otherwise uneventful lp in an 18-year - old girl .

INTRODUCTION CASE REPORT DISCUSSION

PMC3214698

repetitive sequences , such as trinucleotide repeats ( tnrs ) , are spread all over the genome and can be found in intergenic regions , 5 regulatory regions , promoters , introns , or exons . such sequences are particularly prone to mutate , and their rate of mutation can be several orders of magnitude higher than that of bulk dna . a change in the repeat number of a given repetitive sequence can influence gene expression [ 24 ] , allowing morphological evolution or generation of diverse social behaviors . on the other hand , a dozen genetic diseases ( e.g. , myotonic dystrophy , huntington 's disease , and a variety of ataxias ) are caused by expansion of a tnr sequence located at a specific locus of the genome [ 711 ] . similarly , a variety of cancers are due to frameshift mutations in the repetitive sequence of a given gene [ 12 , 13 ] . the molecular mechanism underlying repetitive sequence instability has not been completely unraveled , and current models are based on strand slippage during replicative or repair dna synthesis . strand slippage is made possible by the repetitive nature of the sequence and leads to the formation of a hairpin on the template or newly synthesized strand . a recent study has provided direct evidence for hairpin formation during tnr replication in vivo although the precise time at which hairpin forms is still unknown . many of the in vitro studies published so far in the field of tnr instability relied on primer extension assays combining tnrs with different sequences and lengths and various dna polymerases . if primer extension assays are good models of gap repair dna synthesis , they are not fully adapted to study replicative dna synthesis . the use of replication miniforks containing a defined tnr sequence on which a reconstituted replisome of various complexities can assemble should be very helpful in dissecting the mechanism involved in tnr instability during dna replication . for instance , the activity of each component of the replisome can be easily examined and the experimental conditions changed without difficulty . the t4 replication machinery is an ideal system , since it is the simplest replication system that uses essentially the same set of regulatory proteins also used by higher organisms ( for reviews , see [ 1520 ] ) . it requires only seven proteins to initiate and catalyze coordinated in vitro leading and lagging strand dna synthesis with a speed , processivity , and fidelity similar to those measured in vivo . among the t4 replisomal proteins are ( 1 ) the dna polymerase , the gene product ( gp ) 43 , with a 53 dna synthesis activity and a 35 exonuclease or proofreading activity that removes misincorporated dnmps , ( 2 ) the helicase , gp41 , with a 53 double - stranded ( ds ) dna strand separation activity that unwinds the parental duplex by sterically excluding the ss leading strand template and encircling the ss lagging strand template , ( 3 ) the processivity factor , gp45 , a homotrimeric ring - shaped and noncatalytic protein that confers processivity to the dna polymerase , ( 4 ) the clamp loader , gp44/62 , a hetero - oligomeric complex that loads the processivity factor at a primer - template ( p - t ) junction , ( 5 ) the single - stranded ( ss ) dna binding protein ( ssb ) , gp32 , that binds and protects the naked ss dna exposed by the helicase , and finally ( 6 ) the primase , gp61 , that synthesizes small rna primers complementary to the ss lagging strand template that the lagging strand dna polymerase extends . an eighth t4-encoded protein called the helicase loader protein , gp59 , is required to load the helicase on ss dna covered by ssb . in this paper , we present a method to build replication miniforks containing a tnr of defined sequence and length . each minifork consists of three strands , namely , primer , leading , and lagging strand template . the primer anneals to the 3 end of the leading strand template to create a specific binding site , a p - t junction , for the dna polymerase . the lagging strand template is complementary to the leading strand template except in its 5 extremity . the ss tail at the 5 end of the lagging strand template constitutes the assembly site of the replicative helicase . the method relies on the preparation of each strand of the minifork separately followed by the hybridization of the three strands of the minifork . the dna synthesis activity of simple reconstituted bacteriophage t4 replisomes across specific ds tnr sequences was characterized . we show that a minimal replisome constituted by the t4 helicase ( gp41 ) and the t4 dna polymerase ( gp43 ) can replicate the leading strand template of random , structure - forming or nonstructure - forming tnr sequences . the t4 helicase loader ( gp59 ) increases the efficiency of strand displacement dna synthesis of the minimal homologous couple and may reduce slippage of the helicase that is , hydrolysis of atp nonassociated with ds dna unwinding . in contrast , a heterologous couple constituted by the t4 helicase and klenow fragment undergoes uncoordinated leading strand dna synthesis , even in the presence of the t4 helicase loader . coupled dna synthesis at various concentrations of dntps was performed and surprisingly revealed that the strand of the structure - forming 5cag/5ctg tnr that hinders the progression of the reconstituted t4 replication trio ( gp41-gp43-gp59 ) depends on the concentration of dntps . for instance , at high [ dntps ] , the greatest impediment to progression of the t4 replication trio is measured when the 5cag sequence is on the leading strand template . in contrast , at low [ dntps ] , the 5ctg sequence on the leading strand template creates a greater impediment to t4 replication trio progression than the 5cag sequence due to a very low efficiency of incorporation of damp across tmp of the 5ctg repeat . herculase - enhanced dna polymerase was from stratagene ; phusion high - fidelity dna polymerase was from finnzymes ; t7 exonuclease , t4 polynucleotide kinase ( pnk ) , and klenow fragment were from new england biolabs ( neb ) . t4 helicase ( gp41 ) , t4 dna polymerase ( gp43 ) , and t4 helicase loader ( gp59 ) were prepared as described [ 2125 ] . the proteins purified in our laboratory were estimated to be at least 90% pure by coomassie blue staining . their concentration was measured by uv spectroscopy using an extinction coefficient of 7.6 10 , 1.3 10 , 3.8 10 mcm for monomeric gp41 , monomeric gp43 and monomeric gp59 , respectively . the three plasmids derived from pcdna3 ( invitrogen ) and containing a tnr of defined length and sequence are listed and described in table 1 . oligonucleotides were from eurogentec and their sequence is listed in table 1 as is also their annealing position with respect to the repetitive sequence . the oligonucleotides ( 50t/4ps)/p867 and 1033/4ps carry four phosphorothioate linkages at their 5 extremity that make them resistant to the 53 t7 exonuclease . their concentrations were determined by uv spectroscopy using the extinction coefficients provided by the manufacturer . polymerase chain reactions ( pcrs ) were performed in the buffer of the dna polymerase provided by the manufacturer . the concentrations of plasmid , primers , dntps , and enzyme were as recommended by the manufacturer , except for the oligonucleotide that contains the phosphorothioate linkages whose concentration is half the concentration recommended by the manufacturer . the cycling conditions when using the phusion high - fidelity dna polymerase were as follows : initial denaturation ( 3 min at 98c ) ; 25 cycles of the three following steps ( 10 sec at 98c , 10 sec at 63c , 15 sec at 72c ) ; final extension ( 10 min at 72c ) . the cycling conditions when using the herculase - enhanced dna polymerase were as follows : initial denaturation ( 3 min at 98c ) , and 25 cycles of the three following steps ( 40 sec at 98c , 30 sec at 60c , and 30 sec at 72c ) , and final extension ( 10 min at 72c ) . after pcr , the nucleospin extract kit ( macherey - nagel ) was used to remove unused primers as recommended by the manufacturer . the pcr products were analyzed by electrophoresis on a 2% agarose gel in 1x tbe . t7 exonuclease digestion was performed in 1x neb buffer 4 ( 50 mm koac , 20 mm tris - oac , 10 mm mgoac2 and 1 mm dtt ph 7.9 at 25c ) at 25c . prior to large scale digestion , the enzyme concentration and the duration of the digestion were adjusted by performing a small - scale digestion . the large - scale t7 exonuclease digestion was stopped by adding edta ( final concentration of 80 mm ) and proteinase k ( final concentration of 1.5 mg / ml ) , and incubated for 10 min at 37c and 10 min at 65c . radiolabelling of the oligonucleotide p821 at its 5 extremity was performed by incubating 1 m oligonucleotide with 1 m { p}-atp , 1 m atp and pnk ( 0.2 u / ml ) in 1x pnk buffer ( 70 mm tris - hcl , 10 mm mgcl2 and 5 mm dtt ph 7.6 at 25c ) for 45 min at 37c . nonincorporated { p}-atp and atp were removed using a biospin 6 column ( biorad ) equilibrated in te buffer . miniforks were prepared by mixing the radiolabelled p821 primer with the ss leading and lagging strand templates in a buffer containing 40 mm tris - hcl ph 7.5 , 20 mm mgcl2 , and 50 mm nacl . strand hybridization was performed by heating ( 5 min at 95c ) and slow cooling . the mass ratio of acrylamide to bisacrylamide was 29 : 1 and native gels were in 1x tbe . except for klenow fragment , protein concentrations dna synthesis was performed in the buffer used to prepare the miniforks supplemented with 250 m each dntp ( unless indicated otherwise ) , 2.5 mm atp , and 2.5 mm dtt . the dna , gp43 , gp41 and gp59 concentrations were 10 nm , 30 nm , 200 nm , and 200 nm , respectively . klenow fragment was either at 1 mu/l or 10 mu/l as indicated in the figure . dna and dna polymerase ( gp43 or klenow fragment ) were preincubated for 3 min at 37c before adding gp41 premixed or not with gp59 . at the indicated times , the reaction was quenched by adding edta to 50 mm . proteinase k and sds were added to a final concentration of 3 mg / ml and 0.05% , respectively , and proteolysis performed for 20 min at 37c . one volume of denaturing blue ( 100% formamide , 0.01% bromophenol blue , and 0.01% xylene cyanol ) was added to the samples that were then heated for 5 min at 95c and loaded onto a 10% acrylamide sequencing gel ( mass ratio of acrylamide to bisacrylamide = 19 : 1 ) in 1x tbe . after at least 10 hours of exposure , the screen was scanned with a storm 820 ( ge healthcare ) . the dna substrates used for sequencing the ss leading strand templates were p - t junctions prepared in the 1x sequenase version 2.0 reaction buffer ( 40 mm tris - hcl ph 7.5 , 20 mm mgcl2 and 50 mm nacl ) and consisted of the radiolabelled p821 annealed to the ss leading strand template . the p - t junctions were treated for sequencing as recommended in the sequenase version 2.0 t7 dna polymerase kit ( usb ) . assays were performed in the buffer used to prepare the miniforks supplemented with 250 m of each dntp , 2.5 mm atp , 0.1 m { p}-atp and 2.5 mm dtt . the dna , gp43 , gp41 and gp59 concentrations were 10 nm , 30 nm , 200 nm and 200 nm , respectively ( concentrations expressed in monomeric units ) . when present , gp43 was preincubated with the minifork for 3 min at 37c before adding gp41 premixed or not with gp59 . at the indicated times the reaction was quenched by spotting an aliquot of the reaction on a pei cellulose thin layer chromatography ( tlc ) plate . atp and inorganic phosphate were separated by running the tlc plate in 0.35 m potassium phosphate buffer ( ph 3 ) . after at least 10 hours of exposure , the screen was scanned with a storm 820 ( ge healthcare ) . the strategy used to build replication miniforks of defined sequences is shown in figure 1(a ) and can be divided into three steps . the ss leading and lagging strand templates are prepared separately by a two - step procedure . in a first step , a pcr is performed with a plasmid containing a random or a tnr sequence ( table 1 ) and two oligonucleotides , one of them carrying a track of four phosphorothioate linkages at its 5 extremity to make it resistant to the 53 t7 exonuclease . the oligonucleotide couples for the leading and lagging strand templates are ( p821 , p1033/4ps ) and ( ( 50t/4ps)/p867 , p1033 ) , respectively ( see table 1 for oligonucleotide sequences and annealing positions ) . in a second step , the pcr fragments are treated with t7 exonuclease to specifically degrade the strand that is devoid of phosphorothioate linkage . miniforks are assembled in a third step by annealing the radiolabelled p821 primer , the ss leading and the lagging strand templates by a heating and slow cooling procedure . each minifork ( shown in a rounded rectangle in figure 1(a ) ) carries a p - t junction on which the dna polymerase can bind to initiate dna synthesis and a 5 ss tail on which the helicase with or without the assistance of the helicase loader can assemble . the 15 nucleotide ( nt ) gap of ss dna that exists between the 3 end of the p821 primer and the base of the ss tail of the lagging strand template ( figure 1(a ) ) makes it possible for the dna polymerase to assemble and initiate dna synthesis by filling the 15 nt - long gap of ss dna before starting coupled dna synthesis with the helicase . in what follows , the name of the tnr associated with a minifork refers to the tnr sequence of the leading strand template . for instance , the minifork containing a 5ctg repeat has the 5ctg repeat unit located on its leading strand template ( figure 1(b ) ) . two different dna polymerases were tested , the herculase - enhanced dna polymerase and the phusion high - fidelity dna polymerase , and both gave suitable results . to use all the oligonucleotide that carried the phosphorothioate linkages , its concentration was half the concentration recommended by the manufacturer . each plasmid ( p - empty , p-5gtt16 , p-5ctg17 , and p-5cag23 ; table 1 ) was used with the oligonucleotide couple specific of the leading or lagging strand template . a pcr fragment of expected size was synthesized in each case ( figure 2(a ) ) . the pcr fragments were next treated with t7 exonuclease to generate ss dna . as shown in figure 2(b ) ( data shown for the production of the ss leading templates containing 17 repeats of ctg and 23 repeats of cag ( left panel ) and the ss lagging strand templates containing 17 repeats of cag and 23 repeats of ctg ( right panel ) ) , a dna band with a slower mobility and a weaker intensity than the untreated ds pcr fragment appeared after t7 exonuclease treatment , indicative of the production of ss dna . first , radiolabelled p821 was annealed to the ss leading strand template to generate a p - t junction . hybridization of the two dnas was checked by electrophoresis on a native gel , because the free p821 primer and the p - t junction do not have the same electrophoretic mobility . free p821 indeed migrated faster than the p - t junctions ( figure 2(c ) ) . second , the ss lagging strand template was annealed to the p - t junction , and strand hybridization similarly checked by electrophoresis on a native gel ( figure 2(d ) ) . to test the quality of the miniforks , we first characterized the activity of the t4 dna polymerase ( gp43 ) alone or assisted by the t4 helicase ( gp41 ) and t4 helicase loader ( gp59 ) on a minifork carrying a random sequence . gp43 was incubated for 3 min with the minifork before adding gp41 premixed or not with gp59 to initiate strand displacement dna synthesis . as expected , during the first 3 min , gp43 filled the ss gap of the minifork up to the base of the ss lagging tail ( figure 3(a ) , lane 2 ) . gp43 was unable to efficiently synthesize through the dna duplex due to a very weak intrinsic strand displacement activity , and a dna intermediate corresponding to the p821 primer extended by 15 nts ( labelled ( b ) in figure 3(a ) ) accumulated . addition of gp41 allowed coupled dna synthesis across the duplex part of the minifork to take place ( figure 3(a ) , lanes 38 ) as pointed out by the accumulation of the full - length product ( labelled ( c ) in figure 3(a ) ) over time . coupled leading strand dna synthesis was highly stimulated by gp59 as a significantly higher amount of full length product accumulated over time when gp59 was part of the reconstituted replisome ( figure 3(a ) , lanes 915 ) . in addition , in the presence of gp43 , gp41 , and gp59 , three intermediate dna synthesis products ( indicated by a backward arrow in figure 3(a ) ) near the end of the parental dna duplex transiently accumulated to a significant extent ; they might correspond to pause sites for the minimal reconstituted replisome from where dna synthesis successfully resumed . similarly , klenow fragment has weak strand displacement dna synthesis activity that allowed it to synthesize through the duplex part of the minifork ( figure 3(b ) , lanes 2 and 5 ) . however , contrary to the gp43-gp41 replication couple , dna synthesis performed by klenow fragment was not stimulated by gp41 ( figure 3(b ) , compare lanes 2 , 3 , 5 , and 6 ) . under both conditions ( with or without gp41 ) , dna synthesis was highly distributive as indicated by the numerous pause sites that were clearly visible along the template strand . the addition of gp59 to the klenow fragment - gp41 replication couple stimulated dna synthesis ( figure 3(b ) , compare lanes 3 , 4 , 6 , and 7 ) , but dna synthesis remained distributive , suggesting uncoordinated dna synthesis with this heterologous replication system . miniforks containing 16 repeats of 5gtt , 17 repeats of 5ctg , or 23 repeats of 5cag on their leading strand template were built and the activity of minimal reconstituted t4 replisomes was tested on these miniforks . similarly to the minifork containing a random sequence , the gp41-gp43 replication couple synthesized dna quite efficiently across a nonstructure - forming ( e.g. , 5gtt ) or structure - forming tnrs ( e.g. , 5cag and 5ctg ) ( figure 4(a ) , result shown only for the minifork containing 17 repeats of 5ctg , lanes 3 and 4 ) . addition of gp59 stimulated leading strand dna synthesis fivefold ( figure 4(a ) , compare lanes 3 , 6 , 4 , and 7 ) . a similar stimulation of dna synthesis by gp59 , was measured with miniforks containing 16 repeats of 5gtt or 23 repeats of 5cag ( data not shown ) . the effect of gp59 on the atpase activity of gp41 during coupled leading strand dna synthesis was also evaluated . the gp41 atpase activity during coupled leading strand dna synthesis was measured in the presence or absence of gp59 and was compared to the activity measured in the absence of gp43 ( figure 5 , result shown only for the minifork containing 17 repeats of 5ctg ) . as expected , in the presence of the minifork as nucleic acid cofactor , gp59 stimulated the gp41 atpase activity in the absence of gp43 ( figure 5 , compare black and blue curves ) . this stimulation level is higher than reported in the absence of ss dna ( 2 , ) , indicating that under our experimental conditions , gp59 stimulates both the intrinsic and the ss dna - dependent atpase activity of gp41 . gp43 by itself was also able to stimulate the gp41 atpase activity ( figure 5 , compare black and red curves ) , but to a slightly lower extent than gp59 ( figure 5 , compare red and blue curves ) . nevertheless , the addition of gp59 to the gp41-gp43 replication couple led to a gp41 atpase activity comparable to the one measured for the gp41-gp59 couple ( figure 5 , compare green and blue curves ) . a similar trend of stimulation of the gp41 atpase activity by gp59 was measured with miniforks containing 16 repeats of 5gtt or 23 repeats of 5cag ( data not shown ) . the fact that gp59 stimulates the dna synthesis activity of the gp41-gp43 replication couple to a higher extent than it does the atpase activity ( fivefold ( figure 4(a ) , compare lanes 3 , 6 , 4 , and 7 ) versus twofold ( figure 5 , compare red and green curves ) ) possibly reflects a reduced amount of slippage of gp41 in the presence of gp59 during coupled strand displacement dna synthesis . dna synthesis activity of the reconstituted gp41-gp43-gp59 replication trio across the 5cag and 5ctg repeat unit was quantified . the rates of the minimal t4 replisome prior to , and across the tnr unit were estimated using the method previously described . briefly , the speed of the minimal reconstituted replisome before the tnr was determined by estimating the loss of signal intensity of primers extended by 14 to 16 nts ( indicated by the bracket labelled prior to tnr in figure 4(a ) ) . the intensity of primers of these lengths decreased with time , since they were intermediate dna synthesis products of larger extension products . to assess the effects of passage through the tnr region , the amount of elongation products that contained the full length tnr unit ( indicated by the bracket labelled past tnr ) increased over time , since these products accumulated as coupled leading strand dna synthesis proceeded . the results indicated that before reaching the tnr unit , the reconstituted gp41-gp43-gp59 replication trio assembled on the minifork containing a 5cag unit synthesized dna at a speed very similar to that of the reconstituted gp41-gp43-gp59 replication trio assembled on the minifork containing a 5ctg unit ( figures 4(b ) and 4(d ) ) . however , replicating through a 5cag repeat created a greater impediment to the minimal t4 replisome than replicating through a 5ctg sequence . the amount of synthesized dna containing the full - length tnr unit was indeed lower when the gp41-gp43-gp59 replication complex replicated through a 5cag template than when it replicated through the 5ctg repeat unit ( figures 4(c ) and 4(d ) ) . this result is in a perfect agreement with the relative degree of impediment created by 5cag and 5ctg repeats when these tnr sequences are presented to gp43 in a ss context . at high dntp concentrations , the reconstituted gp41-gp43-gp59 replication trio copied quite efficiently a leading strand template containing random , structure - forming or nonstructure - forming tnr sequences , and full - length product accumulated over time . however , such experimental conditions did not provide the opportunity to characterize the activity of the minimal replisome across the tnr unit itself . therefore , the speed of the gp41-gp43-gp59 replication trio was reduced by lowering the concentrations of the dntps and the dna synthesis pattern was examined under these conditions . as expected , reducing the concentrations of dntps decreased the size of the dna products that were synthesized by the minimal reconstituted t4 replisome during leading strand dna synthesis ( figure 6(a ) ) . the dna synthesis profile prior to , across , and beyond the tnr unit was established at 2.84 m dntps for the miniforks containing 5ctg ( figure 6(a ) , lane 4 ) and 5cag ( figure 6(a ) , lane 10 ) repeats . the results showed that the dna synthesis profiles prior to the tnr unit were very similar for both miniforks ( figure 6(b ) ) indicating that the sequence downstream of the tnr unit did not influence the activity of the reconstituted replication complex . in contrast , a strong blockage of dna synthesis was observed as soon as the gp41-gp43-gp59 replication trio hit the 5ctg sequence ( figure 6(c ) , left panel ) . this strong block was specific to the minifork containing the 5ctg sequence as a smooth profile of dna synthesis was observed with the minifork containing the 5cag sequence ( figure 6(c ) , right panel ) . these results showed that at low concentrations of dntps , the 5ctg repeat created a greater impediment to the minimal reconstituted t4 replication complex than the 5cag repeat . consequently , the situation at low concentrations of dntps differed from that observed at high concentrations of dntps , because at high concentrations of dntps , the 5cag repeat created a greater impediment to the minimal reconstituted t4 replication complex than the 5ctg repeat ( figures 4(b)4(d ) ) . all together , our data suggest that the steps that control the dna synthesis reaction at low and high concentrations of dntps are different . at low concentration of dntps , the 5ctg repeat located on the leading strand template created a greater impediment to the reconstituted gp41-gp43-gp59 replication complex than the 5cag repeat . to investigate which dnmp incorporation reaction was responsible for the impediment of the dna polymerase , a coupled leading strand dna synthesis assay was performed with one of the four dntps at a low concentration . we first established the dna synthesis profiles of the gp41-gp43-gp59 replication trio across a ds random sequence . as shown in figure 7(a ) , for a minifork containing a random sequence , the dna synthesis profile was specific to each reaction condition . for instance , the reaction performed at low concentration of datp ( figure 7(a ) , lane 2 ) gave shorter dna synthesis products than the reaction performed at low concentration of dgtp ( figure 7(a ) , lane 5 ) . to quantify this aspect of the reaction , we counted the number of each dnmp to be incorporated up to the tnr insertion site ( indicated by a backward arrow in figure 7(a ) ) and measured the amount of dna that was synthesized past the tnr insertion site under each of the four reaction conditions ( low [ datp ] , low [ ttp ] , low [ dctp ] , or low [ dgtp ] ) . to facilitate comparisons of efficiency of dna synthesis between the miniforks , the amount of dna synthesized under a given condition there was a clear inverse correlation between the number of dxmp to be incorporated up to the insertion site and the quantity of dna that was synthesized past the tnr insertion site when this dxtp was present at a low concentration ( figure 7(b ) ) . for instance , 20 damp and 9 dgmp had to be incorporated up to the insertion site , and the amount of dna synthesized beyond the insertion site at low datp concentration was 16 + / 5% of that formed at low concentration of dgtp . the same inverse correlation applied to the minimal reconstituted t4 replisome synthesizing through the non structure - forming 5gtt tnr sequence ( figure 7(c ) ) . in the case of a minifork containing a given tnr sequence , the number of dxmp to be incorporated up to the end of the tnr unit and the amount of dna synthesized beyond the tnr unit when this dxtp was present at a low concentration were compared . surprisingly , this inverse correlation no longer held when the gp41-gp43-gp59 replication trio replicated through a structure - forming tnr ( 5cag or 5ctg , figures 7(d ) and 7(e ) ) . for instance , the incorporation of dgmp opposite dcmp of the 5cag or 5ctg sequence gave the highest yield of dna synthesis although the number of dgmp to be incorporated up to the end of the tnr unit ( 32 in case of the 5cag containing minifork and 27 in case of the 5ctg containing minifork ) was not the lowest among the four dnmp to be incorporated . in addition , for both miniforks containing a structure - forming tnr , the same amount of damp or tmp had to be incorporated up to the end of the 5ctg or 5cag unit , respectively ( figures 7(d ) and 7(e ) ) , but the relative amount of dna synthesized past the 5cag unit under low concentration of ttp ( 40 + / 13% ) was roughly twice that synthesized past the 5ctg unit under low concentration of datp ( 22 + / 6% ) ( figures 7(d ) and 7(e ) ) . this result suggested that the incorporation of damp across tmp in the 5ctg sequence context was a more difficult reaction than the incorporation of tmp across damp in the 5cag sequence context , thus giving an explanation for the greater impediment to the gp41-gp43-gp59 replication trio created by a 5ctg unit than a 5cag unit at low dntp concentrations . repetitive sequences , including tnrs , are more prone to mutate than random sequences ellegren . recently , we proposed a new model ( called the template - push model ) for the dependence of tnr instability on the orientation of the replication fork and the deletion bias observed in vivo for these repetitive sequences . in this model , the tnr sequence that the replisome must replicate creates a greater hindrance for the progression of the leading than the lagging dna polymerase ; as a consequence , the replicative helicase and the leading dna polymerase transiently uncouple their activities , and a short gap of ss dna between the two proteins appears . to restore its coupling with the moving helicase and save time for dna synthesis , the leading dna polymerase passes over the small track of naked leading strand template without synthesizing dna . by this mechanism , polymerase - helicase coupling is maintained but at the expense of a hairpin that is formed on the template strand after protein coupling has been re - established . if it is left unrepaired or is repaired in an error - prone manner , the hairpin can induce a deletion of the tnr unit at the next round of replication . in this paper , we describe a method to build replication miniforks suitable for testing the template - push model , and we show how the use of these miniforks brought insights into the mechanism of tnr instability . although very simple to prepare , the miniforks assembled from purchased oligonucleotides are limited in size by the length of the oligonucleotides that can be chemically synthesized ( around 100 nts ) . our method of preparation of replication miniforks overcomes this limitation since the production of ss leading and lagging strand templates relies on pcr followed by the specific degradation of one strand of the pcr product by the t7 exonuclease ( figure 1 ) . as a consequence any sequence carried in a ds dna can be used , making it possible to assemble a replication minifork of any given sequence . the herculase - enhanced dna polymerase and the phusion high - fidelity dna polymerase the leading strand template indeed carries the expected number of repeats ( see sequencing lanes in figures 3 , 4 , 6 , and 7 ) . it is possible that the herculase - enhanced dna polymerase becomes more appropriate than the phusion high - fidelity dna polymerase when dealing with longer repeats because the former dna polymerase can faithfully and efficiently cope with long g - c rich targets ( stratagene ) . the striking conservation of the dna replication apparatus in bacteriophage t4 and in human cells make the t4 dna replication machinery an ideal simple model system to test in vitro the miniforks prepared by the method described above and investigate the activity of reconstituted replisomes of increasing complexities . a functional minimal replication complex composed of the t4 dna polymerase and the t4 helicase can perform strand displacement dna synthesis across a random sequence , nonstructure - forming and structure - forming tnrs ( figures 3 and 4 ) . the t4 helicase loader stimulates both the dna synthesis and the atpase activities of the helicase - dna polymerase replication couple , but interestingly to different extents . for instance , the twofold stimulation of the atpase activity of the t4 helicase by the t4 helicase loader ( figure 5 ) is associated with the fivefold increase of leading strand dna synthesis ( figure 4 ) . as indicated by its name , the t4 helicase loader stimulates the loading of the t4 helicase around naked or ssb - covered ss dna . whether this factor dissociates after loading of the helicase is unclear , and it is possible that the t4 helicase loader remains part of the t4 replisome . if the t4 helicase loader travels with the replication complex and if most of the atp is used to unwind the parental ds dna ( which we believe is true as under our experimental conditions , gp59 stimulates both the intrinsic and the ss - dependent atpase activity of gp41 ) , our result suggests that the t4 helicase loader prevents the slippage of t4 helicase , by reducing the amount of atp hydrolyzed that is not associated with forward translocation . using the method that quantifies the extent of impediment created by various tnr sequences to the progression of dna polymerases and by applying it to minimal reconstituted t4 replication complexes , we found that at high concentrations of dntps a 5cag leading strand template creates a greater impediment to the gp41-gp43-gp59 replication trio than a 5ctg leading strand template ( figure 4 ) . a similar ranking of these two sequences was reported when naked 5cag and 5ctg sequences were tested in a primer extension assay that did not required the t4 helicase . this result suggests that the ss dna exposed either by the t4 helicase or by a chemical denaturing treatment is similarly converted into ds dna by the t4 dna polymerase . in contrast , when the concentrations of all dntps are low , a 5ctg leading strand sequence more dramatically hinders the progression of the gp41-gp43-gp59 replication trio than a 5cag leading strand sequence ( figure 6 ) , suggesting a change in the rate - limiting step when the concentration of dntps varies . it is possible that at low dntp concentrations , the binding of the incoming complementary dntp by the dna polymerase becomes the rate - limiting step of the coupled dna synthesis reaction , whereas at high dntp concentrations another step , such as the chemical incorporation of dnmp or the translocation of the dna polymerase after the dnmp incorporation , becomes rate limiting . by keeping a single ( out of the four ) dntps at a low concentration , it was found that the incorporation of damp across tmp of the 5ctg sequence is a more difficult reaction than the incorporation of tmp across damp of the 5cag sequence context ( figure 7 ) , thus giving an explanation for the greater hindrance to progression of the minimal reconstituted t4 replisome created by a 5ctg sequence than a 5cag sequence under low concentration of dntps ( figure 6 ) . it is well known that dntps are not all four present at the same concentration in the cell , ttp being the most abundant dntp [ 2830 ] , and that specific imbalanced pools of dntps can be mutagenic . in e. coli , the pool of dntps drops during cell growth and when the cell transits from exponential growth to stationary phase . in addition , in eukaryotic cells , the pool of dntps is tightly regulated during the cell cycle and peaks during the s , g2 and m phases [ 28 , 3133 ] . during the g1 phase , the amount of dntps is low and the use of dntps is restricted to mitochondrial dna synthesis and repair . the fact that the concentration of dntps can regulate the progression of the gp41-gp43-gp59 replication trio across a 5cag/5ctg tnr unit in a strand - dependent manner suggests that the repair of a lesion or a gap located in a 5cag/5ctg sequence context , can have different outcomes if it takes place in the g1 or outside the g1 phase of the cell cycle . for instance , during base excision repair , after removal of a 2-hydroxyadenine located in a 5cag sequence context , damp needs to be incorporated opposite tmp of the ctg repeat . the one - nt gap intermediate formed after the excision of the lesion may have more chance to slip and to give rise to frameshift mutations during the g1 phase than early in the s phase before the passage of the replisome or during the g2 phase . at this stage of the cell cycle , the pool of dntps is indeed low and , as pointed out by our experiments , incorporation of damp across tmp is difficult . our results therefore point to a specific role of the phase of the cell cycle ( g1 versus g2 or s ) and the state of the cell ( dividing versus nondividing ) at which dna lesions are repaired .

instability of repetitive sequences originates from strand misalignment during repair or replicative dna synthesis . to investigate the activity of reconstituted t4 replisomes across trinucleotide repeats ( tnrs ) during leading strand dna synthesis , we developed a method to build replication miniforks containing a tnr unit of defined sequence and length . each minifork consists of three strands , primer , leading strand template , and lagging strand template with a 5 single - stranded ( ss ) tail . each strand is prepared independently , and the minifork is assembled by hybridization of the three strands . using these miniforks and a minimal reconstituted t4 replisome , we show that during leading strand dna synthesis , the dntp concentration dictates which strand of the structure - forming 5cag/5ctg repeat creates the strongest impediment to the minimal replication complex . we discuss this result in the light of the known fluctuation of dntp concentration during the cell cycle and cell growth and the known concentration balance among individual dntps .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

PMC4047246

approximately 8085% of the adult population has experienced or suffers from lower back pain , which results in difficulty while walking and turning in bed1 . in physical therapy in clinical settings , measures to alleviate pain frequently include manipulative physiotherapy in addition to physiotherapy and guidance for everyday management and exercise2 . however , some individuals who experience sudden onset of pain have a good prognosis in which the pain is alleviated without the cause of the pain becoming known . this is known as nonspecific lower back pain , and this pain may originate in the sacroiliac joints , the intervertebral joints , the muscles , or fascia or may arise from acute trauma to the annulus fibrosus of the intervertebral disc3 . of these , pain originating from the intervertebral joints occurs most commonly , and accounts for 7080% of all cases of lower back pain4 . the first - line treatment for nonspecific lower back pain is conservative therapy , with the main component being physical therapy5 . typically , conservative therapy includes rest , drug therapy , nerve blocks , traction therapy and physiotherapy . williams flexion exercises , exercises that focus on strengthening the abdominal muscles , and stretching are also performed in combination with pain relief5 . since conservative therapy for acute lumbago does not increase the sensation of physical restraint or fear of moving compared with aggressive exercise therapy , some reports state that it is best to continue everyday activities within acceptable pain limits2 . on the other hand , conservative therapy for chronic lower back pain involves therapeutic exercises consisting mainly of exercises for the trunk muscles and stretching , which are reported to be more effective than anti - inflammatory analgesics6,7,8 . when a noxious stimulus is applied to the area surrounding the intervertebral joints , referred pain associated with muscle spasms occurrs at a high frequency from the gluteal region to the tensor fasciae latae muscle ( including the iliotibial band ) . if left untreated , this can lead to muscle contraction , which exacerbates lower back pain . additionally , in the case of patients with chronic lower back pain who have no neurological symptoms in the lower limbs , performing ds of the tflm and hamstrings can result in an immediate improvement in posture and gait . however , while there are several studies regarding physical therapy for acute lower back pain that have focused on lower back pain exercises and physiotherapy , there are very few studies focusing on interventions targeting specific muscles , and we found no reports of 3-month follow - ups of subjects . in this study , we adopted a single patient with acute nonspecific lumbago as a subject . the subject performed ds of only the tflm , and the immediate effects thereof were analyzed using a single - case study design . we also conducted a 3-month follow - up study in order to investigate the usefulness of this therapy . the subject was a 60-year - old woman ( height , 157 cm ; weight , 58 kg ; body mass index , 23.6 ; hairdresser ) diagnosed with acute lumbago ( nonspecific lower back pain ) originating in the intervertebral joints . the patient had not experienced any back pain before and reported that the pain had occurred in the muscles near the 4th and 5th lumbar vertebrae due to long periods of standing ( as more than 12 hours ) , and that the pain increased the following day , rendering the patient unable to stand in the same position for longer than 5 minutes without sitting down . the purpose of this study was fully explained to the patient and her consent to participate was obtained in writing based on the principles of the helsinki declaration . the subject voluntarily agreed to participate in the experiment after listening to the purpose and methods of the study . on examination by the doctor , the patient was found to be negative for lasgue s sign , and was deemed to have no neurological symptoms such as weakening of the lower limbs or sensory disturbance . acute lumbago is defined as lower back pain that develops suddenly and that is not associated with neurological symptoms or pain in the lower limbs either during the initial examination or during the course of the illness . patients complain of pain that develops within a single day and a temporary inability to move the body or corresponding pain , with the patient seeking medical advice within 14 days of onset9 . because there are no specific diagnostic criteria for lower back pain originating from the intervertebral joints , diagnosis is made comprehensively using physical findings and imaging findings based on previous research and plain x - rays . diagnostic criteria9 included findings of localized tenderness approximately 2 cm from the midline in the area of the intervertebral joints , particularly at the height of the interspinous processes , pain in the multifidus muscle , an absence of trigger points in the affected and painful muscles , and anteflexion of the trunk with notably restricted torsion and retroflexion . differential diagnoses , which included neurological symptoms , tumors , or general medical conditions , were all excluded . the subject did not exhibit any serious neurological symptoms or clinical symptoms ; thus , the need for x - ray imaging was deemed to be low . but 2 days after the examination , the back pain remained unchanged , and the subject visited our offices . the single - case study design systematically measures and evaluates patient performance by providing planned treatment and is said to be a case study method with experimental elements that allows new intervention methods to be assessed on a pilot study - basis10 . the ab - type design involved repeating an a period ( primary basic significance level period ) and a b period ( treatment introduction period ) , which is the most basic method of verifying a treatment and its effectiveness . various dependent variables can be assessed by establishing periods in which conventional treatment is performed or not performed ( periods a and b ) and by establishing a period in which a new treatment is performed . in this study design , a position that alleviated the lower back pain was adopted for 10 minutes during period a , and ds of the left and right tflm was performed for approximately 10 minutes during period b. during this time , treatment was performed in the first 5 minutes , and the remaining 5 minutes were used for rest . during the treatment , a thumb was applied to the muscle being treated while exerting as little pressure as possible , and the opposite hand was used to press down on the thumb from above , which caused extension . the main areas for treatment were the tflm and the myotendinous junction of the iliotibial band . the amount of pressure applied did not exceed 5 on the vas . with the subject in the prone position , the hip joint was abducted to 45 degrees with the knee flexed at 90 degrees . pain was measured using the visual analog scale ( vas ) , finger - to - floor distance ( ffd ) , and degree of lumbar kyphosis . after intervention in the a and b periods , the changes in the vas , ffd , and lumbar kyphosis indices were measured 5 times a minute . a 100 mm scale was used for the vas measurement , with the left end of the scale indicating no pain at 0 mm and the right end of the scale indicating pain at 100 mm . during ffd measurement , the subject was instructed to attempt to reach as far as possible toward the tips of their toes with their fingertips without bending the knees , and to stop bending forward when pain appeared in the lumbar region . at this point , the posterior lumbar flexibility ( plf ) test was used to assess the range of posterior lumbar mobility . the test starts with both hips at 45 degrees of flexion ; the hip joint of the leg placed superiorly is then flexed , and the thigh is brought as close as possible to the chest until resistance is felt . during flexion , the angle at which pain was reported was recorded and was used as an indicator of lumbar flexibility . we also used the same indicators for the entire 3 months and performed a follow - up study of physical function . no interventions were added , and changes in the vas , ffd , and plf indices were measured 5 times in 1 minute . the patients were advised to stay active and continue ordinary daily activity as tolerated . she was also advised to maintain the pelvic tilt at all times , so that her lower back did not sway forward . when doing any task where she would be standing for a long period of time , such as ironing or doing dishes , shampooing , or , face sled , she was advised to put step stool or to sit on a high stool . her most common sleeping position was on her side , with her legs and hips aligned and flexed . because this position leaves her upper leg unsupported , the top knee and thigh tend to slide forward and rest on the mattress , rotating the lower spine . this slight rotation may contribute to back or hip pain . to prevent that problem , she was advised to place a pillow between her knees and thighs . to determine the efficacy of the intervention , a statistical analysis of changes in the vas , ffd and plf obtained during periods a and b was performed using the probability of binomial distribution . the probability of binomial distribution used in this study involved creating a regression line using the least squares method from a data point in period a , extending this regression line to period b , and statistically testing the number of data points that fell above ( or below ) the line11 . the binomial distribution was calculated using the formula p(r)=n!/r!(n - r)!pq . in this formula , n was the total number of data points in period b and p and q were the probability that the data points lay above or below the regression line . after the first treatment ( first day ) , we noted a significant decrease in the vas and ffd and a significant increase in the plf test results .. after the second treatment ( first week ) , we observed a significant decrease in the vas and ffd and a significant increase in the plf test results . after the third treatment ( second week ) , we observed a significant decrease in the ffd and a significant increase in the plf test results . after the fourth treatment ( third week ) and fifth treatment ( fourth week ) , no significant differences were observed in either the ffd or the plf test results . the vas was 0 mm from the third treatment ( second week ) onwards ( table 1table 1 . effects of direct stretching of the tensor fasciae latae musclefirst treatmentafter 1 weekafter 2 weeksafter 3 weeksafter 4 weekstermvasffdplfvasffdplfvasffdplfvasffdplfvasffdplf851511024101200101300514505145821511028101200101350514505145a861511023101200101350514505145891511022101200101300514505145841511024101200101300514505145251012005135051450514505145231012005135051450514505145b231012505135051450514505145251012505135051450514505145211012005135051450514505145a : primary basic significance level period , b : treatment introduction period , vas ( mm ) : visual analog scale , ffd ( cm ) : finger - to - floor distance , plf ( ) : posterior lumbar flexibility ) . , no significant differences were noted between the ffd and plf results in the second and third months in period b ( table 2table 2 . results from the 3-month follow - uptermvasffdplfvasffdplfvasffdplf051450514505145051450514505145a051450514505145051450514505145051450514505145a : primary basic significance level period , vas ( mm ) : visual analog scale , ffd ( cm ) : finger - to - floor distance , plf ( ) : posterior lumbar flexibility ) . a : primary basic significance level period , b : treatment introduction period , vas ( mm ) : visual analog scale , ffd ( cm ) : finger - to - floor distance , plf ( ) : posterior lumbar flexibility a : primary basic significance level period , vas ( mm ) : visual analog scale , ffd ( cm ) : finger - to - floor distance , plf ( ) : posterior lumbar flexibility in order to determine the effect on lower back pain , we used the vas as the pain scale , ffd for flexibility , and the plf test for posterior lumbar flexibility . the vas and ffd are also easy to use in clinical settings and are easily for patients to understand . the plf test is used to measure the range of motion of the lumbar spine , and negative conversion is only considered to have occurred when sufficient posterior inclination of the pelvis is permitted , at which point at which the flexibility of the lumbar spine first starts to improve . the plf test is a simple test of contracture to assess mobility of the lumbar spine . noxious stimulation of the intervertebral joints is the cause of reflexive spasms of the multifidus muscle , and findings of multifidus muscle tenderness are important for the assessment of lower back pain originating in the intervertebral joints11 . the sudden onset of lower back pain in our subject was thought to have been caused by mechanical stimulation of the intervertebral joints due to long periods of standing triggering reflexive spasms of the multifidus muscle . most cases of acute lumbago recover spontaneously , but when the pain is inappropriately neglected , in addition to persistent spasms of the multifidus muscle , the withdrawal reflex is enhanced due to increased secondary hyperalgesia at the level of the spinal cord , which creates a new movement pattern and ultimately results in a vicious cycle of pain . severe pain associated with reflexive spasms in the multifidus muscle due to noxious stimuli leads to immobility of the lumbar region . furthermore , a new movement pattern associated with enhanced withdrawal reflex is formed , resulting in functional compensation by the hip joints below the lumbar spine , and forcing the hip joints to move excessively . the contraction and muscle spasms of the tflm increase the tension in the iliotibial band , thereby promoting anterior inclination of the pelvis . contractions and muscle spasms in the hamstrings act to promote posterior inclination of the pelvis . in other words , simultaneous contraction of both muscle groups reduces the degree of freedom of the pelvis , and this increases the amount of stress placed on the lumbar region during everyday activities . the compensatory tflm spasms that result from this can be understood as a secondary disorder , which enhances the lower back pain and is assumed to be one cause of chronic lower back pain . the reason the lower back pain not progressing to chronic lower back pain in our subject is likely due to the compensatory tflm spasms being assessed at an early stage and thus improving . in the present study , by only applying manipulative extension to the tflm , the range of motion of the hip joint and pelvis significantly improved , which may have led to decreased pain in the lumbar region . kippers et al.13 noted a correlation with anteflexion of the trunk in a standing position when the lower limbs were extended and elevated . however , the correlation with lumbar flexion was weak , and trunk anteflexion in a standing position was found to reflect the extensibility of the hamstrings . in the present study , ffd improved immediately , irrespective of the fact that no extension of the hamstrings was performed . the tflm is commonly connected to the hamstrings through the fasia14 . as a result the tflm fibers penetrate the fascia lata , the gluteus maximus muscle , the gluteus medius muscle , and the vastus lateralis muscle14 . we assume that the degree of tension on the tflm may indirectly influence the muscle tension of the muscle groups that surround the thigh . a significant increase was also observed in the mean plf values . in general , in cases of lower back pain originating from the lumbar intervertebral joints , posterior lumbar flexibility is reduced due to persistent muscle spasms of the multifidus muscle and restriction of the intervertebral joints themselves . accordingly , multifidus muscle relaxation and improvement of the range of motion of the lumbar intervertebral joints have been suggested in order to increase the range of posterior lumbar flexibility . in this study , ds of the tflm had an immediate effect on acute lumbago , and results of a 3-month follow - up study showed that no recurrence of pain occurred during the 3 months , the minimum period required to determine efficacy . the above results suggest that ds of the tflm not only has an immediate effect on acute , non - specific lumbago , but also has a long - term effect .

[ purpose ] a 3-month follow - up study was conducted on a patient diagnosed with acute nonspecfic lumbago . direct stretching ( ds ) of the tensor fasciae latae muscle ( tflm ) was performed , and an immediate effect was confirmed . [ subjects ] the case subject was a 60-year - old woman diagnosed with acute nonspecific lumbago . [ methods ] we used a single - case study design and an ab - type study structure , in which the leg was placed in positions that relieved the back pain in period a and ds of the tflm was performed in period b. the evaluation indices were the visual analog scale ( vas ) , finger - to - floor distance ( ffd ) , and posterior lumbar flexibility ( plf ) , which were analyzed using the binomial test . [ results ] the vas , ffd , and plf in period b showed significant improvement when compared with period a. additionally , complaints of lower back pain ceased after 2 weeks , and the results of the follow - up study showed no recurrence of back pain during the 3 months . [ conclusion ] the results of this study suggest that ds of the tflm has an immediate effect on acute nonspecific lumbago in addition to long - term pain relief effects .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3654563

g protein - coupled receptors ( gpcrs ) form a large and important protein family with 390 members ( excluding olfactory receptors ) in the human genome . gpcrs are critically involved in cell signaling in response to a wide range of endogenous ligands , including hormones , neurotransmitters , cytokines , odorants , and light . they fall into three major classes ( families a , b , and c , of which family a , the rhodopsin family , is the largest ) . historically , the gpcr arena has been a fruitful one for drug discovery , with a large number of small molecule drugs being successfully developed in multiple therapeutic areas , together with a smaller number of new biological entities . gpcr drug discovery continues to be an area of significant focus for the pharmaceutical industry , with over 60 new gpcr drugs , almost a quarter of the total number of approved new chemical entities ( nces ) in this period , being launched in the past 10 years . despite this success , only one new gpcr target per year on average has been drugged in the past decade , indicating that gpcr research remains a difficult area and one in which there are many targets with clinical relevance and validation that are currently underserved by drug discovery efforts . historically , the vast majority of gpcr drug discovery efforts have relied upon cell - based assays coupled with high - throughput screening of large compound libraries for hit identification . this approach has resulted in limited success for challenging , clinically valuable targets such as neuropeptide receptors , chemokine receptors , family b peptide - hormone receptors and family c metabotropic glutamate ( mglu ) receptors . until recent years , in contrast to soluble protein classes such as enzymes , x - ray crystal structures of gpcrs had been lacking with only the structure of the visual pigment rhodopsin , first reported in 2000 , being available to guide structure - based drug discovery efforts . after a gap of 7 years , structures of the 2-adrenergic receptor ( 2ar ) were published , rapidly followed by the revelation of medium to high resolution crystal structures of 14 additional receptors ( 1-adrenergic ( 1ar ) , adenosine a2a , sphingosine 1-phosphate 1 ( s1p1 ) , chemokine cxcr4 , dopamine d3 , histamine h1 , muscarinic acetylcholine m2 and m3 , neurotensin , opioid receptors , , , nociceptin , and protease - activated receptor 1 ( par1 ) . it has also recently been disclosed that the first example of a family b gpcr structure , that of the corticotropin releasing factor ( crf-1 ) receptor , has been solved . the significance of this recent upsurge in gpcr crystallographic information to drug discovery has been recently reviewed . the success in obtaining new x - ray crystal structures has been due to technical advances that have stabilized purified receptors in detergent solution and locked them in specific conformations , both of which are necessary for the formation of well - diffracting crystals . two key approaches to enable the determination of gpcr structures have been devised in recent years . first , insertion of the enzyme t4 lysozyme ( t4l ) into intracellular loop 3 ( icl3 ) of the receptor to promote the formation of crystal contacts has been used . the high resolution ( 2.4 ) structure of the 2ar , previously solved at 3.4 in complex with an antibody fragment , was the first demonstration of this strategy . the t4l approach has been used in combination with the lipidic cubic phase ( lcp ) crystallization method . the second approach introduces a small number of point mutations into the receptor construct , significantly increasing thermostability of the protein and thereby enabling purification in the short chain detergents required for crystallization by vapor diffusion . thermostabilized receptors form well - ordered crystals , as first demonstrated in the structure determination of the turkey 1ar at 2.7 resolution . the thermostabilization approach has the advantage of locking the receptor into a single homogeneous conformation , determined by the pharmacology of the ligand used in the stabilization process , greatly facilitating purification and structure determination . stabilized receptors formed via this approach are also known as stars and typically contain a small number of thermostabilizing mutations , six in the case of the 1ar star ( 1ar - m23 ) , which is stabilized in an antagonist conformation . adrenergic receptors ( adrenoceptors ) are family a gpcrs that activate intracellular g proteins upon binding of endogenous catecholamine ligands such as adrenaline and noradrenaline . the adrenergic receptors are split into and classes , with the latter having 1 , 2 , and 3 subtypes . more than 4 decades of research and development in the area of -adrenergic receptors have resulted in a legacy of clinically important agonist and antagonist molecules with varying degrees of selectivity . antagonists of -adrenergic receptors are frequently used in cardiovascular medicine , as well as in other areas such as migraine and anxiety , and -agonists are used in the treatment of asthma and chronic obstructive pulmonary disease ( copd ) . crystal structures of the human 2ar and turkey 1ar in complex with the inverse agonist carazolol 1 and antagonist cyanopindolol 2 , respectively ( chart 1 ) , were reported in 2007 and 2008 . subsequently , structures with the full agonists carmoterol 3 and isoprenaline 4 and the partial agonists salbutamol 5 and dobutamine 6 in the 1ar were published ( chart 1 ) , as were structures with carazolol and the antagonist iodocyanopindolol in the same receptor . 2ar crystal structures with the inverse agonist ici118,551 and agonist bi-167107 , the latter in a nanobody - stabilized active state of the 2ar , have been published . more recently , structures of the stabilized 1ar bound to the blockers bucindolol and carvedilol , which are classified as biased agonists because they stimulate g protein - independent signaling , have been reported . in comparison to other 1ar structures , both of these ligands make additional contacts to helix 7 and extracellular loop 2 ( el2 ) of the receptor , an observation that may provide an insight into the structural requirements of biased ligands . taken together , the 1ar and 2ar structures provide a wealth of structural understanding within the -adrenergic area , including insights into ligand receptor interactions that impart antagonist , partial agonist , or full agonist pharmacologies . the advent of gpcr crystal structures has initiated a new era of structure based drug design ( sbdd ) for this important target class . in concert with sbdd , fragment - based drug discovery ( fbdd ) as a strategy for identifying small ( 100250 da ) , efficient hit molecules is now a well established technique . fragment hits , when coupled with a structural understanding of how they interact with their target protein , represent excellent starting points for medicinal chemistry , and many examples of how fragments have been successfully optimized to potent leads have been published . indeed , multiple agents have been progressed into clinical trials in recent years and the first fragment - derived drug , vemurafenib , has reached the market . biophysical methods such as spr , nmr , and x - ray crystallography are among the mainstays of fbdd approaches used for soluble protein targets , but their application to the gpcr field has been highly challenging because of low expression and poor stability of the target receptor when isolated from the cell membrane . for these reasons , fbdd has been rarely utilized for gpcrs to date . the engineering of stars with significantly increased thermostability has recently enabled fragment screening of gpcrs to be validated for the first time using a variety of biophysical and biochemical approaches . screening of the 1ar and adenosine a2a receptors by target - immobilized nmr screening ( tins ) and spr techniques , respectively , was reported in 2011 , and fragment screening of the adenosine a2a receptor has been recently validated using the capillary electrophoresis ( ce ) approach . additionally , capture of the 1ar star and evaluation of the binding constants of small - molecule antagonists by spr have been described , and by use of this approach , a subset of the heptares fragment library ( approximately 650 fragments ) was screened in tandem against the 1ar star and adenosine a2a receptor star . figure 1 shows the responses obtained from the 1ar surface plotted against responses from the a2a receptor surface . most responses ( some of those shown in gray squares ) cluster around the origin . a number of responses ( also shown in gray squares ) showed binding to both a2a and 1ar . positive control compounds for each receptor were screened periodically and gave responses that cluster near the axes , confirming that these two compounds were selective binders to either 1ar or a2a receptor . similarly , a subset of fragments appeared to bind more significantly to one receptor or the other ; hits highlighted in red were selective a2a binders , those highlighted in yellow were selective 1ar binders . the selective 1ar hits were retested in concentration series to obtain affinity information . among the selective binders to the 1ar star were 1-[3-(trifluoromethyl)phenyl]piperazine and 2-(piperazin-1-yl)quinoline ( 7 and 8 , chart 2 ) , which were estimated to have encouraging binding affinities ( kd = 16 m ( pkd = 4.80 ) and 5.6 m ( pkd = 5.25 ) , respectively ) and good ligand efficiency ( le = 0.41 and le = 0.48 , respectively ) . plot of spr responses of heptares fragment library against the adenosine a2a star and 1ar star proteins : orange circles , 1ar selective hits ; blue squares , 1ar control compound ; red circles , a2a selective hits ; green squares , a2a control compound . substituted phenylpiperidines have ample history within gpcr drug discovery and have been described as privileged structures for this target class . the analogous phenylpiperazines also have precedent within gpcr chemical space , for example , in the atypical antipsychotics aripiprazole and lurasidone ( chart 2 ) . despite this significance , phenylpiperazines have little precedent in the field of -adrenergic receptors , where the vast majority of clinically relevant molecules are typified by an ethanolamine side chain extending from an aromatic or heteroaromatic moiety ( e.g. , chart 1 , 15 ) . encouraged by the affinity and efficiency of spr hits 7 and 8 , we embarked upon a fragment hit - to - lead exercise to explore these phenylpiperazine fragments as novel hits for the 1ar . screening in an orthogonal assay format is a routine approach to confirm hits during fbdd , and in line with this strategy we assembled a set of commercially available analogues of similar size and complexity compared to 7 and 8 ( see modeling section ) . the set was screened in a radioligand membrane binding assay with human wild - type 1ar and [ h]dihydroalprenolol , and affinity data ( together with ligand efficiency ( le ) , ligand lipophilicity efficiency ( lle ) , and clogp values ) for selected analogues are shown in table 1 . all of the commercially available analogues tested have high ligand efficiencies and bind with moderate to high affinity . unsubstituted phenylpiperazine 9 has good affinity , with the more polar 2-pyridyl and 2-pyrimidinyl analogues of 7 ( 10 and 11 , respectively ) being weaker and less ligand efficient than 9 . disubstituted phenyl groups yielded the highest affinities , with chloro ( 1214 ) and methyl ( 15 , 16 ) variants yielding higher affinities than the trifluoromethyl or methoxy derivatives 17 and 18 . the chloro or methyl disubstituted analogues , where 2,3- or 3,5-regiochemistry is preferred over the 3,4-isomers , bound with moderate to high affinity and exceptional ligand efficiencies ( > 0.60 ) . ligand lipophilicity efficiency ( lle = pki clogp ) is another measure of druglikeness ; in general lles for the fragments are moderate to good ( with the exception of the highly lipophilic 17 ) . the le and lle values for several compounds in table 1 compare favorably to those of the well characterized 1ar ligands carazolol 1 ( le = 0.62 , lle = 6.4 ) , cyanopindolol 2 ( le = 0.56 , lle = 6.8 ) , and carmoterol 3 ( le = 0.38 , lle = 5.6 ) . selected quinoline derivatives to explore initial spr hit 8 were screened ; 8 was included as a control and returned highly comparable affinity in the radioligand binding assay to that from the spr screen ( pki = 5.20 vs pkd = 5.25 , respectively ) . installation of a methyl group at the 4-position ( 20 ) or substitution at the 8-position ( 21 ) yielded compounds with approximately 25-fold greater affinity than 8 , whereas interchanging the methyl and piperazinyl groups of 20 to yield isomer 22 was only moderately effective . compounds 23 and 24 were purchased to loosely mimic the carbazole core of carazolol ; closer analogues were not readily available . nevertheless , 23 and 24 returned submicromolar affinity , albeit with lower lle than the parent hit 8 . selected compounds ( 12 , 13 , 19 , and 20 ) were profiled in muscarinic m1m4 acetylcholine receptor membrane binding and agonist functional assays to provide an initial indication of selectivity of the piperazine fragments against related family a gpcrs . all four compounds were inactive in the functional assay for each isoform and had only weak affinity at best in the binding assays ( 13 , 19 , and 20 m1m4 pki < 4.7 ; 12 m1m3 pki = 5.15.4 , m4 pki = 4.5 ) . given the precedent for related piperazines such as aripiprazole and lurasidone to have complex pharmacology with activities against -adrenergic , dopaminergic , and serotonergic receptors , characterization of optimized compounds in the series against these gpcr subfamilies will be relevant . the advent of gpcr crystallographic information presents opportunities for structure - based design and discovery . we sought to use the available 1ar crystal structure data to drive progress with the piperazine fragment hits . as it is not currently feasible to perform crystallography on the human 1ar because of its instability , these data were obtained with the turkey 1ar . however , the two receptors are 82% identical in the transmembrane and loop regions ( with the exclusion of icl3 ) and the residues that comprise the ligand binding pocket are 100% identical . the turkey receptor therefore provides an excellent model for studying ligand receptor interactions . receptor interactions observed in the 1ar structures with cyanopindolol , carmoterol , and carazolol bound were of notable interest . during the hit - to - lead process , molecules were routinely docked into the liganded structure of the 1ar and examined in relation to inverse agonist carazolol 1(8 ) and agonist carmoterol 3 . because of the small size of fragments and the multitude of ways they can fit into a receptor binding site , it is notoriously difficult to dock these ligands with any great confidence without examining the site in question in significant detail . a complete druggability analysis of the binding site was therefore carried out to identify the region most likely to be occupied by the fragment . water and ch aromatic probes show the regions most favorably occupied by polar and hydrophobic portions of a ligand , respectively . optimization of overlap of the docked molecule along with displacement of the least energetically favorable waters ( shown in red in figure 2 ) has been shown previously to be predictive for highly ligand efficient molecules . thus , this strategy was also used in our analysis of the solutions obtained from dockings , as described below in further detail for representative hit 16 . additional compounds were selected from similarity and substructure searching approaches to complement the structure - based strategy and expand sar in the series . druggability analysis of the 1ar ligand binding site , showing water molecules and their energies relative to bulk solvent ( red , high ; blue , low ) combined with surfaces depicting water probe ( green ) and ch aromatic probe ( yellow ) hotspots , with the docking pose of 16 selected for comparison to the crystal structures . in the dockings of 16 with carazolol 1 and carmoterol 3 the carbon atom of the 3-methyl substituent of 16 overlays clearly with the carbazole nitrogen of 1 and is in the region of the quinolinone nitrogen of 3 , suggesting an opportunity to introduce similar polar interactions with the receptor in this area of the molecule ( figure 3 ) . this possibility is more clearly visualized in the structure of cyanopindolol 2 in the 1ar stabilized receptor ( figure 4 ) , where hydrogen bond donation of the indole nitrogen to ser211 ( superscripts indicate ballesteros weinstein nomenclature ) , a nonpolar interaction of the indole phenyl ring with phe307 , and interaction of the charged ethanolamine portion with asp121 and asn329 are observed . inspired by these observations , we extended our studies to include a number of molecules with larger and more polar substituents in order to promote further interactions with residues in the 1ar ligand binding site and in particular polar interactions with ser211 on helix 5 . the strategy most notably resulted in identification of the simple indole derivative 19 ( table 1 ) , which has both high affinity and ligand efficiency and reduced lipophilicity compared to parent molecule 16 , a finding that validated our structure - driven approach and represents a very promising lead compound for further optimization . piperazine 16 ( pink carbons ) docked into the crystal structures of ( a ) 1ar complexed with the inverse agonist carazolol 1 ( orange carbons ) ( pdb code 2ycw ) and ( b ) 1ar complexed with the agonist carmoterol 3 ( cyan carbons ) ( pdb code 2y02 ) . ( a ) x - ray crystal structure of the antagonist cyanopindolol 2 in complex with 1ar ( pdb code 2vt4 ) . the purple arrows and their directions represent hydrogen bonds between the ligand and the protein . the green line represents the stacking arrangement seen between the aromatic core and the aromatic residue phe307 . the identification of a series of piperazine - based fragments with high affinity and high solubility provided an invaluable opportunity to obtain co - structures of the molecules with the 1ar , as detailed structural information would confirm the modes of binding of the fragments , facilitate comparisons with existing structures , and indicate opportunities for further development . on the basis of a consideration of affinity and ligand efficiency , indole 19 and the structurally dissimilar quinoline 20 cocrystallization of thermostabilized 1ar with fragments 19 and 20 was performed as previously described and resulted in structures determined to resolutions of 2.8 and 2.7 , respectively ( supporting information table 2 ) . the structures show that the piperazine rings in the two molecules are located between asp121 on helix 3 ( h3 ) and asn329 on h7 , and their phenyl substituents are positioned adjacent to h5 ( figure 5a and figure 5b ) . the piperazine ring therefore substitutes for the ethanolamine core of conventional adrenergic receptor ligands , and the structural roles of the phenyl substituents are similar to those of the indolecarbonitrile and carbazole headgroups of cyanopindolol and carazolol ( figure 5c and figure 5d ) . however , the total number and nature of ligand receptor interactions observed in the 1ar-19 and 1ar-20 structures are reduced when compared to those observed in the crystal structures with cyanopindolol and carazolol ( supporting information table 3 ) . with cyanopindolol and carazolol , the ethanolamine secondary amine and -hydroxyl groups both form hydrogen bonds to the asp121 and asn329 side chains . in contrast , with 19 and 20 , only one secondary amine nitrogen in the piperazine ring is suitably positioned to interact with asp121 and asn329 , and the interactions are mostly weaker , polar interactions rather than hydrogen bonds ( supporting information table 3 ) . possibly as a consequence of the weaker interactions of fragments 19 and 20 , there is greater variation in the orientation of the piperazine rings than is shown by the ethanolamine groups in carazolol and cyanopindolol . this can be observed in the superpositions of the different ligands and fragments complexed with the receptor depicted in figure 5e . the variation in the positions of the piperazine rings in the two structures results in some differences in how 19 and 20 interact with asp121 , asn329 , trp303 , and tyr333 ( supporting information table 3 ) . the structures of 1ar bound to 19 and 20 do not show either the change in rotamer conformation of ser215 observed when full agonists are bound or a contraction of the ligand binding pocket ( supporting information table 3 ) characteristic of the binding of both full and partial agonists . these observations suggest that both of the fragments are antagonists , although data to support this hypothesis have not yet been generated . however , the interactions of the phenyl substituent headgroup with ser211 on h5 differ between the two fragment molecules , as only 19 can form a hydrogen bond to ser211 . in the 1ar-20 structure , the conformation of ser211 is similar to that observed in the 1ar - carazolol structure ( figure 5b and figure 5d ) , whereas in the 1ar-19 structure the conformation of ser211 corresponds to that observed in the 1ar - cyanopindolol structure ( figure 5a and figure 5c ) . the differences in pharmacological activity between cyanopindolol ( a partial agonist with sympathomimetic activity ) and carazolol ( an inverse agonist ) may in part result from the differences in the conformation of ser211 observed in the structures with these ligands ( figure 5c and figure 5d ) , and there may be similar differences in pharmacological activity between 19 and 20 . because of their relatively small size , fragments 19 and 20 interact with a subset of the residues that comprise the ligand binding pocket . this suggests that by use of the detailed structural information that has been made available , the targeted addition of further substituents to these fragments could serve to further increase their affinities , as well as to modulate their pharmacological activities . 1ar structures are shown in cartoon representation as viewed in the membrane plane with all or part of h1 , h2 , h3 , and h4 obscuring the ligand binding site removed for clarity , and the extracellular side is uppermost : ( a ) 1ar-19 ; ( b ) 1ar-20 ; ( c ) 1ar - cyanopindolol ( pdb code 2vt4 ) ; ( d ) 1ar - carazolol ( pdb code 2ycw ) . atoms are colored accordingly . for ligands / fragments : c , cyan , gold , silver , yellow ; o , red ; n , blue . for selected receptor side chains : c , green ; o , red ; n , blue . potential hydrogen bonds and polar contacts are shown as red dashes . for a full list of receptor ligand interactions see supporting information table 3 . . global alignment of the receptor structures ( monomers selected as in supporting information table 3 ) was performed using pymol ( superpose ) , and the resulting positions of the ligands are depicted . fragment screening is a well - established and powerful approach to the discovery of a new lead series that is now starting to be utilized for membrane - bound receptor targets . recent virtual screening efforts have demonstrated that , at least for aminergic family a gpcrs , fragment hits can be identified , now that the details of how small molecules bind to these receptors are better understood . furthermore , studies on , for example , the histamine h1 receptor ( h1r ) have shown , in a manner analogous to the work exemplified here , that fragment - sized ligands can be bound to the receptor , crystallized and their binding modes established . in the research presented here , biophysical fragment screening of 1ar using spr was enabled by receptor stabilization and led to the identification of arylpiperazine hits 7 and 8 . selection of analogues for screening in an orthogonal wild - type radioligand membrane binding assay through similarity and substructural searching approaches was complemented by the use of -adrenergic receptor crystallographic information , allowing a parallel structure - based design strategy . high affinity and ligand efficient fragments were identified , including indole 19 and quinoline 20 , which were identified and subsequently cocrystallized with the stabilized 1ar , yielding structures at 2.8 and 2.7 , respectively . overall , these results demonstrate , for the first time , that a true fragment based drug discovery paradigm , encompassing biophysical screening with a direct - binding platform of a diverse fragment library , structure - guided fragment optimization , and cocrystallization of fragment hits , can now be applied to gpcr targets . hek293 cells were transiently transfected with cdna encoding human 1ar using genejuice ( novagen ) according to the manufacturer s instructions to achieve approximately 2 10 post - transfected cells . after 48 h of incubation the cells were dissociated using tryple express cell dissociation fluid and washed with pbs , pelleted down , and stored at 80 c until required . the pellet was resuspended in chilled buffer consisting of 20 mm hepes , 10 mm edta containing 1 complete protease inhibitor cocktail tablet per 50 ml of buffer ( ph 7.4 ) . the suspension was homogenized for 15 s and centrifuged at 40000 g for 15 min . the pellet was suspended in buffer containing 20 mm hepes and 0.1 mm edta ( ph 7.4 ) and homogenized for 30 s. after centrifugation at 40000 g for 45 min , the pellet was resuspended in the same buffer and homogenized for a further 30 s. protein concentration was estimated against the standard bovine serum albumin using the bca method ( pierce , socochim , lausanne , switzerland ) and then frozen to 80 c at 2 mg / ml prior to use . cell membranes were incubated with [ h]dihydroalprenolol in assay buffer ( 50 mm hepes , 15 mm mgcl2 , 150 mm nacl , ph 7.4 ) in a total assay volume of 0.25 ml with a final dmso concentration of 1% . after 90 min of incubation at room temperature the reaction was terminated by rapid filtration through gf / b 96-well glass fiber plates with 5 0.25 ml washes with doubly distilled h2o using a tomtec cell harvester . bound radioactivity was determined through liquid scintillation using lablogic safescint and detected on a microbeta liquid scintillation counter . nonspecific binding was determined as that remaining in the presence of a 10 m saturating concentration of the antagonist alprenolol . competition binding was performed by incubating membranes ( 1.25 g of protein / well ) with 1.5 nm [ h]dihydroalprenolol and a range of concentrations of the test compound . ic50 values were derived from fitting to a four - parameter logistic equation in prism ( graphpad software , san diego , ca , u.s . ) . binding affinities are expressed as pki values , where pki = log10 ki . surface plasmon resonance ( spr ) binding studies were performed at 25 c using biacore t200 and s51 optical biosensors equipped with nickel - charged nta chips and equilibrated with running buffer ( 20 mm tris - hcl , 350 mm nacl , 0.1% dodecylmaltoside , 5% dmso , ph 7.8 ) . aliquots of the affinity - purified , his - tagged 1ar and a2a receptors were both capture - coupled on the sensor surface to densities of > 9000 resonance units ( ru ) . in an initial screen , the entire library was tested at 50 m with two control compounds , l-748,337 ( a 1ar positive control , cas 244192 - 94 - 7 ) and psb 1115 ( an a2a receptor positive control , cas 409344 - 71 - 4 ) , tested periodically at 10 m throughout the screen to track the activity of the receptor surfaces . for a second screen , fresh receptor surfaces were prepared and the compound library was retested at 11 m ( after omitting compounds that bound to the reference surface or bound with nanomolar affinity to a2a ) . potential selective 1ar hits were analyzed in concentration series ( 2-fold dilutions typically starting at 150 m , with each concentration tested in duplicate ) to confirm their selectivity and determine their affinities . the crystal structure of 1ar bound with cyanopindolol , pdb code 2vt4 , was used as the basis for dockings of fragments . the protein preparation workflow was used to add hydrogens , cap the n - terminus and c - terminus groups , optimize the orientation of hydroxyl groups , asn , and gln , and the protonation state of his , and then perform a number of constrained refinements on the hydrogens alone and the protein with a maximum rmsd tolerance of 0.30 . dockings were undertaken using the glide extra precision ( xp ) protocol within maestro . grid analyses of the binding sites were used to evaluate potential docking poses and drive the designs ( using the csp3 probe ( c3 ) for shape , aromatic ch probe ( c1= ) for lipophilic hotspots , water probe ( oh2 ) for water hotspots , carbonyl group probe ( o ) for hydrogen bond acceptor hotspots , and amide nh probe ( n1 ) for hydrogen - bond donor hotspots ) . the turkey ( meleagris gallopavo ) 1ar ( star ) construct that was used in crystallization experiments , 44-m23 , contains six thermostabilizing point mutations and truncations at the n terminus , inner loop 3 , and c terminus . for crystallization , the detergent was exchanged to hega-10 ( 0.35% ) on an alprenolol affinity column . receptor was competitively eluted from the alprenolol affinity column with 100 m ligand ( 19 or 20 ) and concentrated to 25 mg / ml in 10 mm tris - hcl , ph 7.6 , 100 mm nacl , 0.1 mm edta , 0.35% hega-10 . before crystallization , hega-10 and cholesteryl hemisuccinate crystals were grown at 4 c by vapor diffusion in sitting drops with 150 nl of receptor + 150 nl of precipitant ( 0.1 m bicine , ph 9.0 , 24% peg 600 in both cases ) and cryoprotected by addition of 60% peg 600 for 1 min before mounting on hampton crystalcap ht loops and cryocooling in liquid nitrogen . for both 1ar-19 and 1ar-20 structures , diffraction data were collected from a single cryocooled crystal ( 100 k ) using a 10 m focused beam at i24 , diamond light source , oxford , u.k . both structures were solved by molecular replacement with phaser using the structure of 1ar with carvedilol bound ( pdb code 4amj(39 ) ) as a starting model . refinement , rebuilding , and validation were carried out with refmac5 , coot , and molprobity . in both the 1ar-19 and 1ar-20 structures there is a distortion of the ligand binding pocket in monomer a due to lattice contacts , and monomer b represents the more physiologically relevant conformation . the 1ar-19 and 1ar-20 crystal structures were determined at a resolution of 2.8 and 2.7 , respectively . this resolution was more than adequate to provide clear omit electron densities that enabled unequivocal placement of the ligands in the structures ( supporting information figure 1 ) , as well as further details such as specifically bound detergent and lipid molecules and ordered internal water molecules and sodium ions ( supporting information table 2 ) .

biophysical fragment screening of a thermostabilized 1-adrenergic receptor ( 1ar ) using surface plasmon resonance ( spr ) enabled the identification of moderate affinity , high ligand efficiency ( le ) arylpiperazine hits 7 and 8 . subsequent hit to lead follow - up confirmed the activity of the chemotype , and a structure - based design approach using protein ligand crystal structures of the 1ar resulted in the identification of several fragments that bound with higher affinity , including indole 19 and quinoline 20 . in the first example of gpcr crystallography with ligands derived from fragment screening , structures of the stabilized 1ar complexed with 19 and 20 were determined at resolutions of 2.8 and 2.7 , respectively .

Introduction Results and Discussion Conclusions Experimental Section

PMC3195985

blunt traumatic diaphragmatic rupture ( btdr ) is a life - threatening condition with an incidence of 0.8%1.6% in blunt trauma [ 13 ] . the diagnosis often happens to be late due to the absence of typical symptoms or other major injuries dominating the clinical aspect . an isolated btdr is rare and thus might be followed by a period of weeks or months not revealing any symptoms [ 2 , 5 ] . most btdr are located on the left side in the musculotendinous intersection [ 1 , 3 , 4 ] . herniation of colon , small bowel , or liver may occur and result in ileus , necrosis , and perforation [ 4 , 7 ] . we present a rare case of a 68-year - old female hospitalized in the neurological department due to parkinson disease . the examination showed a slightly reduced breath without any signs of pneumothorax or dyspnea , a decent pressure pain and a bruise . the chest radiograph displayed a herniation of bowel into the right hemithorax with consecutive ileus signs ( figure 1 ) . we performed a laparoscopic approach and found a 4 5 cm rupture of the right diaphragm with herniation of 1 meter small bowel . the transdiaphragmatic thoracoscopy displayed a collapsed lung and a dislocated rib fracture ( figure 2 ) . after irrigation of the thoracic cavity we made a direct laparoscopic strainless running suture with nonabsorbable tie ( 0/0 ethibond ) . the delay of the diagnosis was 4 days indicating the variety of the clinical appearance of the btdr . the missing typical symptoms can mask the severity of the injury and lead to increasing morbidity . although large prospective long - term studies regarding outcome after laparoscopic approach are still missing , laparoscopy was the method of choice in our case and safely performed , particularly when severe side injuries are absent . we could show , that laparoscopy has advantages in isolated diaphragmatic ruptures compared to traditional laparotomy , which is still preferred by some authors [ 8 , 9 ] . laparoscopy mostly allows easy reposition of herniated organs , sufficient inspection of the thoracic , and abdominal cavity and immediate laparotomy if necessary .

blunt traumatic diaphragmatic rupture ( btdr ) is a life - threatening condition with an incidence from 0,8%1,6% in blunt trauma , mostly located on the left side . the main prognostic factors are severe side injuries and the delay of diagnosis . we present a rare case of a 68-year - old female , with an isolated right diaphragm rupture . the diagnosis was done with a delay of 4 days by thoracic radiographs , which showed a herniation of small bowel into the right thoracic cavity . a reposition of the small bowel and a closure of the diaphragmatic defect by running suture were carried out laparoscopicly . although large prospective studies concerning the outcome of laparoscopic approach to right btdr are still missing , we could show , that laparoscopy can be performed safely in right traumatic diaphragm rupture .

1. Introduction 2. Narrative 3. Discussion

PMC2878938

in december 2008 , the journal of cell biology ( jcb ) launched the jcb dataviewer , an online repository for original image data in the life sciences ( fig . 1 ) . to our knowledge , this system is the first open repository that enables routine archiving and sharing of original image datasets supporting published scientific articles . one key attribute of the jcb dataviewer that distinguishes it from past and current data repositories is that the original binary data and metadata , additional information captured by acquisition software about an image , such as the instruments used , acquisition settings , image size , and resolution , are preserved and accessible by the community . as of this writing , the jcb dataviewer contains 6,446 multidimensional ( 5d ; including space , channel , and time ) images in support of 186 published articles . the jcb dataviewer is a customized application based on the open source and open development open microscopy environment ( ome ) remote objects ( omero ) and bio - formats projects , released by the ome consortium ( http://openmicroscopy.org ) . an example of original image data associated with this paper , viewed in the jcb dataviewer . the image shows the following : a 3d stack of a fixed hela cell stained with dapi ( blue ) , anti - incenp ( red ) , and anti - tubulin ( green ) , recorded using a wide - field microscope ; a time - lapse video of a c. elegans embryo expressing gfp - tubulin , recorded using a multiphoton microscope ; a transmission electron microscope ( tem ) image of bacteriophages visualized using negative stain ; a 3d stack of a fixed hela cell stained with anti - tubulin , recorded using an omx 3d structured illumination microscope ; a tem image of rb bound to dna ; and a 5d image of gfp - coilin and yfp - histone h2b in a hela cell , recorded by wide - field microscopy ( platani et al . , 2000 ) . note that available metadata differ substantially between the different images , depending on the metadata that are stored in the original files . these images and their associated metadata are available at http://jcb - dataviewer.rupress.org / jcb / browse/2859/. one goal of the jcb dataviewer was to initiate the development of a functional , scientifically valuable online image repository . the first step was to make original data available alongside a publication , available for examination by reviewers and readers of a submitted or published manuscript . currently , the jcb dataviewer allows access to original data for viewing , simple measurement , and review , but users can not download the original data files , and sophisticated image analysis and querying tools are not included in the application . in the next update , users will be able to download video versions of data stored in the jcb dataviewer , and original image data will be available in an open , standardized data format that preserves the original image metadata ( ome tagged image file format [ tiff ] ) . authors will also retain access to their original data , thereby making the jcb dataviewer an archive where authors can store their own published data . these updates represent one more step toward the development of a fully functional data repository . the data in the jcb dataviewer are freely available to the public immediately upon publication , without a subscription to the jcb . in the future , as image repositories mature , we plan to merge the data held in the jcb dataviewer with whatever resources emerge as the definitive public repository of image data in the life sciences . the jcb dataviewer is one of a growing number of image data repositories that are now available , each focused on providing access not only to results but also to some combination of sophisticated visualization , analysis , and mining of these complex data ( table i and fig . each of these efforts has emphasized specific applications and functionality and reflects the simple fact that the diversity of scientific exploration and images can not yet be addressed by a single resource . however , there are ongoing efforts to align data models where possible , and perhaps most importantly , simplify submission and subsequent processing through the definition and use of file formats that support standardized metadata . these are examples of real progress toward the goals that many have discussed and that have recently been reiterated ( cosepup , 2009 ; field et al . , 2009 ; schofield et al . , scientific image data repositories for cell and developmental biology slif , subcellular location image finder ; pslid , protein subcellular location image database . in development as of 2010 . recommendations for ome compliant image metadata . the image and instrument elements from the ome data model , with attributes and hierarchies shown in diagrammatic form . the image element contains core metadata that can be used for display and processing of the associated binary image data . currently , an ome compliant image completes all of the metadata in the image element . by the end of 2010 , we aim to include the instrument element in the ome compliant specification . the bio - formats library provides support for writing ome - xml either as a stand - alone file or within the header of an ome - tiff file . updates to the ome data model are announced on the project s roadmap site ( http://ome-xml.org/roadmap ) . in summary , significant effort by peer - reviewed , competitively funded groups in the us and europe has produced image informatics tools that the research community uses . the tools and resources are by no means finished , and our current status seems analogous to the state of the genomics resources in the mid-1980 s , when individual authors submitted their own sequence data to genbank , swissprot , and others . the diversity of imaging platforms , experiments , techniques , and data makes this analogy only partially correct and undoubtedly makes the challenge of building and running scientifically useful image repositories harder . regardless , the sophistication of centralized scientific image resources is growing , and as a result , so will the value they deliver to the scientific community . those resources that depend on submissions from the community will require the development , adoption , and use of standardized file formats that support as rich a metadata structure as possible . this is why the development and use of standardized image data and metadata formats are so important . many laboratories have at least one sophisticated imaging system , and many large shared - use facilities provide access to an array of imaging systems . after many years of innovation and development , modern digital imaging systems enable temporally and spatially resolved , multichannel measurement and visualization of molecular and ion concentrations in cells and tissues . emerging imaging techniques such as multispectral , polarization , fluorescence lifetime , and fluorescence correlation are extending the complexity of analysis of biological cells and tissues . however , most digital microscope imaging systems , whether commercial products or laboratory prototypes , are usually run by custom software that saves and processes data using a pff . in general , every new imaging platform comes with a new pff , so rapid advances in imaging simultaneously make data exchange and access more difficult . to realize the dream of open data access and sharing , we first must solve the basic problem of accessing the data contained in pffs . any solution will not directly lead to new scientific insights , but it is a prerequisite for submission to repositories and the discoveries they enable through reanalysis . for example , if the data from cell - based phenotypic screens were available , they could be reanalyzed for aberrations that were not of interest to the investigators who did the original screen . generally speaking , image data are written in formats that include the binary data and the actual image measurement , along with some representation of the metadata : the size of the binary data , its dimensions , acquisition system settings , and any other information that the developer of the acquisition software considered useful . in our experience , storage of binary data in many commercial microscopy formats is based on common formats ( tiff , hdf5 , and ole2 , etc . ) or other formats that most software tools can read ( although there are some notable , extreme exceptions ) . because standards are not yet agreed upon , microscope and imaging companies define their own metadata formats in their pffs , and these are often incompatible with those from competing companies . since 2000 , the ome has been dedicated to building tools for specification , management , and sharing of biological light microscopy data ( swedlow et al . , 2003 , 2009 ; 2 ) , which covers most of the metadata in pffs from many sources and includes most of the fundamental imaging metadata in cell and developmental biology . this specification , used within the context of a tiff file ( ome - tiff ) , provides a simple , easy to use format for microscope imaging data that can be used by any software that reads the tiff file format . a popular tool ( > 13,000 installations worldwide ) is bio - formats , a software library that interfaces with a large number of software tools ( such as imagej ) , enables the reading of > 75 pffs , and supports output to ome - tiff . for many years , the imaging community has expressed a desire to move away from the current ad hoc approach toward more defined standards for metadata representation ( goldberg et al . , 2005 ) . however , creating a reasonable standard takes years of community discussion and effort . for the standard to be successful , it must be widely used and functional enough to be worth the effort of conformance , and it takes time for the snowball effect to occur . given the diversity and rapid evolution of imaging applications in biology , we do nt believe that standards can be mandated by any one entity . instead , we argue that standards for biological imaging must be supported and developed , and once they are valuable for scientific discovery and data sharing , and have demonstrated the ability to rapidly adapt to new technologies , the community must demand the support of these formats in the commercial platforms they purchase . under the umbrella of the ome , we have been collecting community feedback for several years now , and our recommendations for this process are detailed in box 1 . recommendations for use of pffs image metadata must be associated with the binary image data , preferably as a single file . microscope systems must not store metadata in proprietary databases that are available only on the data acquisition system . metadata must be readable by third party software using a common , openly accessible software package or library . pff developers must work with developers of open translation libraries to ensure their format is correctly interpreted . if compression is supported , the user must be given the option of saving uncompressed or losslessly compressed images ( which allows the exact original data to be reconstructed after compression ) . if compression or encryption is used , the algorithm and parameters must be stated and stored in the metadata . commercial software programs must provide data export to an open metadata specification . to ensure that commercial software writes these formats correctly , open public and charity funding for imaging systems must include a requirement that the system writes data in an open , accessible format , wherever possible . when pffs must be used , new versions must be announced to the scientific community , and users and funding bodies must predicate their purchases on this type of support for the scientific community . once a standardized repository is available , journals must require deposition of original data supporting scientific manuscripts . in some cases , however , in our experience with bio - formats , omero , and the jcb dataviewer , most of the data we have seen could be recorded in an open , standardized , multidimensional file format . as the number of imaging systems and the rate of innovation grows , maintaining a tool like bio - formats , simply because commercial vendors do not use standardized file formats , becomes increasingly untenable . reverse engineering is slow and inherently error prone , as metadata stored in pffs are decoded and translated . as popular as bio - formats is , it is time to reconsider the value pffs deliver for a specific commercial product against the costs , which are paid for by public and charity funding : lost time for scientific researchers , inhibited collaborations , and impeded access to data using the aforementioned emerging data repositories . many scientific funding bodies now require the published output from the work they fund to be deposited in open access repositories . the same open access principle should be extended to the data generated through their funding to enable broad dissemination and further analysis . as with other forms of data , there is no requirement to publish all images associated with a paper , just the ones that form the definitive representation of the reported discovery . the ome , international society for advancement of cytometry ( http://www.isac-net.org ) , and digital imaging and communications in medicine ( http://medical.nema.org/dicom/ ) formats are all well developed , supported , and available for use . it may be that no single format can satisfy every requirement or data type , but our experience demonstrates that the vast majority of the data used to support scientific publications can be properly stored in these formats . we can support a range of open file formats with bio - formats , thus allowing interconversion between open file formats where necessary . we have developed the ome metadata standards through extensive direct experience and discussion with the user and commercial developer communities . we plan to use them as we progress to the development of a public repository but remain open to suggestions about how they can be improved . as noted in the box 1 , the use and adoption of these file formats wo nt happen by itself , the community must work to drive their adoption . individual scientists and their funding bodies must require support for these formats when they purchase or fund new imaging systems . the argument for this concerted action is based on a simple , practical goal : scientific data , funded by the public and nonprofit charities , must be publicly available . over the next few years , the technical capabilities in image repositories will mature . data to fill these repositories must be open , accessible , and ready for use .

data sharing is important in the biological sciences to prevent duplication of effort , to promote scientific integrity , and to facilitate and disseminate scientific discovery . sharing requires centralized repositories , and submission to and utility of these resources require common data formats . this is particularly challenging for multidimensional microscopy image data , which are acquired from a variety of platforms with a myriad of proprietary file formats ( pffs ) . in this paper , we describe an open standard format that we have developed for microscopy image data . we call on the community to use open image data standards and to insist that all imaging platforms support these file formats . this will build the foundation for an open image data repository .

Scientific image data repositories for the life sciences Microscopy file formats Future directions and recommendations

PMC5260523

while the tests of basic motor abilities such as speed , maximum strength or endurance are well recognized , testing of complex motor abilities such as agility remains unresolved in the current literature . the term agility itself has undergone changes , resulting in the current definition : the ability to change speed and direction of a movement rapidly and accurately ( jeffreys , 2011 ; sheppard et al . , 2014 ) , usually in response to a highly specific stimulus . the specific stimuli include different situations during sport competitions that vary in intensity , with the condition of space - time uncertainty being met . the term agility has evolved significantly , with the main factors determining agility also changing . the most current model divides agility into cognitive , physical and technical factors ( young et al . , 2015 ) , instead of the perceptual one with decision - making factors and change - of - direction speed ( cods ) factors that were used in the previous model ( young et al . , 2002 ; the result of this inconsistency created additional specific terms to describe specific parts of agility , such as reactive agility , quickness or reactiveness . however , no quantitative description is currently available to describe and test agility itself or its partial factors . models of main factors determining agility * factors evaluated by principal component analysis or factor analysis - exploratory approach . empirically accepted tests of agility have been screened to determine their relationships between each other and to specific subtests , which delineate any agility sub - factors . l agility test ( r = 0.73 ) and moderately with the reactive agility test ( r = 0.50 ) ( gabbett et al . , 2008 ) , and the factor of straight sprinting represented by the 36 m dash correlated with the t agility test ( r = 73 ) ( pauole et al . , 2000 ) . in these cases , the authors determined whether the performance on one test was related to another , indicating that if an athlete performed well during one test , he may perform well in another test . these types of findings resolve the specific relationships among agility sub - tests and are useful to explain the role of specific tests in sports training . however , this approach does not offer an explanation of the structure of the main factors that determine agility ; for example , how does one explain how significantly the 36 m dash , l agility test or reactive agility test predicts overall agility ? moreover , the choice of an agility test should clearly fit into an agility structure and not cross between its sub - factors with unknown factorial strengths ; therefore , it is currently not known whether five or ten tests are enough to estimate agility performance . it may be advantageous to reduce the seemingly infinite number of agility tests when aiming to determine agility performance . moreover , it may be possible to test independent sub - factors of agility that can effectively estimate agility performance . the empirical aspect of agility testing is necessary as the choice of the test can differ with respect to the specificity of the sport discipline or performance level . since the relationships among individual agility tests are already known for numerous sports and athletic populations ( sheppard et al . , 2014 ; young et al . , 2015 ) , quantifying to what extent the underlying sub - factors of agility ( i.e. , leg strength , change - of - direction speed , linear sprint speed , anthropometrics and other quantifiable variables ) directly impact agility performance could be useful . although uncommon within sport science literature , structural equation modeling ( sem ) is common within behavioral research ( maccallum and austin , 2000 ; mcneill et al . , 2006 ; marsh et al . , sem seeks to explain the relationships between independent and dependent variables , which can be either continuous or discrete ( schreiber et al . , 2006 ) . from this perspective , sem is often understood as a combination of factor analysis and multiple regression analysis or path analysis ( hox and bechger , 1998 ; ullman , 2001 ; magiera et al . therefore , sem is often used in situations when studies aim to empirically establish the structure of a certain behavioral domain without any evidence or information about relationships and their strengths ( kline , 2011 ) . for example , intelligence is a latent variable that does not have an observational or quantifiable result ; rather , it is inferred based on quantifiable data from the underpinning subfactors of intelligence that have observational outcomes based on previously established testing norms ( spatial , kinesthetic , musical , linguistic , etc . ) . much like intelligence , agility is a latent variable with underlying sub - factors ( leg strength , linear sprint speed , reaction time , etc . ) . therefore , the evidence and information about relationships between manifest and latent variables are important for modeling of agility structure and further development of valid and reliable tests . the models of agility s main underlying sub - factors ( jeffreys , 2011 ; sheppard et al . , 2014 ; were specifically based on the empirical approach , while the explanation and application of the agility tests were derived mainly from the following : 1 ) validity and reliability of indicators that were considered criteria of agility performance ; 2 ) correlations revealed between these indicators and other manifest variables such as the t agility test , straight sprint , countermovement jump or skinfold thickness ( gabbett et al . , 2008 ; therefore , the aim of this review was to determine which main factors could quantitatively determine agility performance . in an attempt to explain and describe the relationships between manifest and latent variables and distinguish them from the underlying factors of agility , different approaches were used , such as principal component analysis ( pca ) , factor analysis and sem . the results provide an overview of agility structure modeling , shed light on scientifically unresolved portions of defined agility structures and discuss directions for future research . this study utilized the preferred reporting items for systematic reviews and meta - analyses ( prisma ) ( moher et al . , 2010 ) the database search was conducted in may 2016 , and the web of science , pubmed and scopus databases were screened from 1976 to 2015 . the title and abstract screening was focused on studies that included tests of agility or cods , testing agility or cods , relationships between agility and cognition , the role of agility in team sport games , agility or cods in relation to sport performance and any factors influencing agility . the selection criteria were as follows : ( 1 ) the abstract and the article itself were published in a reviewed journal in english ; ( 2 ) the topic was exclusively concerned with agility or cods in sport ; ( 3 ) the aim of the study was to explain or improve agility performance , describe the agility structure or recognize the main factors determining agility ; and ( 4 ) the participants of the research studies were recreational , collegiate , amateur or professional athletes . a team of independent evaluators was appointed for full - text screening to choose relevant research studies and hand search through the reference lists of articles and books . each evaluator was tasked with reviewing the articles and making a decision on accepting the articles based on the following inclusion criteria : ( 1 ) the number of study participants reached the minimal biological sample ( 30 people ) and ( 2 ) the agility factorial structure or its sub - factors were evaluated by either pca , factor analysis or sem . the database search resulted in 2260 studies after removing duplicates during title and abstract screening . a total of 60 papers were followed up by selection criteria for full - text screening ( figure 2 ) . finally , four research studies that focused on cods met the defined inclusion criteria . using a desired methodological approach , no quantitative empirical research was found that attempted to verify the quality of the whole suggested model of the main factors determining agility using the sem approach or confirmatory factor analysis . during full - text screening of numerous studies concerning agility , factorial models were rejected because they reported only correlation coefficients across different tests and did not apply appropriate statistical methods . moreover , these studies had already been summarized in previous reviews ( sheppard et al . , 2014 ; young et al . , 2015 ) . ( metiko et al . , 2003 ) was rejected because it was not available in english . additionally , other studies that used multiple regression ( mr ) were excluded ( delextrat et al . although mr principles are directly linked to the sem approach , its core is in analyzing the observed variables only ( tabachnick et al . , 2001 ) . therefore , mr does not evaluate relationships among multiple dependent factors ( tests ) and can not recognize the level and strength of their dependency in structural models simultaneously ( muthn and muthn , 2010 ) . three of the studies used pca with kaiser - guttman criterion and one study used factor analysis ( exploratory approach ) ( efa ) along with regression analysis . articles evaluating the agility factorial structure or change of direction cods = change of direction speed , * physical education student , 1rm = one repetition maximum , ssc = stretch shortening cycle , cj = countermovement jump , slj = standing long jump , sj = squat jump , dj = drop jump , ls = lateral stepping , sq = squat , sbf = sprint with backward and forward running , pca = principal component analysis , efa = factor analysis - exploratory approach only 4 studies out of 60 relevant titles discussing agility involved research attempting to determine the main factorial model of agility , which means that the current understanding of agility is based strongly on the empirical approach rather than a quantified hierarchy of manifest variables or tests . this result seems to be appropriate because the concept of agility arose from empirical experiences and practices . moreover , it must be emphasized that three of the studies used a pca approach . although pca belongs to the family of exploratory techniques , it is not based on a common factor model ( fabrigar et al . , 1999 ) . rather , pca is focused on the amount of variance explained by manifest variables instead of an explanation of the correlations between them . therefore , pca is appropriate as a data reduction technique , but not for verifying relationships between latent variables within an evaluated structure . pca is used mainly when it is necessary to reduce a large number of indicators to a smaller and more manageable amount of directly measured tests for subsequent analysis . in our review , we found only one study ( markovic et al . , 2007 ) that used a common factor model from sem , specifically the exploratory factor analyses ( efa ) approach . however , no research has verified the structure of agility using sem , such as confirmatory factor analysis or structural regression models , although many methodologists ( mcdonald , 1999 ; muthn and muthn , 2010 ) showed that approaches from a family of sem enable researchers to specify relationships between latent variables ( constructs ) and a set of measures as well as to determine the relationships between constructs . consequently , a large number of studies described the internal relationship between agility tests , which is the relationship between a dependent variable and defined independent variables . certainly this situation gives an apparent chance for developing new agility tests , which will probably provide a sufficient correlation with previously accepted tests . however , adding or removing different tests can not explain the basic principles that make up agility performance . therefore , it would be highly desirable to verify and establish a factorial model of agility by second generation data analysis techniques ( bagozzi and fornell , 1982 ) to which sem belongs . despite the theoretical background , the original empirical model of agility ( compounding from perceptual and decision - making skills and change - of - direction speed ) might seem to be appropriate , but its usefulness is diminished by the large number of agility test choices that do not have a quantified hierarchy . for example , it has been reported that cods is often used in team sports ; however , performance in sports or activities that involve cods does not predict ( perhaps not with a strong relationship ) complex agility performance ( henry et al . , 2011 ; serpell et al . one of the results from the present study is that only tests of cods and not complex agility have been analyzed to determine the variance in cods performance . ( 2014 ) concluded that common cods tests ( illinois , l - run , pro - agility , t - test , and 505 agility ) were highly reliable and valid measures of cods in collegiate athletes performing in invasive games and could explain 89.5% of the cods latent variable ( factor ) variance . therefore , performance in these tests is a suitable and sensitive assessment of cods . as a result , the above mentioned specific combination of selected cods tests is sufficient to assess the cods performance of athletes at the collegiate level who perform in different invasive games . on the other hand , if elite athletes representing one sport discipline are tested , the need for specific cods tests increases . ( 2010 ) showed that sprinting with backward and forward running had greater factorial validity for cods than a slalom test , a cods sprint 4 x 5 m , a t - test and a sprint with 90 or 180 turns in elite soccer players , but those tests could explain only 57% of cod variance ( table 1 ) . these findings support the empirical knowledge that higher performance levels require more specific tests of agility involving cods . furthermore , in the future , the structure of the specific agility tests for elite athletes should be estimated independently from general agility tests . other studies have confirmed that within the agility structure , there is a cods independent factor ( markovi et al . , 2007 ; salaj and markovic , 2011 ; zajac et al . , 2014 ) . ( 2007 ) revealed three other independent factors ( in addition to cods ) , namely , explosive strength , elastic strength and maximum strength . however , according to their results , these factors explained only 17% of cods performance . later , salaj and markovic ( 2011 ) found that other independent factors could be determined , namely , sprinting ability , concentric / slow stretch and shortening cycle jumping ability and fast stretch and shortening cycle jumping ability , which together with cods explained 80% of the test variance . these results indicated that sprinting along with jumping ability could better determine cods than explosive and maximum strength in collegiate athletes . considering the agility concept , we can estimate that only cods and perhaps some of the technical and physical determinants have been appropriately analyzed in current literature ( figure 2 ) . on the other hand , some studies have focused on creating complex agility tests that also include cognitive or decision - making aspects ( serpell et al . , 2011 ; czuba et al . , 2013 ; spiteri et al . , 2014a ) ; currently , there is poor quantitative evidence validating the previously established factorial model of agility . only four studies used exploratory techniques such as pca or efa , and no study used structural equation modeling or confirmatory approaches for determining the relationships between indicators and concepts within the agility factor structure . therefore , we strongly suggest that future research should verify and establish a factorial agility model using a second generation data analysis technique to which the family of structural equation modelling ( sem ) belongs . from the whole structure of agility , the combination of common cods tests is reliable and useful to estimate cods performance in subelite athletes ; however , for elite athletes , specific cods tests that are relevant to the needs of particular sport disciplines must be constructed . sprinting and jumping tests are stronger factors for cods than explosive and maximal strength tests . future research should focus on the following aspects : performing factor analyses of the agility factor structure for each agility classification.identification of independent factors for general and sport - specific agility tests.identification of sport - specific agility tests for elite athletes.analysis of the factorial strength of complex agility tests such as the test of reactive agility . performing factor analyses of the agility factor structure for each agility classification . identification of independent factors for general and sport - specific agility tests . analysis of the factorial strength of complex agility tests such as the test of reactive agility .

abstractwhile tests of basic motor abilities such as speed , maximum strength or endurance are well recognized , testing of complex motor functions such as agility remains unresolved in current literature . therefore , the aim of this review was to evaluate which main factor or factor structures quantitatively determine agility . in methodological detail , this review focused on research that explained or described the relationships between latent variables in a factorial model of agility using approaches such as principal component analysis , factor analysis and structural equation modeling . four research studies met the defined inclusion criteria . no quantitative empirical research was found that tried to verify the quality of the whole suggested model of the main factors determining agility through the use of a structural equation modeling ( sem ) approach or a confirmatory factor analysis . from the whole structure of agility , only change of direction speed ( cods ) and some of its subtests were appropriately analyzed . the combination of common cods tests is reliable and useful to estimate performance in sub - elite athletes ; however , for elite athletes , cods tests must be specific to the needs of a particular sport discipline . sprinting and jumping tests are stronger factors for cods than explosive strength and maximum strength tests . the authors suggest the need to verify the agility factorial model by a second generation data analysis technique such as sem .

Introduction Material and Methods Results Discussion Conclusion

PMC2935962

mechanical testing is frequently used to characterize tendons , and to describe the effects of interventions such as exercise or medication on their mechanical properties . force , deformation and stiffness , derived from an ultimate tensile strength test , provide sample specific information about the mechanical behaviour of a tendon sample . from these values , stress , strain and elastic modulus are regularly derived in order to provide quantitative data concerning material properties , with the intention of characterizing the material irrespective of sample dimensions . however , when comparing the results of different studies , it is evident that the reported material properties , in particular the strain values , vary hugely , e.g. from 610% ( rigby et al . , 1959 ; tamiwa , 2007 ) to 1720% ( almeida - silveira et al . , 2000 it is well documented that the mechanical properties of tendons are influenced by a range of intrinsic and extrinsic factors including anatomical site ( haraldsson et al . , 2005 ) , age ( nakagawa et al . , 1996 ) , loading history ( simonsen et al . , 1995 ) or hormonal status ( inhofe et al . , 1995 ) . in addition , results can also be influenced by experimental setup , including environmental conditions and test protocol ( schatzmann et al . , 1998 ; however , even taking these factors into account , there is a remarkably wide range of reported material properties for tendon , with particularly large strain values frequently reported when analyzing the tendons of small mammals such as rats . a recent study by tamiwa et al . ( 2006 ) suggested that stress is not able to accurately normalize force data for rat tail tendon fascicles , whilst two brief articles published in the 1980s describe an effect of specimen length on stress strain characteristics in rat tail tendon ( haut , 1986 ; sanjeevi et al . , 1982 ) . a number of reasons for these variations have been hypothesized , including inhomogeneity of samples ( atkinson et al . , 1999 ) and end - effects ( lam et al . , 1988 ) . the influence of each of these effects is important , as it highlights the difficulties in simply using stress and strain parameters to derive true material properties for non - homogenous soft tissues . the aims of our study were to highlight the influence of specimen length and csa on apparent material properties , investigate why these occur , and determine if they differ between two functionally different tendons . we hypothesize that the variations recorded in material properties with specimen dimensions are largely influenced by gripping the specimens . however , we further suggest that variations in micro - structure between different sized samples may lead to different material properties . tendon fascicles were dissected according to an established protocol ( screen , 2003 ) from five rat tails and five bovine foot extensors , all from healthy animals sacrificed for other , unrelated reasons . for each fascicle , the diameter was determined by a laser micrometer at multiple points along a 1 cm region in the middle of the fascicle . fascicles were secured in a materials testing machine ( bionix100 , mts , 50n load cell ) by pneumatically driven grips with a serrated surface , exerting a gripping pressure of 3 gpa . fascicles were loaded to failure at room temperature at a strain rate of 1%/s . during dissection and testing , specimens were kept moist by continually spraying with phosphate buffered saline solution ( pbs , sigma ) . grip - to - grip length was varied and at each length 34 fascicles per animal were tested : rat tail fascicles were tested at 5 , 10 , 20 , 40 , 60 , 80 and 100 mm ( 103 fascicles in total ) ; bovine extensor fascicles were tested at 5 , 10 , 20 and 40 mm ( 67 fascicles in total ) . force and deformation were both continuously recorded from the bionix100 at 50 hz and engineering stress and strain calculated using the csa and length of the sample at the start . from the resulting data , the point at which a 0.1 n load was detected was located , and defined as the test start point . strain data were smoothed with a 5-point moving average filter , before calculating the modulus over every 8 values . the modulus across the most linear region of the stress strain curve and the maximum modulus were found ( fig . 1 ) and referred to as the linear and maximum modulus , respectively . the end - effect was calculated to establish the effective specimen length , which is longer than the daylight length . compliance ( elongation / linear moduluscsa ) against specimen length was plotted , and the regression line extrapolated to cut the x - axis and indicate the magnitude of the end - effect ( ker , 1981 ) . material properties were then corrected for end - effects , to determine how they influence the measured data . to determine the strain distribution along the length of the fascicles , markers ( ink ) were drawn every 5 mm along bovine extensor fascicles of 20 and 40 mm lengths . the quasi - static tests to failure were filmed ( olympus c-740 , 15 hz ) and the strain distribution determined at the beginning of the test , 50% of strain to failure , and in the last few frames prior to failure . the grip - section was defined as the distance of the second highest marker to the upper grip and the second lowest marker to the lower grip , resulting in a constant grip - section length of 15 mm for both the 20 and 40 mm long samples . the remaining distance was defined as the mid - section , which was 5 mm long in the 20 mm sample and 25 mm long in the 40 mm sample . distances between the markers and the grips were measured using an image processing program ( imagej , national institutes of health , usa ) and local strains in each section were determined and expressed as a contribution to total sample strain . a one - way - anova ( post - hoc test : tukey ) was used to determine the effect of specimen length on mechanical parameters . to describe relationships between mechanical parameters and either csa or specimen length , pearsons correlation coefficient was used . bovine extensor and rat tail fascicles at a specific specimen length were compared by an unpaired t - test , as were mid - section and grip - section strains in short and long samples . mid - section and grip - section strains within a group of samples of the same length were compared by a paired t - test . gripping samples during mechanical testing will always lead to stress concentrations at the grips , hence the reported failure properties should be considered a minumum . however , using identical testing methods for all specimens , the values can be compared . failure stress , strain and modulus , derived directly from the bionix100 force and extension data , are compared for different sample lengths or diameters in figs . 2 and 3 , respectively . strain to failure was significantly influenced by specimen length and consistently reduced as the sample length increased . by contrast , specimen length did not influence failure stress in rat tail fascicles , although in bovine fascicles , it was significantly lower in 40 mm than 5 or 10 mm long specimens . the linear modulus increased with increasing specimen length in both sample types . whilst the effect of specimen length on mechanical parameters was similar in rat tail and bovine extensor fascicles , failure strains were consistently significantly lower in rat tail than bovine extensor fascicles , resulting in a significantly higher linear modulus in rat tail fascicles ( fig . 2 ) . strain measurements at longer specimen lengths were very consistent as evident from the particularly small standard deviations . failure strain , stress and linear modulus all correlated significantly with csa at certain specimen lengths . in bovine extensor fascicles , correlations were only apparent in short specimen lengths ( 5 and 10 mm lengths ) , where csa positively correlated with strain and negatively correlated with stress and linear modulus . in rat tail fascicles , failure strain correlated positively with csa at specimen lengths of 5 and 20 mm , whilst stress and linear modulus correlated negatively with csa at the majority of sample lengths ( 1060 and 100 mm and 560 and 100 mm , respectively ) ( fig . the linear regression lines , indicating that compliance is proportional to length , crossed the x - axis at 5.86 mm ( bovine extensor ) and 7.50 mm ( rat tail ) ( fig . the end - effect at each grip can thus be estimated at 2.93 mm in bovine extensor and 3.75 mm in rat tail samples . these data highlight that , having accounted for end - effects , a single linear modulus value can be used to describe each tendon type , found from the reciprocal of the regression lines to be 1000165 mpa for rat rail tendons and 714120 mpa for bovine extensors . correcting failure strains for end - effects reduced differences between short and long sample lengths ; however , significant reductions in failure strain with increase in sample length were still evident ( fig . strain curves was consistently different for bovine extensor and rat tail fascicles , and in the case of the rat tail fascicles it also changed with specimen length . the continuous modulus curves showed a characteristic two - peak course in rat tail fascicles in contrast to one peak in bovine extensor fascicles ( fig . 1b ) . in rat tail fascicles , the height of the first peak , rising to the maximum modulus , increased with increasing specimen length ( r=0.592 ; p<0.01 ) , enlarging the difference between the maximum and linear modulus . by contrast , this difference remained constant in bovine extensor fascicles ( r=0.107 ; p=0.387 ) ( fig . the location of the maximum modulus on the stress strain curve , relative to the strain at failure , appeared to be constant and independent of specimen length in both types of tendon fascicles . however , it occurred significantly earlier in rat tail fascicles at 217% of the strain at failure compared to 3613% of failure strain in bovine extensor fascicles ( fig . 6d ) in the grip - section were consistent for both sample lengths , mid - section strains were significantly smaller in larger specimens , highlighting that the variable strain distribution extended well beyond the gripping regions ( fig . whilst similar trends were unsurprisingly seen at 50% of failure , local strains in the mid - section of short samples were significantly smaller than in the grip - section at this time point only ( fig . 6a ) , highlighting that strains did not increase homogenously throughout the test either , but increased more rapidly in the grip - section for both sample lengths ( fig . 6b and e ) . we have highlighted how the material properties of tendon fascicles appear to be influenced by both specimen length and csa . measurement of grip - to - grip failure strains yielded significantly larger values in shorter specimens , with a subsequent reduction in modulus in these samples , while fig . 3 also highlighted how failure stress and modulus both decreased with increasing sample diameter . increased total strains at short specimen lengths further studies have suggested that this might be due to the end - effect , where extra unseen sample length within the grips contributes heavily to overall strain measurements in short specimens , whereas the response of longer samples more closely reflects true material properties ( bennett et al . , 1986 ; lam et al . , 1988 ) . a correction for end - effects in the current data notably reduced the magnitude of differences in strain between short and long samples , and also enabled us to find a single modulus value for each tendon type , indicating that the apparent changes in sample properties with length are likely to be almost entirely artifactual . one method to avoid this is to use an extensometer , which provides a method of measuring sample length without including the grip -region , and thus eliminating end - effect artifacts . 3 indicates that material properties are also influenced by csa . while this is at odds with fig . 4 , in which a single modulus value was appropriately found for each tendon type , it may explain the large standard deviations for each sample length in this data , if modulus is additionally influenced by csa . a correlation between csa and failure stress it is possible that some of the variation is still related to gripping , as collagen fibers in the centre of a larger fascicle experience less gripping pressure and are therefore more likely to shear and add to the end - effect , thereby increasing strain . however , in rat tail specimens of 10 , 40 , 60 and 100 mm length , no correlation between csa and strain was observed , but csa did correlate with stress and modulus . the smaller stress values of larger diameter samples may also be related to structural differences in the internal organization of samples . this may encompass fiber organization , interactions between subfascicles connected by connective tissue sheaths , or the relative contribution of collagen fascicles and connective tissue sheaths . it has been suggested that the decrease in the amount of areolar connective tissue in smaller samples might lead to an increase in modulus ( danylchuk et al . , 1978 ) . alternatively , the proportion of fiber bundles running parallel to the axis of force might be larger in smaller specimens ( butler et al . , 1986 ) . indeed , on a macro - scale , a study of human patella tendon has shown that small ( 1 mm ) and large ( 20 mm ) sections of tendon do not exhibit the same mechanical properties with larger modulus values in small specimens ( atkinson et al . , 1999 ) . this has been attributed to structures other than the subfascicle , such as the epitenon or other connective tissue components . average fascicle csa has been shown to be negatively correlated with tendon modulus , but positively correlated to the total tendon csa in equine superficial digital flexor tendon , also indicating an effect of structural organization on mechanical properties ( gillis et al . , 1997 ) . indeed , differences in composition and structure of functionally different tendons , such as the rat tail and bovine tendons investigated in the current study , may explain many of the differences seen in their mechanical properties . although failure strain , stress and modulus displayed similar trends with specimen length in both tendons , the shape of the stress strain curve at different specimen lengths changed in rat tail fascicles only , and the effect of csa on mechanical properties was more pronounced in rat tail fascicles . whilst end - effects explain the variation in failure strain with sample length , fig . it has been reported previously that the strains close to the gripping points are higher than in the mid - section of specimens ( butler et al . , 1984 ; devkota and weinhold , 2003 ; wu et al . , 2004 ) . this has been attributed to sample slippage in the grips , or premature failure , resulting from local stress concentrations at the grips ( butler et al . , 1984 ; wu et al . , 2004 ) . while gripping may reduce the sample diameter at these points and lead to greater stress and more extension in these regions , failure occurred throughout the length of the samples , opposing the suggestion of localized damage near the grips , although we can not exclude grip - related damage triggering the rupturing process . furthermore , two studies investigating the failure mechanics of tendons have found no reduction in the ultimate tensile strength of tendons failing at the grips compared with those failing in the mid - section ( ng et al . , 2005 ; smith et al . , the act of gripping is likely to alter the stiffness of the ends of the sample close to the grips . however , the small diameter of the samples would suggest that this effect should only extend a minimal distance into the sample . it does not readily explain a strain distribution along the sample length , evidently extending in excess of 5 mm from the gripping points . considering these findings from a micro - structural perspective , they may indicate discontinuity of fibrils . the applied strain is evidently transferred to those fibrils held within the grips , but our data imply that it may not be fully transferred to the adjacent fibrils near the centre of the sample , resulting in reduced strains in this area . since the grip - section in our experiment was between 5 and 10 mm long at each end ( in sum constantly 15 mm ) , and the mid - section strains were notably higher in the short samples ( 20 mm ) than in the long samples , we conclude that this subunit is on average more than 5 mm and less than 20 mm long . while tendon fascicles are thought to span the entire length of a tendon ( basso et al . , 2001 ) and to be mechanically and functionally independent ( haraldsson et al . , 2008 ) , controversy exists regarding the length and continuity of collagen fibrils in skeletally mature tendons and ligaments ( provenzano and vanderby , 2006 ) . fibrils are clearly discontinuous during tendon development ( birk et al . , 1997 ; provenzano and vanderby , 2006 ) and the variable csa along the length of some tendons , such as the human achilles tendon ( kongsgaard et al . , 2005 ) , suggests that discontinuous subunits are necessary to allow for this change in csa . however , in mature tendon only two fibril ends have been spotted using transmission electron microscopy to analyze 5639 fibrils ( trotter and wolfsky , 1989 ) , and no ends have been found in a study analyzing 7275 fibrils ( provenzano and vanderby , 2006 ) . the extremely high aspect ratio and interweaving of collagen fibrils make imaging complex ( craig et al . , 1989 ) , and with a fibril length in excess of 5 mm , the likelihood of seeing an end is low . since the large aspect ratio of collagen fibrils complicates the experimental assessment of fibril length , theoretical approaches have also been used , estimating a mean collagen fibril length in mature rat tail tendon of 6.412.7 mm ( craig et al . , 1989 ) . this corresponds positively with our estimation of > 5 and < 20 mm length of a discontinuous subunit . our findings have implications for the mechanical testing of tendon tissue : while it is not always possible to control for fascicle length and/or csa , these parameters have to be taken into account when comparing samples of different dimensions . it seems advisable to use longer specimens whenever possible to reduce the variability within a given subgroup of a defined fascicle length , and to avoid end - effects by using an extensometer in a portion of the specimen well clear of the clamps .

stress , strain and modulus are regularly used to characterize material properties of tissue samples . however , when comparing results from different studies it is evident the reported material properties , particularly failure strains , vary hugely . the aim of our study was to characterize how and why specimen length and cross - sectional area ( csa ) appear to influence failure stress , strain and modulus in fascicles from two functionally different tendons . fascicles were dissected from five rat tails and five bovine foot extensors , their diameters determined by a laser micrometer , and loaded to failure at a range of grip - to - grip lengths . strain to failure significantly decreased with increasing in specimen length in both rat and bovine fascicles , while modulus increased . specimen length did not influence failure stress in rat tail fascicles , although in bovine fascicles it was significantly lower in the longer 40 mm specimens compared to 5 and 10 mm specimens . the variations in failure strain and modulus with sample length could be predominantly explained by end - effects . however , it was also evident that strain fields along the sample length were highly variable and notably larger towards the ends of the sample than the mid - section even at distances in excess of 5 mm from the gripping points . failure strain , stress and modulus correlated significantly with csa at certain specimen lengths . our findings have implications for the mechanical testing of tendon tissue : while it is not always possible to control for fascicle length and/or csa , these parameters have to be taken into account when comparing samples of different dimensions .

Introduction Methods Results Discussion

PMC4144136

during tooth preparation for indirect restorations such as inlays , onlays , veneers , and crowns , a significant area of dentin is exposed . to avoid problems such as dentin contamination by provisionalization and hybridization failure sensitivity , a technique called immediate dentin sealing ( ids ) was suggested in the early 1990s . this technique consists of the application of an adhesive system immediately after tooth preparation and before taking the impression . another ids technique was developed in which a sealing film is applied to the dentin surface immediately after tooth preparation using an adhesive system and a low - viscosity composite resin . it is believed that this layer of low - viscosity composite resin isolates the underlying hybrid layer and consequently , aids in preserving the dentin seal . therefore , ids techniques are based on the principle that adhesive systems bond better to freshly prepared dentin , thus protecting the dentin pulp complex and preventing or decreasing sensitivity and bacterial leakage during the provisional stage . when using the ids techniques , the impression is taken after the application of the resin material on the dental substrate . this step is critical because the impression material can interact with the outer resin layer , which is unpolymerized due to the oxygen - inhibition of the radicals that initiate the polymerization reaction . different techniques have been suggested to reduce or eliminate the oxygen - inhibition layer ( oil ) , such as the application of glycerin jelly followed by an additional light cure or the use of a cotton pellet soaked in alcohol . however , no study has compared these two techniques before an impression with vinyl polysiloxane and polyether . the aim of this study was to qualitatively evaluate the interaction between the resin materials used in the ids techniques and impression materials when two different techniques to reduce / eliminate the oil are applied . this study was based on the hypothesis that these techniques do not eliminate the interaction between impression materials and resin materials . thirty - five unerupted human third molars , which were extracted for therapeutic reasons , were obtained from the tooth bank after the approval of the ethics committee of the pontifical catholic university of rio grande do sul . the water was changed every week , and the teeth were used in the study within 6 months . the roots were mounted in self - cured acrylic resin , and the occlusal enamel surface was removed with a diamond disc mounted in a low - speed laboratory cutting machine ( labcut 1010 , extec corp . the rest of the enamel was removed with 400-grit silicon carbide abrasive paper in a polishing machine ( dpu-10 , panambra , so paulo , sp , brazil ) under water . the superficial dentin was exposed and finished with 600-grit silicon carbide abrasive paper in the polishing machine , and a flat dentin surface was obtained . after polishing , the teeth were randomly divided into seven groups ( n = 5 ) to be used in group 1 ( impression with express xt ) . the teeth were used again for group 2 ( impression with impregum ) after remotion of the resin material with 600-grit silicon carbide abrasive paper . materials used in the study express xt vinyl polysiloxane impression material was used in group 1 , and the following subgroups were assigned [ figure 1 ] : group 1a : unsealed tooth surfacegroup 1b : ids with clearfil se bond ( cse ) . se primer was first applied to the tooth surface for 20 s and gently air dried . se bond was then applied , mildly air dried , and light cured for 10 s using a conventional halogen light curing unitgroup 1c : ids with cse and glycerin jelly . the polymerization of the adhesive was followed by the application of an air - blocking barrier with glycerin jelly and was then light cured for an additional 10 s. the glycerin jelly was rinsed under running tap watergroup 1d : ids with cse and alcohol . the surface of the adhesive system was wiped with a cotton pellet soaked in 70% alcohol for 10 sgroup 1e : ids with cse and protect liner f ( plf ) . after application of the adhesive , plf was placed on the adhesive surface using a brush - on technique and was then light cured for 20 sgroup 1f : ids with cse and plf + glycerin jelly . the polymerization of the cured low - viscosity composite resin was followed by the application of an air - blocking barrier with glycerin jelly and light curing for an additional 10 s. the glycerin jelly was rinsed under running tap watergroup 1 g : ids with cse and plf + alcohol . the surface of the cured low - viscosity composite resin was wiped with a cotton pellet soaked in 70% alcohol for 10 s. group 1a : unsealed tooth surface group 1b : ids with clearfil se bond ( cse ) . se primer was first applied to the tooth surface for 20 s and gently air dried . se bond was then applied , mildly air dried , and light cured for 10 s using a conventional halogen light curing unit group 1c : ids with cse and glycerin jelly . the polymerization of the adhesive was followed by the application of an air - blocking barrier with glycerin jelly and was then light cured for an additional 10 s. the glycerin jelly was rinsed under running tap water group 1d : ids with cse and alcohol . the surface of the adhesive system was wiped with a cotton pellet soaked in 70% alcohol for 10 s group 1e : ids with cse and protect liner f ( plf ) . after application of the adhesive , plf was placed on the adhesive surface using a brush - on technique and was then light cured for 20 s group 1f : ids with cse and plf + glycerin jelly . the polymerization of the cured low - viscosity composite resin was followed by the application of an air - blocking barrier with glycerin jelly and light curing for an additional 10 s. the glycerin jelly was rinsed under running tap water group 1 g : ids with cse and plf + alcohol . the surface of the cured low - viscosity composite resin was wiped with a cotton pellet soaked in 70% alcohol for 10 s. schematic design of the experimental groups a monophase polyether impregum was used in group 2 , and subgroups similar to group 1 ( 2a , 2b , 2c , 2d , 2e , 2f , 2 g ) were assigned based on the materials and techniques used [ figure 1 ] . individual trays with self - cured acrylic resin ( jet clssico , so paulo , sp , brazil ) were prepared . the adhesive was applied to the tray and was permitted to dry for 10 min . in groups 1a to 1 g , the putty / wash one - step technique was applied using express xt . the tray was filled with the heavy - body material , and then the tray was placed over the tooth . in groups 2a to 2 g , the one - step technique was applied using impregum . the medium - body material was injected over the tooth and in the tray , and the tray was placed over the tooth . the impression materials were allowed to set for 10 min before being removed from the tooth . each tooth surface was photographed using a digital camera nikon coolpix p100 ( nikon , melville , ny , usa ) with a tl3 model light source ( olympus , center valley , pa , usa ) . the images were saved in jpeg format and were used to examine the presence of unpolymerized and/or residual impression materials left on the treated tooth surface . the interactions between the impression materials and the resin materials are described in table 2 . interactions between the impression materials and the resin materials in the control groups ( groups 1a and 2a ) , no interaction was observed between the impression materials and the tooth structure [ figure 2 ] . photo of the tooth surface - control group ( without immediate dentin sealing ) and impression with vinyl polysiloxane : there is no impression material attached to the dentin surface . the same was observed for polyether for the vinyl polysiloxane impression material , the group that received ids with the cse ( group 1b ) and the group with plf ( group 1e ) had observable interactions in three and five impressions , respectively . a small quantity of unpolymerized impression material remained attached to the adhesive system [ figure 3 ] or to the low - viscosity composite resin . the application of glycerin jelly followed by an additional light cure ( group 1c ) and the use of cotton pellet soaked in alcohol ( group 1d ) prevented the interaction between the vinyl polysiloxane and the adhesive system [ figure 4 ] . however , neither treatment was completely effective with the low - viscosity composite resin , as a small quantity of unpolymerized impression material remained attached to the plf in one impression . photo of the tooth surface - immediate dentin sealing with clearfil se bond and impression with vinyl polysiloxane : small areas of unpolymerized impression material ( arrow ) are attached to the surface of the resin material . the same was observed for protect liner f photo of the tooth surface - immediate dentin sealing with clearfil se bond , alcohol application and impression with vinyl polysiloxane : there is no interaction between the impression material and the resin material . the same was observed for glycerin jelly application , as well as for ids with protect liner f and impression with polyether after glycerin jelly or alcohol application for the polyether impression material , the group that received ids with cse ( group 2b ) and the group with plf ( group 2e ) had observable interactions in five and three impressions , respectively . a small quantity of polymerized impression material remained attached to the adhesive system or to the low - viscosity composite resin [ figure 5 ] . the same interaction was observed for the groups that received ids with cse and the application of glycerin jelly ( group 2c ) and a cotton pellet soaked in alcohol ( group 2d ) , occurring in three and two impressions , respectively . when both treatments were applied to the low - viscosity composite resin ( groups 2f and 2 g ) , photo of the tooth surface - immediate dentin sealing with clearfil se bond , glycerin jelly application and impression with polyether : polymerized impression material is attached to the cse . the hypothesis of this study was partially rejected , as the prevention of interactions following the proposed treatments depended on the impression material and the resin materials employed . in the control groups , in which the ids was not applied , there was no interaction between the impression material and the tooth structure . however , in the groups in which the ids was applied , there was an observable interaction of vinyl polysiloxane and the polyether with the resin materials , either with the adhesive systems or the low - viscosity composite resin . the cse adhesive system and the plf low - viscosity composite resin are composed of methacrylates and when light cured , these materials present a superficial layer of approximately 40 m that does not polymerize with air - oxygen contact . the oil has a jelly - like consistency and is composed mainly of residual monomers that do not react after the polymerization of the resin material . it is likely that the difference between the chemical composition of the vinyl polysiloxane and the polyether caused the impression materials to react in the different ways to the resin materials . the permanence of the unpolymerized vinyl polysiloxane over the cse adhesive system was observed in three impressions and was also observed in every impression with the plf low - viscosity composite resin . it is speculated that the monomers presented in the oil may have reacted with the platinum salt , which is the catalyst in the reaction of polymerization of the vinyl polysiloxane , and a small portion of the light impression material remained unpolymerized over the resin materials . however , the amount of unpolymerized material is negligible and is likely functionally irrelevant ; therefore , it is believed that this interaction does not render the use of the impressions impractical . for the polyether , the polymerized material remained joined to the resin materials in all the impressions with cse and in three impressions with plf . this interaction may have occurred due to the polymerization reaction of the polyether ( ionic polymerization ) , in which the initiator agent of the reaction is an ion ( cation ) that can react with the free radicals of the monomers from the resin materials on the surface . moreover , the hydrophilicity , as well as the higher stiffness and the low resistance to the tearing of the polyether when compared to the vinyl polysiloxane , may have favored the superficial adhesion and the tearing of the impression material . this type of interaction renders the use of the impressions impractical . taking into account the principles of molecular interaction , it is believed that the oil was critical for the adhesion between the increments of composite resin and with the adhesive system . however , currently , it is known that oil is not essential for the chemical adhesion of the layers of the composite resin . additional polymerization with glycerin jelly over the layer of resin material , as well as the use of alcohol , both procedures were effective when applied over the cse adhesive system and the impression was made with vinyl polysiloxane , as no interaction was observed between the adhesive and impression material . however , it is speculated that there is a certain amount of residual unpolymerized monomers on the surface of the adhesive after the application of glycerin jelly or alcohol because the polyether remained joined to the adhesive surface , subsequently tearing and rendering the impression unusable . protect liner f has a higher percentage of filler than cse and consequently , should present fewer residual monomers after the light curing . therefore , the application of glycerin jelly and alcohol were effective in preventing interactions with plf when the polyether was used , as there was no interaction of this impression material with the low - viscosity composite resin . the application of glycerin jelly and alcohol on the low - viscosity composite resin was effective in four impressions with vinyl polysiloxane , and an interaction was observed only in one impression . the interaction was characterized by incomplete polymerization and the permanence of unpolymerized impression material adhering to the surface of the plf . however , the amount of unpolymerized material was insignificant , and therefore the resulting impressions remain usable . despite using standardized procedures , small variations , such as the final thickness of the low - viscosity composite resin , the thickness of the glycerin jelly applied on the low - viscosity composite resin , and the application pressure of the cotton pellet soaked in alcohol , likely contributed to the lack of effectiveness when removing or eliminating the oil in the sample in which the interaction was observed . both the application of glycerin jelly and alcohol affected the oil , as the results were similar for the same impression material when comparing the two techniques . this in vitro study presents some limitations , such as the shape of the samples . the application of ids was evaluated using flat dental surfaces . however , the preparations for indirect restorations are geometric and irregular , making it difficult to apply resin materials and remove oil . thus , additional studies are necessary to determine the nature of the interactions between the resin materials and the impression materials and to solve this potential clinical problem . despite the limitations of this in vitro study , the following conclusions can be drawn : the ids using the cse adhesive system or the plf low - viscosity composite resin produces interactions with the express xt vinyl polysiloxane and with the impregum polyetherthe application of glycerin jelly and alcohol prevented the interactions between the cse and the express xt and between the plf and the impregum . however , these treatments were not completely effective in preventing the interactions of cse with the impregum or the interactions of the plf with the express xt . the ids using the cse adhesive system or the plf low - viscosity composite resin produces interactions with the express xt vinyl polysiloxane and with the impregum polyether the application of glycerin jelly and alcohol prevented the interactions between the cse and the express xt and between the plf and the impregum . however , these treatments were not completely effective in preventing the interactions of cse with the impregum or the interactions of the plf with the express xt .

objectives : the objective of the following study is to evaluate the interaction between the resin materials used in immediate dentin sealing ( ids ) techniques and impression materials with two different techniques to eliminate the oxygen - inhibition layer.materials and methods : the occlusal dentin surface of 35 human molars was exposed . the teeth were used in two groups : group 1 impression with express xt ; group 2 impression with impregum . groups 1 and 2 were divided into 14 subgroups : groups 1a and 2a control groups ; 1b and 2b ids with clearfil se bond ( cse ) ; 1c and 2c ids with cse + additional polymerization with glycerin jelly ; 1d and 2d ids with cse + alcohol ; 1e and 2e ids with cse and protect liner f ( plf ) ; 1f and 2f ids with cse and plf + additional polymerization with glycerin jelly ; and 1 g and 2 g ids with cse and plf + alcohol . five teeth were used in each experimental group , and the tooth surface was photographed using a digital camera.results:small quantity of unpolymerized impression material remained attached to the cse or to the plf in groups 1b and 1e . groups 1c and 1d prevented the interaction . small quantity of polymerized impression material remained attached to the cse or to the plf for groups 2b and 2e . the same interaction was observed for groups 2c and 2d . for groups 2c and 2f , no interactions were observed.conclusion:resin materials interacted with impression materials . the application of glycerin jelly and alcohol prevented the interaction of cse with express xt and plf with impregum ; however , these treatments were not completely effective in preventing the interaction of cse with impregum and plf with express xt .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

PMC4393056

at the beginning of the 21st century , an explosion of information was discovered from the living organisms , especially in areas of molecular biology and genetics . the focus of bioinformatics deals with this flood of information , which comes from academy , industry , and government labs , and turning it into useful knowledge . as the genetic information being organized into computerized databases and their sizes steadily grow , molecular biologists need effective and efficient computational tools to store and retrieve the cognate information such as biological information from the databases , to analyze the sequence patterns they contain , and to extract the biological knowledge the sequences contain . the field of bioinformatics computing is advancing at an unprecedented rate . for people working with genomics and high - throughput sequencing data analysis , it is a serious challenge to analyze the vast amounts of data coming from the next generation sequencing ( ngs ) instruments . for example , there were approximately 126 , 551 , 501 , and 141 bases in 135 , 440 , and 924 sequence records in the traditional genbank divisions as of april 2011 . the tendency is likely only to be reinforced by new generation sequencers , for example , illumina hiseq 2500 generating up to 120 gb of data in 17 hours per run . the draft of the human genome has given us a genetic list of what is necessary for building a human : approximately 35,000 genes . for a genome as large as the human genome , it may take many days of cpu time on large - memory , multiprocessor computers to analyze . to handle this much data , computational strategies are important to tackle this vital bottleneck , which can aid scientists in the extraction of useful and important biological data . algorithms for biological sequence comparison can be categorized into two groups : exhaustive and heuristic . exhaustive algorithms based on dynamic programming give optimal solutions , and well - known search algorithms like the smith and waterman , needleman and wunsch , and hmm ( hidden markov models ) are of the dynamic kind . examples of heuristic algorithms are the blast , fasta , and feng and doolittle algorithms . heuristic algorithms are statistically driven sequence searches and alignment methods , and not as sensitive as the exhaustive algorithms such as the smith and waterman algorithm and hmm . an overview given in this paper concentrates on the computational capabilities and achievable performance of the systems discussed . to do full justice to all aspects of present high - performance implementations of the sequence analysis the methods we obtained from the entries of the individual implementations may be useful to many other bioinformatics applications . we believe that such an overview is useful for those who want to obtain a general idea about the various means by which these implementations achieved at high performance and high throughput with the most recent computing techniques . although most computer architecture and parallelization terms are familiar to many technical readers , we think it is worthwhile to give some concise information about high - performance computer architectures and the various processors employed in these research works in section ii , in order to better appreciate the systems information given in this paper . the majority of parallel systems are computing clusters of reduced instruction set computing ( risc ) based symmetric multi - processing ( smp ) nodes which in turn are connected by a fast network . shared and distributed - memory simd ( single instruction multiple data ) and mimd ( multiple instruction multiple data ) implementations which are described according to their macroarchitectural class are discussed in section 3 . the bioinformatics computing research is a very dynamic field and is especially true for the hardware - accelerated cluster world that has emerged at a tremendous rate in the last few years . the amount of research work that is related to hardware - accelerated biocomputing has boomed correspondingly . we comment on hardware characteristics and their position relative to other methods in section 4 , such as gpus ( graphics processing units ) , fpgas ( field programmable gate arrays ) , and cell be ( cell broadband engine ) architecture . an hmm is a statistical modeling method that has been widely used in the area of computational biology since the early 1990s . hmms were originally used in speech recognition and then borrowed to predict protein structures and analyze genome sequences . , qn}. each state can transition to another state according to state - transition probabilities { pi , j}. pi , j is defined as the probability that state qi at time t transmits to state qj at time t + 1 . one state can also emit a set of symbols ( residues ) { v1 , v2 , , vn } based on emission probabilities { pivk}. pivk represents the probability that state qi emits symbol vk . all the possible state transitions and their associated transition probabilities are shown by arrows in the figure , where the set of symbols ( i.e. { a , c , g , t } ) that can be emitted by that state as well as the emission probability for each symbol is shown in the state boxes ( figure 1 ) . starting from an initial state , a sequence of states can be generated by moving from state to state according to the state transition probabilities . then , a symbol sequence can be produced by allowing each state to emit symbols according to its emission probability distribution . the following gives an example of a possible transmission sequence and symbol sequence for the hmm in figure 2 . given this example , we can easily calculate the probability of the above state transition sequence . if we assume that the probability of being in state q1 at time t = 0 is pq1(0 ) = 0.7 ( i.e. , the initial state ) , we can get ( 1)p[q1 q2 q2 q1 q1]=0.70.20.60.40.8=0.02688 . we can also calculate the probability of the above symbol sequence given the above transition sequence : ( 2)p[(actgc ) ( q1 q2 q2 q1 q1 ) ] = 0.30.50.30.40.1=0.0018 . the probability of obtaining the above transition sequence and the above symbol sequence is ( 3)p[(actgc)(q1 q2 q2 q1 q1 ) ] = 0.026880.0018=4.8384105 . one can see that hmms provide a mathematical model for linear problems like sequences or time series . in practice , only the output symbol sequence is observable and the state transition sequence is hidden from us . hence , we must infer the state sequence by given observed sequence data ( e.g. , dna sequences ) . please also note that while an hmm has a finite number of states , it can generate infinite number of possible sequences . hmms have been successfully employed to represent profiles of multiple sequence alignment in computational biology . a profile is defined as a consensus primary structure model consisting of position - specific residue scores and insertion or deletion penalties . various hmm architectures have been proposed to model a profile in the literature . while traditional pairwise alignment strategies such as smith - waterman , blast , and fasta use position - independent scoring , these hmm - based models can capture position - specific alignment information . the plan 7 model is a representative profile of hmm architecture in figure 3 . a multiple sequence alignment of homologous protein sequences can be precisely represented by such a model . there are three major types of states in this architecture : matching states ( m ) , insertion states ( i ) , and deletion states ( d ) . the matching states were used to model the distribution of residues allowed in the columns of a multiple sequence alignment . that is , each state has an emission distribution that reflects the frequency of each residue observed in the corresponding column of that alignment . one can see that each matching state can transit to another matching state and different types of other states ( e.g. , i , d ) according to its transition probabilities not only transits to a matching state but also transmits to itself which allows one or more symbols to be inserted . since there exist 20 different amino acid residues for protein sequences , each matching state or insertion state carries a set of 20 emission probabilities . the plan 7 model starts from state b and ends at state e. in addition , it contains five special states ( i.e. , s , n , c , t , and j ) that deal with alignment specific features ( e.g. , global or local alignment ) . given a profile hmm like the plan 7 model , researchers are typically interested in three problems : ( 1 ) how are different parameters of the model learned given some observed sequence data ? that is , given the architecture or topology of a hmm and observed data , we want to find the optimal model that maximizes p ( observationshmm ) ; ( 2 ) given an existing optimized hmm and an observed sequence , what is probability that the model produces that sequence ( i.e. , p ( sequencehmm ) ) ? ( 3 ) what is the most likely state sequence that the hmm would use to generate a sequence ? that is , we want to find the optimal state sequence q1 , q2 , , qn such that p ( q1 , q2 , , hmmer [ 21 , 22 ] is a software tool that relies on the plan 7 model . it consists of a collection of programs that can be employed to solve the above three problems . due to the huge amount of genomic sequence data , exploring acceleration techniques there are various accelerating platforms that allow performing multiple computations in parallel so as to reduce the total computing time . while the hardware includes processors and memory , the software consists of computer programs and data stored in memory . in addition , special - purpose hardware can be specifically designed for a particular type of bioinformatics computing ( e.g. , sequence searches ) . parallelism can be achieved both within one computer and among a group of computers . within one computer , modern compilers can detect independent instructions and pack them together in vliw ( very long instruction word ) for parallel execution . during runtime , cpus ( central processing units ) can reorder the execution of instructions such that those independent instructions can be executed simultaneously . moreover , intel 's htt ( hyper - threading technology ) or smt ( simultaneous multithreading ) supports parallel execution of multiple instructions from different hardware threads - running programs ' control flows . in recent years , the multi / many - core computer architecture allows several processing units to be built inside a single cpu chip where each core can host multiple threads . furthermore , multiple cpu chips can be connected inside a single box , called smp ( symmetrical multiprocessors ) , for processor level parallelism . also , a group of computers can be connected through fast networks and work as a virtual supercomputer . each node has its own memory and cache but can not directly access another processor 's memory . each node also has an ni ( network interface ) for all communication and synchronization . please note that a node can be either a single - cpu computer or an smp . to effectively and efficiently utilize various hardware resources at different granularity levels , software needs to decompose data and programs , map them onto multiple processing units , and support communication in order to coordinate different subtasks . the shared address space programming paradigm ( i.e. , multithreading ) is a widely used approach on single cpu machines or shared memory multiprocessors . with this programming paradigm , posix ( portable operating system interface ) threads and openmp ( open multi - processing ) are popular software packages that support multithread programming . however , it can not be directly applied on clusters . on distributed memory machines or clusters , the message passing programming paradigm is more effective and commonly used . with this approach , multiple programs are developed and executed concurrently on different computers . each program runs independently unless it needs to communicate with another program in order to share and synchronize data . if the overhead of such communications can be under control , this approach will utilize the otherwise idle processors in multiprocessors or idle computers in clusters . mpi ( message passing interface ) is a representative library for this paradigm . using mpi or posix to accelerate sequence alignment tools like hmmer requires high - performance multiprocessor computers or large - scale computing clusters . an alternative approach is to design special - purpose hardware that can operate on normal computers . since such hardware is specifically tailored for the sequence alignment problem , good performance can be easily achieved . fpga has been used to build special - purpose hardware for accelerating hmmer [ 19 , 27 , 28 ] . an fpga is an integrated circuits that contains a lot of programmable interconnected logic blocks . the advantage of this approach is that it can easily achieve good performance since the hardware is especially tailored for the sequence alignment problem . its disadvantages include high initial design cost and programming complexity of the hardware description language . in addition , the special - purpose hardware might not be employed to solve other problems . gpu cards have also been applied to accelerate hmmer as well as other sequence alignment algorithms [ 15 , 29 ] . today 's commodity gpu cards can provide tremendous memory bandwidth and computational horsepower . moreover , software tools such as general - purpose computation on gpu , nvidia 's cuda ( compute unified device architecture ) and apple 's opencl have made parallel programming on gpus much easier than before . there are various metrics , for example , speedup , efficiency , and cost , which are used to measure the performance of a certain parallel program . no single method is usually preferred over another since each of them reflects certain properties of the parallel code . a straightforward measure of the parallel performance would be the ratio of the execution time on a single processor ( the sequential version ) to that on a multicomputer . amdahl 's law describes the relationship between the expected speedup of accelerated implementations of a program relative to the original nonaccelerated program . it states that the maximum expected performance improvement to be gained from using a faster mode is limited by the fraction of the time the faster mode can not be applied to . for example , assume that we have a program that needs 10 hours using a single - processor computer and a particular portion of 1 hour can not be accelerated . that is , only the remaining portion of 9 hours can be accelerated , either using multiple processors or special - purpose hardware . intuitively , no matter how many processors or hardware units we employ to accelerate this program , the overall execution time can not be less than the critical 1 hour . theoretically , amdahl 's law is depicted by the following equation : ( 4)overall speedup=1(1p)+(p / s ) , where p represents a fraction of a program that can be accelerated and s is the corresponding speedup of the enhanced portion . in the above example , p is equal to 9/10 = 0.9 . if we assume the portion of 9 hours can be mapped onto 4 processors or hardware units and executed simultaneously , then s is 4 , assuming no extra overheads are introduced . in this case , the overall expected speedup is ( 5)overall speedup=1(10.9)+(0.9/4)=3.08 . one can see that , given limited hardware resources ( e.g. , 4 processors ) , the most important thing for accelerating a program is to detect the time - consuming portion that can be parallelized or accelerated . note that the acceleration process often introduces additional overhead such as data transfer to and from the external hardware accelerators , data redecomposition and reformatting , and communication among different hardware components . therefore , the actual achievable speedup is usually less than the theoretical value obtained by amdahl 's law . various software approaches have been applied to accelerate the original hmmer programs which were developed by eddy and his coworkers [ 21 , 22 ] . most of these approaches focus on accelerating one or more of the three programs : hmmpfam , hmmsearch , and hmmcalibrate . hmmpfam is used to search a database of profile hmms against a given query sequence . hmmcalibrate takes a profile hmm and determines its statistical significance parameters which make the database search more sensitive . in the literature , various strategies have been applied to modify and parallelize these programs in order to take advantage of different levels of commodity hardware components and achieve much better performance . modern general purpose processors often have 16-byte - wide registers that can hold and process multiple data items within one single instruction . this simd ( single instruction multiple data ) technique can be used to explore fine - grained instruction level parallelism . a variety of commodity cpus such as amd opteron , amd turion 64 , intel xeon , and intel core solo / duo provide simd instructions and corresponding large registers . these instructions have a great potential to accelerate sequence analysis in biological computing due to its data - intensive nature . sse2 ( streaming simd extensions 2 ) instructions from intel are a representative set of simd instructions that extends intel 's previous mmx ( multimedia extension ) and sse technologies . they provide a series of packed integer operations and double precision floating point operations that work on 128-bit data . walters and his colleagues have attempted the use of sse2 instructions to accelerate hmmer . since reimplementing the entire hmmer program using sse2 instructions is costly and time consuming , they only focused on the innermost loop of the viterbi function used by both hmmpfam and hmmsearch when performing a search . that loop consumes more than 50% of the execution time for both programs . moreover , only additions and maximum value selections over 32-bit integers are included in this short segment of code . since sse2 instructions operate on 128-bit data , four integer operations are allowed to be performed in parallel . therefore , the ideal speedup of this loop is 4x . according to the amdahl 's law introduced in section 2.3 , the ideal overall speedup would be 1/((1 0.5 ) + 0.5/4 ) = 1.6 . however , it is very difficult to achieve the above speedup because there are several types of overhead during the reimplementation of the code of that loop . therefore , the code in the loop must be rearranged and split into several loops in order to remove the dependencies . second , the sse2 does not have direct max / min instructions which are required in order to reimplement the loop . the max / min operations must be implemented using the existing instructions , which introduces further overhead . third , extraoperations , such as data alignment and the moving of data into the 128-bit registers , also bring in additional overhead . experiments were performed on a 2.66 ghz intel xeon processor with 2.5 gb of memory . both hmmpfam and hmmsearch were tested using various samples from the pfam and nr databases . while the pfam database contains many multiple sequence alignments and hmms represent different protein families , the nr database is a sequence database from ncbi ( national center for biotechnology information ) . the major advantage of using sse2 to accelerate hmmer is that it does not require new hardware upgrades and the cost is almost free . but the critical segment of code that consumes most of the execution time must be identified . if that segment of code is short , reimplementing the code is trivial and does not need much development time . however , its disadvantage is that the modified codes from c to intrinsic / assembly are not portable but architecture dependent . that is , moving the modified code from one platform to another requires reimplementing the code again for the new platform . while simd instructions exploit instruction - level parallelism within one cpu , in order to take advantage of multiple cpus or cpus with multiple cores in a computer system , users must develop multithreading programs . as a widespread programming model , multithreading allows a process to generate multiple software threads that share the same virtual address space . these threads inherit many resources from the hosting process and , at the same time , have their own stacks and registers . programmers need to partition source code or data into different threads that will be mapped onto different hardware units . then , these threads execute concurrently and finish their own jobs in parallel . one of the most widely used thread libraries is pthread ( posix threads ) . to develop multithreaded programs , users have to insert library calls to create , delete , and synchronize threads . however , it requires openmp compliers to compile programs and it is often difficult to deal with irregular problems , such as problems inherent in sparse and unstructured computations . the original hmmer package provides pthread implementations of three programs : hmmsearch , hmmpfam , and hmmcalibrate . to run these programs on a multiprocessor machine , reimplemented the hmmpfam program using openmp directives and tested its performance on various intel 86 architecture - based shared memory multiprocessor ( smp ) systems . with 600 hmm models and 250 sequences , the speedup is 3.36 on a four - processor smp system ( 2.0 hz ) . its performance was also compared on two smp systems that both have sixteen x86 processors . but one of the systems has 8 gb of memory and its processors are 3.0 ghz . the other system has 16 gb of memory and its processors are 2.2 ghz . the speedup of the former system is 14 whereas the speedup for the latter system is about 10.3 . this comparison indicates that the multithreaded hmmer hmmpfam program is more sensitive to cpu speed than memory size . another multi - threading implementation of the hmmpfam program was done by zhu et al . . they reimplemented hmmpfam on earth ( efficient architecture for running threads)an event - driven fine - grain multithreaded program execution model . through its threaded - c language ( an extension of c ) , the earth - based multithreaded hmmpfam achieved about 30 speedups on a cluster of 18 nodes , each containing two 1.4 ghz amd athlon processors . on a much larger cluster that consists of 128 nodes , each with two 500 mhz pentium iii processors , the achieved speedup is as high as 222.8 for a data set containing 50 hmm profiles and 38,192 sequences . while sse2 allows users to exploit instruction level parallelism within one single processor , distributed memory parallelism provides opportunities to execute programs in parallel across different computers . a group of processes can be started simultaneously and mapped onto multiple machines , each of which accomplishes a subtask . since processes do not share the address spaces , communication approaches such as mpi and pvm ( parallel virtual machine ) must be utilized across machines in order to send / receive messages and coordinate and manage different tasks . clusters . a cluster can be viewed as a parallel computer system that consists of an integrated collection of independent nodes . these nodes can work together closely but each of them can perform independent operations and may be derived from products developed for other standalone purposes . with a fast network , common users can easily build their own clusters . there exist several variations of cluster computing such as internet computing , grid computing , and cloud computing . clusters are very suitable for implementing distributed memory parallelism and have been widely used in bioinformatics computing . it typically takes a master - slave model where a master node distributes subtasks to different slave nodes , and slave nodes do the real computing work and return results to the master node . under the supervision of professor vijay pande at stanford university , the folding@home project is such an example where volunteer computers form one of the largest clusters in the world . a volunteer computer always contacts the server ( i.e. , master ) for protein folding work , and every time they finish their local work , they contact the server again for extra work . this large distributed computing platform allows us to do extremely challenging computation that could not be achieved before . that is , the communication overhead over the long distance to the master node is trivial when compared to the computing time . both pvm and mpi have been adopted for implementing parallel versions of hmmer that can run on distributed clusters . the original hmmer package provides a pvm implementation of three programs : hmmsearch , hmmpfam , and hmmcalibrate . in this implementation , the computation for one sequence is executed concurrently , and the master node dynamically assigns one profile to a specific slave node for comparison . upon finishing its job , the slave node reports the results to the master , which will respond by assigning a new profile . when all the comparison regarding this sequence is completed , the master node sorts and ranks all the results it collects and outputs the top hits . reimplemented hmmsearch and hmmpfam using mpi and compared their performance to their corresponding pvm implementations . second , since pvm does not truly support asynchronous sends , implementing a nonblocking , double - buffering strategy using mpi has the potential to hide communication latency and thus achieve better performance . the double - buffering scheme allows a slave node to compute on a buffer while at the same time receiving another sequence into a second buffer . the basic idea of this strategy is to overlap the computation time and communication time as much as possible . to further reduce the communication time , the researchers employed a database chunking technique to minimize the number of sends and receives . this technique allows a node to receive and work on batches of sequences simultaneously before returning its results to the master node this combined version of hmmer can take advantage of both the parallelism between computing nodes and the instruction level parallelism within a single workflow . the performance of three hmmer implementations ( i.e. , pvm , mpi , mpi + sse2 ) was compared on a university cluster in which each node consists of two 2.66 ghz intel 4 xeon processors and different nodes communicate through 100-mbit ethernet . experiments have shown that the mpi implementation outperformed the pvm implementation , and the mpi + sse2 implementation gained even better performance . for example , the speedups of hmmsearch on 16 cpus for the three implementations are 4.56 , 5.90 , and 7.71 , respectively , using a 100 mb database . with various software tools , however , there are several other issues such as scalability , i / o , and quality of code that can also affect the performance of the accelerated hmmer . if these issues are not handled appropriately , the computing power of a large system can not be fully utilized and thus the speedup is only limited . scalability is a very important issue for a massively parallel system / cluster that contains hundreds or thousands of nodes . a system is considered to be scalable if its performance improves proportionally to the hardware added . the scalability of a large parallel system can be affected by many factors such as communication latency , data dependency , and organization of involved processes . jiang and his colleagues studied the techniques that can improve the scalability of the three hmmer programs ( i.e. , hmmsearch , hmmpfam , and hmmcalibrate ) on the blue gene / l ( bg / l ) massively parallel supercomputer from ibm . the mpi implementation of hmmcalibrate is found to be scalable and its scalability is up to 2,048 nodes . however , the observed scalability of hmmsearch and hmmpfam is no higher than 64 nodes ; that is , no improvement was observed beyond 64 nodes . bioinformatics applications like hmmer are not only computationally intensive but also highly i / o demanding . data intensive input and output operations can become the performance bottleneck like the hmmer program , especially when large databases are searched . studied the i / o problems in hmmer and enhanced its mpi implementation ( called mpi - hmmer ) using parallel i / o and a parallel file system . in this new version ( called pio - hmmer ) , the database distribution mechanism was modified so that the master node , instead of directly reading data and sending them to slave nodes , only distributes sequence indexes ( each containing sequence offsets and lengths ) to slave nodes and slave nodes read from the database in parallel . in addition , several new optimizations were implemented to further improve the overall performance : ( 1 ) enhanced postprocessing reducing the number of messages being sent to the master node by only returning those messages resulting in hits ; ( 2 ) a database chunking technique similar to the one implemented in mpi - hmmer , but with larger chunks of the database for parallel i / o ; ( 3 ) asynchronous i / o for returning scores to the master node ; ( 4 ) a load - balancing scheme for hmmsearch achieved by allocating database based on the lengths of sequences instead of assigning equal number of sequences to each slave node ; ( 5 ) a database caching scheme similar to the one implemented by jiang et al . . various experiments were performed on a cluster with 1056 nodes , each equipped with two 3.2 ghz intel xeon processors and 2 gb ram . with parallel i / o and the above optimizations , the overall speedup achieved by pio - hmmer is 221x for hmmsearch and 328x for hmmpfam , while mpi - hmmer is only able to achieve 55x speedup for hmmsearch and 27x speedup for hmmpfam . the performance impact of some individual optimizations ( including parallel i / o ) was also tested . as shown by experiments , the parallel i / o represents the single greatest performance impact of all optimizations . it improved the speedup from 42x to 190x for hmmsearch ( searching a 236 state hmm against a large sequence database ) . for example , the mpi - hmmer 's nonlinear scalability is only 64 nodes whereas pio - hmmer 's scalability is extended to 256 nodes . experiments also demonstrated that both mpi - hmmer and pio - hmmer can achieve better scalability for large data sets ( i.e. , large hmm database and sequence database ) . this is consistent with the results obtained by srinivasan et al . who tested the scalability of an openmp implementation of hmmer using various datasets . their experiments revealed that the scalability of multithreaded hmmer improves when the size of the input data increases . the background reason of this observation is that proportion of time spent in the sequential code decreases with respect to the time spent in the parallel portion when large data sets are used . the quality of source code may affect compiler / runtime optimizations and cache misses and as a result influence the performance of a program . made minimal changes ( e.g. , removing unnecessary intermediate variables , breaking iteration dependencies ) of the loop in the p7viterbi routine and achieved 1.8x speedup for hmmcalibrate . by splitting the loop and breaking the intraiteration dependence in the viterbi algorithm , srinivasan et al . all these changes , while minor , can have a big effect on the compiler optimization and result in modest performance improvements . also attempted to reduce the number of cache misses by dynamically allocating the size of the dp_matrix used by hmmpfam so that it matches the size required by a profile hmm . on a 4-processor machine , the achieved performance of different software approaches for accelerating hmmer is summarized in table 1 . some researchers or research groups did various experiments using various database sizes or hardware configurations . in this case , only the best achieved speedup is primarily focused on scalability , and explicit speedups of their experiments were not provided . even though the achieved speedups can not be directly compared due to the variations in experimental setups and database sizes , several observations can be made from this table . first , the overall speedup achieved by instruction level parallelism is limited since it only exploits internal parallelism within one single processor . but this approach does not require any new hardware and can be easily combined with other approaches like mpi . second , the performance achieved by shared memory parallelism is generally moderate since the number of processors sharing the same memory is often limited . however , good performance can be obtained under the assistance of earth since it provides an efficient architecture for running threads on large distributed memory systems like clusters . finally , compared with instruction level parallelism and shared memory parallelism , distributed memory parallelism has the greatest potential to achieve the best performance after carefully taking care of i / o issues and other communication bottlenecks . programmers need to explicitly divide databases , map computing tasks onto different nodes , and handle various synchronizations among different tasks . as an alternative to software accelerated computing , such as shared and distributed memory computing systems , hardware - accelerated computing has been advocated in bioinformatics applications by combining powers of special - purpose hardware and existing computational resources . in this section , we give most hmmer implementation details with an emphasis on the hardware architecture in various forms of which we will discuss some general characteristics . it has become all the more clear that no one type of processor is best for all types of computation . in the recent several years , a wide range of computational hardware accelerators have been applied to bioinformatics research . the developments in this field are rapidly gaining popularity . today , microprocessors are more powerful for general purpose computing , but it is still too slow to perform the hmmer profile search which is extremely data and compute intensive . the hardware structure or architecture determines to a large extent what the possibilities and impossibilities are in speeding up a computer system beyond the capability of a single cpu . bioinformatics users never tend to content themselves with the performance of the machines they own and are continuously seeking new breakthrough to speed up the calculation . presently there is a group of acceleration products which can deliver significant performance gains over traditional approaches on hmmer when properly deployed . before going on to the descriptions of the hmmer acceleration techniques , it is useful to consider some specialized computer architectures , which have been used to increase the computational performance . we follow the main trends in emerging architectures for the heterogeneous parallel systems and discuss the most recent hmm hardware implementations on the three classes of accelerators . gpgpus ( general purpose computation on graphics processing units ) may have been invented to power video games , but today these massively parallel devices harness the computational power to perform nongraphics calculations . the latest gpu architectures provide tremendous memory bandwidth and computational horsepower , with fully programmable vertex and pixel processing units that support vector operations up to full ieee floating point precision . the interconnections can be reconfigured dynamically , thus allowing the hardware datapaths to be optimized for a given application or algorithm . when the logic blocks are full processors , the fpga can be used as a parallel computer . cell be is a heterogeneous architecture containing one general - purpose computer and eight simd - based coprocessors on a single chip . by exploiting the mimd parallelism of the coprocessors and overlapping memory operations with computations , the cell be has been proved to achieve impressive performance on many bioinformatics applications . moreover , hardware accelerators are now used in some of the world 's fastest computers . they can also be seen as a new level in hierarchical machines , where they operate in parallel with the host processors . the top supercomputers have taken advantages of heterogeneous cooperation architecture to scientific calculation in a multiuser environment . 1 supercomputer was roadrunner , which has 129,600 cores , and utilizes ibm cell be processors as accelerators . hmmer has also been accelerated on other special - purpose processor , such as network processors - jackhmmer , which builds on the intel ixp 2850 network processor , a heterogeneous multicore chip consisting of an xscale cpu paired with 16 32-bit microengines . gpus can offer energy - efficient performance boosts to traditional processors since they contain massive numbers of simple processors , which are more energy efficient than a smaller number of larger processors . graphics processing is characterized by doing the same operation on massive amounts of data . to accommodate this way of processing gpus consist of a large amount of relatively simple processors , fast but limited local memory , and fast internal buses to transport the operands and results . the key to accelerating all of these operations is parallel computing , often realized by computing all pixels of a display or all objects in a list independently . the growing popularity of gpu - based computing for nongraphics applications has led to new interfaces for accessing gpu resources . a major challenge in the evolution of gpu programming involves preserving gpu performance levels and ease of use while increasing the generality and expressiveness of application interfaces . with improving programming models , nvidia 's cuda allows programmers to write data - parallel applications for gpus at the kernel newest technique by the portland group offers fortran and c accelerator compilers to accelerate the existing high - level standard - compliant programs for the cuda - enabled nvidia gpus by adding openmp - like compiler directives . it is motivated by gpgpus ' enhanced programmability , attractive cost / performance ratio , and incredible growth in speed . today they are being pressed into high - performance bioinformatics computing . for accelerated hmm search , a considerable amount of research made the use of a gpu for nongraphics high - performance computing more interesting . in the paper , gpu - hammer was implemented by taking advantage of nvidia 8800 gtx ultra gpus with 768 mb ram . the 8800 gtx ultra is composed of 16 stream multiprocessors , each of which is itself composed of 8 stream processors for a total of 128 stream processors . it can maintain 4,096 active threads and all threads run in parallel on a single gpu with each operating on its own sequence . in order to achieve the best performance thus , it is able to achieve a nearly 7x performance over the unsorted database . when the hmmsearch codes were targeted to the 8800 gtx ultra , a variety of optimizations were implemented including database - level load balancing memory layout and coalescing , loop unrolling , and shared / constant memory use . redesigned the hmmsearch program to extract data parallelism out of the serializing data dependencies using 1 and 4 tesla c1060s . the highlight of this work is that each gpu thread block operates on individual sequences and writes the cost of decoding to the global memory independently . they reported that the time grows linearly with the hmm module size and scales linearly with the number of gpus . this work showed a speedup of 5x-8x over gpu - hmmer . with 4 tesla c1060 boards , it could achieve 100 + x speedup compared to a serial implementation on an amd opteron at 2.33 ghz . an fpga is an array of logic gates that can be reconfigured to fulfill user - specified tasks . in this way we can design special purpose functional units that may be very efficient for some specific purpose . in addition , fpga allows easy upgrading and users to explore the applicability of its reconfigurable nature to various scientific problems compared to application - specific hardware . because of their versatility it is difficult to specify where they will be most useful . the clock cycle of fpgas is low as compared to that of present cpus : 100550 mhz which means that they are very power effective . to program fpgas , there are now two industry standard hardware description languages , vhdl ( very high speed integrated circuit hardware description language ) and verilog . vendors , like xilinx and altera , provide runtime environments and drivers that work with linux as well as windows . oliver et al . presented an fpga solution that implements a full plan 7 model . instead of computing the viterbi algorithm on one dataset at a time , they aligned query / subject in separate processing elements ( pes ) they designed in verilog . their design assumes that the same profile hmm has to be aligned to different sequences . the system is connected to the hmmer software running on the host system via an usb port . this strategy outperforms the sequential implementation on a desktop for both hmmserch and hmmpfam by one to two orders of magnitude . another recent research proposed a systolic - array - based implementation of plan 7 hmm on fpgas with a parallel data providing unit and an autorecalculation unit . a speedup of 56.8 with 20 pes on a virtex-5 board was obtained compared to an intel core 2 duo 2.33 ghz cpu . combining the parallel efficiency of a cluster with one or more fpga cards can significantly improve the hmmer 's hmmserch functionality , in some cases achieving near linear speedup . the system is not just designed for running individual fpga , but for scaling codes on a fpga cluster . the cell be was jointly designed for video gaming industry by ibm / sony / toshiba . one cell be contains two different types of processors : one 64-bit ppe ( powerpc processor element ) and eight spes ( synergistic processing elements ) , all running at a clock speed of 3.2 ghz and theoretical peak performance of 204.8 gflops for single precision . ppe acts a controller for the eight spes , which handle most of the computational workload . each spe consists of a spu ( synergistic processing unit ) and a mfc ( memory flow controller ) . in addition , a high - speed memory controller and high - bandwidth bus interface are all integrated into one chip . the nature of hmm , all - to - all comparison , has most potential to gain benefits from porting the entire program to a heterogeneous multicore processor like the cell be . each query search is completely independent and thus can be performed in parallel across the 8 spus of the cell processor . sachdeva et al . implemented the computationally expensive kernel viterbi on the spus and run the reminder of the code on the ppe . unfortunately , their preliminary implementation only worked on a single sequence being compared against a single hmm on a single spu . however , it still showed about 3.5x faster than a 2.4 ghz dual core opteron processor . potentially , the cell be is not used as a screen platform , but also as a complete computing engine with large performance benefits . the performance of hardware acceleration is summarized in table 2 based on the peak performance that the hardware can achieve . note that there are no standard hmm benchmarks and the reported performance was done by individual research group using various databases and hmm models . for example , running an application efficiently on a hardware accelerator often requires keeping the data near the device to reduce the computing time taken up with moving data from the cpu or memory to the accelerator . also , a programmer must decide how to collect results from the accelerator back into the cpu program . this is very cumbersome for the average programmer as one not only has to explicitly define such details as the placement of the configured devices but also the width of the operands to be operated on and so forth . the goal of hardware acceleration or hybrid computing is to reduce the search time from hours to minutes . heterogeneous implementation utilizes a mix of hardware accelerator and compute nodes to achieve the excellent performance over that of a single node and accelerator only implementation . the results show that integrating the parallel efficiency of a cluster with one or more hardware accelerators can significantly increase performance for even the compute / data intensive hmm searches . obviously , a hybrid cluster would be a smart choice to offer bioinformatics users access to a technology that is positioned to change how many traditional applications are written . in this paper , we selected hmmer , a scientific application from the domain of bioinformatics , to evaluate a variety of its implementations by comparing their performance with corresponding sequential , software , and hardware solutions . we have reviewed the above exploratory work on the use of modern computing architectures , ranging from chip level multithreading , multicore architectures , clusters , and grid computing to special architectures . first of all , we emphasized several observations that were verified by various experiments and the techniques might be extended to other bioinformatics applications . ( 1 ) the cpu speed has a bigger impact on the multithreaded hmmpfam program than on memory size . this observation can direct us to select appropriate hardware configurations so as to get better performance and also provide useful implications for bioinformatics computing since many bioinformatics applications are compute intensive . ( 2 ) the scalability of both mpi and multithreaded implementations of the hmmer program is sensitive to the size of the input data . the reason is that the larger the data set , the more time is spent in the parallel portion of the program . thus , a better speedup can be obtained with respect to the amdahl 's law . this observation implies that the performance of parallel version of hmmer including both shared memory and distributed memory parallelism is related to the size of input data set . ( 3 ) experiments on the mpi implementation of both hmmpfam and hmmsearch showed that , relatively speaking , hmmpfam is more i / o - bound while hmmsearch is more compute bound . thus , analyzing each application and carefully handling its performance bottleneck are also very important in bioinformatics computing . second , clock frequencies of chips are no longer increasing , so all future improvements in computer speed will come from parallelism . though many bioinformatics applications are parallelizable like hmmer , parallelism is usually not explicitly expressed in their original codes . users interested in accelerating a bioinformatics application are required to either develop software to distribute data and manage parallel jobs or modify existing codes to make sure of libraries or hardware that facilitate distributed computing . next , the long - term goal of bioinformatics computing is to design a system that is a more efficient , much less expensive and has an immediate impact on the world 's energy consumption . bioinformatics should stand at the front of green computing to reduce energy consumption compared to other commercial systems . in comparison to general purpose cpus , certainly they will do only part of the work in a complete system but still the power savings can be considerable which is very attractive these days . when speaking of special purpose hardware , that is , computational accelerators , we should understand that they are indeed good at certain specialized computations while they may not be able to perform others . despite the inherently parallel nature of modern computer architecture , so , not all applications can benefit from them and those which can , not all to the same degree . for example , the computations execute on external accelerators efficiently only under conditions of massive data parallelism . programs that attempt to implement nondata parallel algorithms perform poorly . fpga technology has been seen by many as an ideal way to handle a lot of the data overhead in bioinformatics . we have reviewed some hmmer implementations accelerated by fpga technology , and generally the performance is a lot better . the board can be installed in any computer , which in turn will provide a huge boost in performance . however , due to the fact that fpgas are relatively difficult to program and optimize , there has never been any major renovations for fpga technology within genomics . currently technology is moving into the spotlight as the premier technology has raised the computational challenges following the rollout of ngs instruments . this is partly due to the fact that gpus and the popular cuda architecture offer a rather easy way to teraflop computing . however , the bioinformatics society still expects to see implementations of known algorithms which truly exploit the cutting - edge computing technology . although the software tools for accelerators have improved enormously lately , for many applications it is still a big challenge to obtain a significant speedup . an important factor is that data must be transferred between the accelerator and cpu ; therefore , the bandwidth of the connecting bus is a severe bottleneck in most cases . the key to high performance lies in strategies that hardware components and their corresponding software components and their corresponding software interfaces use to keep hardware processing resources busy . this can be overcome by overlapping data transport between the accelerator and host with processing . tuning the computation and data transport task can be cumbersome . whether on a compute node , cluster , grid , or cloud software acceleration does not require additional investment , but delivering top performance requires using threads on multiple cores or mpi on multiple nodes with simd . unlike the other technologies mentioned above , simd is based on software acceleration , by utilizing the built - in wide instruction sets that inherently built into all x86 architecture cpus from both intel and amd . there is no doubt that software acceleration , like simd , is cheaper , and its performance is equal to adding more hardware virtually . the fast implementation using simd vectorization is available in the hmmer 3 package . everyone in biology is now affected by parallelism . a computing cluster running accelerated application is now a competitive alternative to a traditional general - purpose cluster running serial code . therefore , it has become a trend to emerge heterogeneous processors within a single architecture . the sign of this trend is the presence of gpgpu , fpgas , cell be , and other computation accelerators in combination with standard processors . as the next generation of high - performance computing technology is coming a heterogeneous parallel architecture , for example , the new generation of gpgpu processors which also support simd a strategy of using simd acceleration along with cluster computing becomes even more attractive . in brief , we have presented a comprehensive analysis of the bottleneck of hmmer algorithm and efficiency of various implementations on modern computing platforms . most parallel bioinformatics applications use just one of these abovementioned forms to express parallelism , but with the deeper hierarchical structure within heterogeneous computing systems , there has been a movement towards hybrid computing models . the goal of hardware acceleration or hybrid computing is to boost performance and extend the range of applications , particularly to bioinformatics computing . hybrid computing involves combining multiple techniques together , which apply a different programming paradigm to different levels of the hierarchical system , for example , mixing mpi for internode communication with openmp for the intra - node parallelization . additional parallelism can be achieved to take advantage of the distinct properties of specialized underlying hardware to gain higher - level efficiencies . finally , cloud computing is another way of handling the data analysis challenges , by renting access to a large cpu pool on a need - to - use basis . while cloud computing surely is a relatively easy and interesting alternative to setting up and running a cluster , generally cloud computing may not give real performance gains over cluster computing , because of the huge overhead of distributing the data and calculations across the leased nodes . we hope that we have shown a clear trend to potential performance improvement in software and hardware acceleration combined with increased compute density , faster i / o access , and higher degree of user control over optimizations , which suggests that a heterogeneous parallel architecture makes a major breakthrough in accelerating bioinformatics applications . we expect this trend will continue in the coming years and make the bioinformatics computing roadmap more diverse and interesting .

this paper focuses on the latest research and critical reviews on modern computing architectures , software and hardware accelerated algorithms for bioinformatics data analysis with an emphasis on one of the most important sequence analysis applications hidden markov models ( hmm ) . we show the detailed performance comparison of sequence analysis tools on various computing platforms recently developed in the bioinformatics society . the characteristics of the sequence analysis , such as data and compute - intensive natures , make it very attractive to optimize and parallelize by using both traditional software approach and innovated hardware acceleration technologies .

1. Introduction 2. Background 3. Software Accelerated HMMER 4. Hardware-Accelerated HMMER 5. Discussion 6. Conclusion

PMC4195381

precise imaging of the cell surface of fluorescently labeled bacteria requires super - resolution methods because the size - scale of these cells is on the order of the diffraction limit . in this work , we present a photocontrollable small - molecule rhodamine spirolactam emitter suitable for non - toxic and specific labeling of the outer surface of cells for three - dimensional ( 3d ) super - resolution ( sr ) imaging . conventional rhodamine spirolactams photoswitch to the emitting form with uv light ; however , these wavelengths can damage cells . we extended photoswitching to visible wavelengths > 400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam . further , an n - hydroxysuccinimide - functionalized derivative enabled covalent labeling of amines on the surface of live caulobacter crescentus cells . resulting 3d sr reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x , y , and z. the distribution of cell stalk lengths ( a sub - diffraction - sized cellular structure ) was quantified for a mixed population of cells . pulse - chase experiments identified sites of cell surface growth . covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 1020 nm .

Supplementary Material

PMC4207070

the nervous tissue is composed of numerous types of cells of neural ( ectodermal : neurones and neuroglia ) and non - neural ( mesodermal : microglia , endothelial cells , pericytes , muscle cells , etc . ) origins organised into tightly coordinated cellular networks . evolution of the nervous system progressed through cellular specialisation , with neurones becoming chiefly occupied with fast information processing and transfer , and neuroglial cells assuming responsibility for housekeeping . neuroglia of the central nervous system ( cns ) is classified into macroglia ( astrocytes , oligodendrocytes and ng2 cells ) and microglia ( which are the descendants of embryonic macrophages invading the brain early in development ) . the systemic function of neuroglia is the preservation of homeostasis at all levels of the cns organisation , from molecular to organ [ 1 , 2 ] . astrocytes , which are arguably the most diversified type of glia , define the architecture of the grey matter being the central elements of the glio - vascular unit . inside these glio - vascular units elaborated processes of astroglia cover synaptic contacts and neuronal membranes , and control molecular composition of the interstitium by regulated transport of water , ions and neuroactive agents such as neurotransmitters and neurohormones [ 3 , 4 ] . astrocytes are indispensable for synaptic connectivity ; astroglial cradle governs synaptogenesis , synaptic maturation and synaptic maintenance [ 5 , 6 ] . astroglia are fundamental for neuro - transmission , being specialised in clearance of neuro - transmitters ( such as glutamate , gaba and adenosine ) and for supplying neurones with glutamine , which is a dual precursor for glutamate and gaba [ 7 , 8 ] . astroglial cells provide neurones with metabolic substrates and protect nerve tissue against reactive oxygen species ( ros ) , being the chief source of ros scavengers such as glutathione and ascorbic acid [ 10 , 11 ] . astroglia are responsible for : i ) cns development and adult neurogenesis ; formation and maintenance of the cns - blood and cerebrospinal fluid - blood barriers ; and iii ) the regulation of local blood flow . in addition , specialised astrocytes appear as central chemoceptors involved in systemic regulation of na , ph and co2 homoeostasis [ 15 , 16 ] , and regulation of sleep . oligodendrocytes contact , support and myelinate axons in grey and white matters , thus , being central elements of the cns connectome . the ng2 glia ( a lineage related to oligodendrocytes ) possibly contribute to adult myelination and may also possess certain homeostatic functions . finally , microglia are highly important for development of the cns and shaping neuronal networks through synaptic stripping and removal of excessive neurones which undergo massive apoptosis at different stages of embryogenesis [ 18 - 20 ] . indeed , brain lesions trigger homeostatic response such as the containment of excitotoxicity through buffering an excess of k and glutamate , and by the release of ros scavengers . in conditions of ischaemia and glucose deprivation astrocytes and oligodendrocytes neuroglial cells are in possession of an evolutionary conserved defensive programme known as reactive gliosis , triggered in response to polyaetiological insults [ 21 , 22 ] . the gliotic response is further sub - classified into reactive astrogliosis , reactive response of ng2 cells and activation of microglia . oligodendrocytes ( as well as schwann cells in the peripheral nervous system ) are also activated in response to axonal damage , this activation being a part of wallerian degeneration . reactive gliosis is a complex and multistage response of glial cells , which is disease- and context- specific , and involves activation of thousands of genes . this glial reactivity is a defensive response aimed at protecting stressed neurones ( and the brain in general ) isolating injured area , removing pathogens , dying cells and cellular debris , and remodelling the nerve tissue after the resolution of pathology . the hallmarks of reactive astrogliosis are hypertrophy and proliferation of astrocytes associated with up - regulation of cytoskeletal components such as glial fibrillary acidic protein ( gfap ) , vimentin or nestin [ 23 , 24 ] . an increased expression of these intermediate filaments are , however , only considered as broad markers of this process , because astrogliotic metamorphosis may produce many different , yet to be fully characterised , reactive phenotypes specific for different diseases . in the process of a productive gliotic response astrocytes undergo a complex remodelling of their biochemistry and function , which generally leads to neuroprotection . in severe lesions astrocytes produce glial scar aimed at isolating the area of damage ; astrogliosis is also critical for regeneration of nerve tissue after resolution of pathology . all in all , the suppression of astrogliotic response is detrimental for nerve tissue viability and exacerbates pathological progression ( for details and exhaustive reference lists see [ 22 - 26 ] ) . morphologically , astrogliosis is broadly divided into isomorphic gliosis in which domain organisation of astrocytes is preserved and anisomorphic gliosis in which astrocytes proliferate and lose their domain organisation with their processes becoming densely overlapped . isomorphic gliosis is fully reversible , whereas anisomorphic gliosis is frequently resolved in the formation of a glial scar . reactivity of ng2 glia has been studied to a much lesser extent ; their response to a pathological insult is represented by shortening and thickening cellular processes and a strong increase in the expression of ng2 . together with astrocytes ng2 glia may contribute to the formation of a glial scar through secreting chondroitin sulphate proteoglycan 4 . in certain conditions ng2 cells may possibly act as stem cells ; in particular , they can generate new oligodendrocytes which in turn can assist in post - lesion remyelination of axons [ 28 , 29 ] . activation of microglia progresses through many stages and cell phenotypes characterised by distinct morphological , biochemical , functional and immunological changes . similarly to astroglia , activation of microglia is a multistage , complex and context - specific process , which produces multiple phenotypes of activated cells , many of which demonstrate prominent neuroprotective features [ 30 - 32 ] . in conditions of severe or specific brain lesions , such as , infectious encephalitis , microglial cells start to proliferate , become motile , accumulate around sites of damage and become phagocytotic , thus , actively removing foreign agents and cell debris . the philosophy of contemporary clinical and experimental neurology is created around neuronal doctrine that regards neurones as a central element for pathological progression . this is reflected by drug development , with most of the agents being specifically aimed at affecting neuronal excitability or neuronal receptors . this neurono - centricity is somewhat surprising in the light of common definition of the disease as a homeostatic failure . in this respect , the homeostatic arm of the nervous system , the neuroglia , shall naturally be considered as a fundamental element for initiation , development and outcome of neurological disorders . indeed , neurones when left to their own devices have limited capacity for self - protection and for meeting environmental challenges ; it is the neuroglia that protect and maintain the nervous system operation . the glio - cenric angle in neurology is still in statu nascendi ( as reviewed recently [ 33 - 45 ] ) , although it is rapidly gaining popularity . conceptually , the glial involvement in a neuro - pathological process could be primary or secondary , i.e. , primary neurogliopathy ( manifested by the loss or change of the glial functions ) and secondary reactivity , respectively . the boundary between these two faces of glial pathology is blurred and often they are present in combination . a striking example of astrogliopathy ( which can be considered as an astroglial asthenia ) is associated with the down - regulation of astrocyte - specific glutamate transporters ( excitatory amino acid transporters 1 and 2 ) , which is a common cause of many neurotoxic ( e.g. , mercury , lead or aluminium encephalopathies ) and neurodegenerative ( e.g. , amyotrophic lateral sclerosis - also called motor neurone disease , wernike - korsakoff encephalopathy or huntington 's disease ) disorders ; a compromised astroglial glutamate clearance acts as a primary mechanism of neurotoxicity , neuronal death and brain atrophy [ 44 , 46 - 51 ] . similarly , toxic damage to astrocytes produced by ammonia that leads to the occlusion of glutamate - glutamine shuttle , exocytotic release of glutamate , failure in glutamate clearance and k buffering is a central element for hepatic encephalopathy [ 52 - 55 ] . atrophy and asthenia of neuroglia have been identified in major neuropsychiatric diseases such as schizophrenia and major depression ; in both pathologies degradation of astrocytes and oligodendrocytes are prominent histopathological features [ 40 , 45 ] . similarly , atrophic astrocytes have been observed in the pre - symptomatic stages of alzheimer 's disease ( ad ) in animal models [ 56 - 58 ] ; the earliest occurrence of this atrophy was found in entorhinal and prefrontal cortices , the most vulnerable regions in ad pathology [ 59 , 60 ] . the asthenic astroglial cells in these two brain regions failed to mount gliotic response to extracellular depositions of amyloid which might be a relevant explanation for this high vulnerability . namely , in the animal models , microglial cells almost doubled their density at pre - plaque stages of the disease , this being very similar to changes found in normal ageing [ 61 - 63 ] . formation of plagues trigger activation and accumulation of activated microglia around plaques [ 38 , 64 ] ; these activated cells , however , are deficient in their phagocytotic function . reactive astrogliosis and activation of microglia usually appear in response to disease - specific lesions . for example , reactive glia in ad is recruited in response to an appearance of senile plaques or perivascular amyloid depositions . similarly , gliotic response accompanies late stage of amyotrophic lateral sclerosis [ 66 , 67 ] ; is detected in fronto - temporal dementia and is prominent in thalamic dementia ( in which astroglial activation has been claimed to be associated with a loss of function , which causes neuronal death ) . in neuronal ceroid lipofuscinosis , also known as batten disease , astroglial reactivity ( manifested by significant increase in gfap expression and hypertrophy ) occurs at the very early stages ; inhibition of astrogliosis ( by genetic removal of intermediate filaments gfap and vimentin ) accelerates disease progression and exacerbates neurodegeneration . unresolved gliotic response , however , may have various detrimental consequences to the outcome of neurological diseases . chronic astrogliosis , for example , suppresses neurogenesis , whereas an astroglial scar prevents axonal regrowth . suppression of astroglial reactivity improved regeneration in lesioned nerves and enhanced regenerative processes in animal models of ischemia , stroke and injury and facilitated integration of retinal grafts , as well as differentiation of transplanted neural stem cells . neuroglial cells are one of the central elements of neuropathology ; loss of neuroglial function as well as neuroglial reactive responses contribute to most ( if not all ) neurological , neuropsychiatric and neurodevelopmental diseases . a multitude of molecules , specifically expressed by neuroglial cells and responsible for their homoeostatic and defensive functions , are potential and legitimate targets for therapeutic management . in this special issue we collected papers specifically dedicated to neurogliopathology with an aim to expand glio - centric views into translational medicine .

neuroglia of the central nervous system ( cns ) , represented by cells of neural ( astrocytes , oligodendrocytes and ng2 glial cells ) and myeloid ( microglia ) origins are fundamental for homeostasis of the nervous tissue . astrocytes are critical for the development of the cns , they are indispensable for synaptogenesis , and they define structural organisation of the nervous tissue , as well as the generation and maintenance of cns - blood and cerebrospinal fluid - blood barriers . astroglial cells control homeostasis of ions and neurotransmitters and provide neurones with metabolic support . oligodendrocytes , through the process of myelination , as well as by homoeostatic support of axons provide for interneuronal connectivity . the ng2 cells receive direct synaptic inputs , and might be important elements of adult remyelination . microglial cells , which originate from foetal macrophages invading the brain early in embryogenesis , shape the synaptic connections through removing of redundant synapses and phagocyting apoptotic neurones . neuroglia also form the defensive system of the cns through complex and context - specific programmes of activation , known as reactive gliosis . many neurological diseases are associated with neurogliopathologies represented by asthenic and atrophic changes in glial cells that , through the loss or diminution of their homeostatic and defensive functions , assist evolution of pathology . conceptually , neurological and psychiatric disorders can be regarded as failures of neuroglial homeostatic/ defensive responses , and , hence , glia represent a ( much underappreciated ) target for therapeutic intervention .

NEUROGLIA CONTROL HOMEOSTASIS OF THE CENTRAL NERVOUS SYSTEM NEUROGLIA MOUNTS BRAIN DEFENCE PATHOLOGICAL POTENTIAL OF NEUROGLIA: NEUROLOGICAL DISEASES AS NEUROGLIO-PATHIES TARGETING NEUROGLIA FOR NEUROTHERAPY

PMC4859103

childhood overweight and obesity have become a worldwide epidemic with significant medical , psychosocial , and economic consequences . the prevalence of childhood overweight and obesity is increasing rapidly worldwide , and there is mounting concern on the need for their early prevention and treatment . overweight and obesity in childhood tend to persist into adulthood and are associated with increased adult mortality and morbidity . several studies that investigated childhood overweight and obesity in saudi arabia have found that lifestyle transformation ( physical activity , leisure , and modernization ) and a change in nutrition related to changing economic , social , and health factors have largely contributed to the increase in the prevalence of overweight and obesity observed in saudi children and youth . however , a fairly recent report indicated that overweight and obesity are the major problems among local children of southwestern highlands of saudi arabia . it was suggested that the difference in the rate of overweight and obesity between saudi highland and lowland children could be related to the difference in physical activity levels since race , socioeconomic conditions , and dietary habits appeared to be similar in the two communities . in general , the relationship between childhood overweight and obesity and physical activity is complex . in the previous prospective studies , however , other studies of the same nature have failed to show any significant relationship . the aim of the present study was to describe the distribution of physical activity and its relationship to childhood overweight and obesity in high and low altitude populations of children in southwestern saudi arabia where both environmental and population genotype differ from other studied areas . this cross - sectional study was carried out in high and low altitude areas of the aseer province in southwestern part of the kingdom of saudi arabia . three villages alsoda , alsoga , and tabab located at 30003150 m above the sea level were selected for the study . for comparison , alraish and al - khatarish villages located at a relatively low altitude of 500 m above the sea level were selected . the weather of alsoda , alsoga , and tabab is cold and rainy in winter and dry with moderate temperatures in summer . winter in al - khatarish and alraish is rainy with relatively moderate temperatures and summer is dry and hot . the permanent residents in the two areas are rural populations who have many modern facilities ( electricity , clean water supply , cars , etc . , ) but who still maintain their basic dietary and social habits . meat , chicken , and rice constitute the major dietary items for people living in the two areas . primary health care in aseer province is provided through a wide network of primary health care centers . secondary and tertiary care services in the province are provided through a network of well - staffed hospitals . data were collected from 145 healthy subjects aged 1015 years who were born and bred at a high altitude ( about 93% of the total population aged 1015 years registered in alsoda , alsoga , and tabab health centers ) and 154 healthy subjects of comparable age who were also born and permanently domiciled at low altitudes ( about 94% of the total population aged 1015 years registered in al - khatarish and alraish health centers ) . subjects were selected on the basis of their acceptance or their parents ' acceptance of the invitation by the medical practitioners and the local people working in the health centers to participate . each child children in whom pathology was detected by clinical examination ( chronic renal , respiratory , cardiovascular , and gastrointestinal ) and those children who were not born and who did not have permanent residence in the designated study areas were excluded from this study . to determine the level of physical activity of children at this age , the international physical activity questionnaire - short form - a ( ipaq - sf - a ) was used . the ipaq - sf - a form was translated into arabic and distributed 1 week before the clinical examination . the form was completed by the child , checked by the parents , and rechecked by the researchers and/or the medical practitioners in the health centers . the ipaq - sf - a assesses physical activity undertaken across a comprehensive set of domains including leisure time , domestic and gardening ( yard ) activities , and work - related and transport - related activities . only the physical activity lasting more than 10 min without rest breaks and within the last 7 days was estimated . during the study , however , the questions on sitting were developed as separate indicators and not as part of the summed physical activity score . vigorous activity was physical activity that caused a high increase in breathing and/or heart rate such as carrying or lifting heavy loads , digging , running , playing football , or construction work . moderate physical activity caused small increases in breathing and/or heart rate , for example , carrying a light load , cycling , swimming , and playing volleyball . for each specific type of activity , the frequency ( measured in days per week ) and duration ( time per day ) were recorded . physical activities were calculated in metabolic equivalent ( met ) . met is the ratio of a person 's working metabolic rate relative to the resting metabolic rate . one met is defined as the energy cost of sitting quietly and is equivalent to a caloric consumption of 1 kcal / kg / h . vigorous activity was given 8 mets , moderate 4 mets , and walking 3.3 mets . ffor each activity the score was calculated by multiplying its mets x frequency ( days per week ) x duration ( time per minutes ) . the subject 's overall physical activity using ipaq - sf - a ( total physical activity met - minutes / week ) was sum of total ( walking + moderate + vigorous ) met - min / week scores . using these values , three levels of physical activity were identified : high physical activity : the two criteria for classification as ; high are vigorous - intensity activity on at least 3 days achieving a minimum total physical activity of at least 1500 met - min / week or 7 or more days of any combination of walking and moderate - intensity or vigorous - intensity activities achieving a minimum total physical activity of at least 3000 met - min / weekmoderate physical activity : the pattern of activity is to be classified as ; moderate in any of the following criteria : 3 or more days of vigorous - intensity activity of at least 20 min per day or 5 or more days of moderate - intensity activity and/or walking of at least 30 min per day or 5 or more days of any combination of walking and moderate intensity or vigorous intensity activities achieving a minimum total physical activity of at least 600 met - min / weeklow physical activity : those individuals who did not meet criteria for high or moderate were considered to have a ; low physical activity level . high physical activity : the two criteria for classification as ; high are vigorous - intensity activity on at least 3 days achieving a minimum total physical activity of at least 1500 met - min / week or 7 or more days of any combination of walking and moderate - intensity or vigorous - intensity activities achieving a minimum total physical activity of at least 3000 met - min / week moderate physical activity : the pattern of activity is to be classified as ; moderate in any of the following criteria : 3 or more days of vigorous - intensity activity of at least 20 min per day or 5 or more days of moderate - intensity activity and/or walking of at least 30 min per day or 5 or more days of any combination of walking and moderate intensity or vigorous intensity activities achieving a minimum total physical activity of at least 600 met - min / week low physical activity : those individuals who did not meet criteria for high or moderate were considered to have a ; low physical activity level . for each subject at high and low altitudes , body weight was measured and recorded using an avery beam weighing scale to the nearest 0.1 kg . subjects were weighed partly dressed and a correction of 0.5 kg was made for clothing . standing height was measured and recorded to the nearest 0.5 cm with a stadiometer ( without shoes ) . the age of the child was calculated from birth certificate and body mass index ( bmi ) was computed from the weight and height ( bmi = weight [ kg]/height [ m ] ) . overweight and obesity were defined as a bmi > 85% according to age- and gender - specific percentiles for bmi from the who - nchs reference population . spss version 20 ( ibm corp . , armonk , ny ) was used for standard statistical analysis . the student 's t - test and z - test were used to compare the two means and two percentages , respectively . spearman 's correlation coefficient was used to test for association between activity scores and bmi . chi - square test was used to test for the association between overweight and obesity and the levels of physical activity . spss version 20 ( ibm corp . , armonk , ny ) was used for standard statistical analysis . the student 's t - test and z - test were used to compare the two means and two percentages , respectively . spearman 's correlation coefficient was used to test for association between activity scores and bmi . chi - square test was used to test for the association between overweight and obesity and the levels of physical activity . table 1 shows the distribution of weight , height , bmi , activity scores , and prevalence of overweight and obesity by altitude and sex . at both high and low altitudes , there were no significant differences in the mean ages between boys and girls at high altitude and their respective groups at low altitude ( p < 0.76 for both ) . the overall prevalence of overweight and obesity was significantly higher among highland children ( 39.3% ) than among lowland children ( 24.0% ) ( p < 0.004 ) . this was true for both boys ( 37% at high altitude and 23.9% at low altitude , p < 0.04 ) and girls . there were no significant differences in the prevalence of overweight and obesity between the two sexes at the same altitude ( p < 0.39 for highlanders and < 0.94 for lowlanders ) . at both high and low altitudes , boys reported significantly more activity than girls ( p < 0.001 for both ) . when the two sexes were compared at different altitudes , lowland boys reported significantly more activity than highland boys ( p < 0.001 ) while there was no significant difference in the level of activity between highland and lowland girls ( p < 0.5 ) . the distribution of weight , height , bmi , activity scores and prevalence of overweight and obesity by sex table 2 shows the number and percentage of subjects in the three activity levels by altitude and sex . for boys of the same age , the proportion of subjects who belonged to the high activity group was significantly greater in lowlanders ( 21.1% ) than highlanders ( 10% ) ( p < 0.03 ) . there were no significant differences in the percentages of subjects who belonged to the moderate activity level between boys and girls at high altitude and their counterparts at low altitude . the proportion of boys whose activity level was low was significantly greater in the group at high altitude than that at low altitude ( p < 0.001 ) while no significant difference was found between the proportions of girls in the low activity group at high and low altitudes . distribution of physical activity levels by altitude and sex significant negative correlations between activity scores and bmi were observed in highland and lowland boys and girls ( r= 0.47 , p < 0.001 ; r= 0.63 , p < 0.001 for high altitude boys and girls , respectively ; r= 0.52 , p < 0.001 and r= 0.35 , p < 0.02 for low altitude boys and girls , respectively ) . analyses were performed separately for boys and girls to test for the association between physical activity and overweight and obesity . for highland and lowland boys , physical activity was inversely related to overweight and obesity [ table 3 ] . in both highland and lowland boys , the proportion of overweight and obese was lowest in the highly active group and highest in the least active group . no significant association was observed between physical activity and overweight and obesity for either highland or lowland girls although the highest proportion of overweight and obese was in the least active group and the lowest was in the moderately active group [ table 3 ] . the prevalence of overweight and obesity according to the level of physical activity the present study demonstrated that the level of physical activity was inversely and significantly associated with overweight and obesity in boys aged 1015 years at both high and low altitudes , but there was no clear trend for girls of the same age at either altitude . we selected the short form ipaq - sf - a instrument , out of various methods available , as a measure of physical activity because it is short , reproducible , easy to administer in surveys and provides a reliable estimation for a range of physical activity domains . more importantly , the method was acceptable to the respondents at both high and low altitudes . in addition , the ipaq - sf - a has been used in a series of international and national studies and has widely accepted reliabilities . in this study , the validity of the ipaq - sf - a was indirectly tested by comparing activity scores with resting pulse rate which was found to be inversely and significantly related to activity score ( r= 0.154 , p < 0.008 ) . the relationship of pulse rate and physical activity levels is shown graphically in figure 1 . the vertical bars represent the standard deviations out of various indices , we selected bmi as an indicator of overweight and obesity because bmi is easy to derive and applies to all populations without any need for a reference population . furthermore , the bmi is highly correlated with other estimates of fatness . in this study , the low level of physical activity observed in girls , especially those at low altitude , may be related to sociocultural factors . girls of this age tend to spend most of their time indoors watching television and playing computer games , and they are usually taken to school by car . the low levels of physical activity observed in highland boys compared to boys at low altitude may be mainly the result of the environment . because of the cold weather at high altitude and the topography of the area , highland boys tend to spend most of their time indoors whereas lowland boys tend to be more active . they spend more time outdoors on farms rearing goats and sheep . the negative association between physical activity and overweight and obesity in both high and low altitudes boys was consistent with previous studies in the field this disparity may be the result of problems associated with the methods used to evaluate physical activity . the bmi of both highland and lowland girls showed significant negative correlation with the activity score . however , no significant association was observed between the level of physical activity and overweight and obesity in either highland or lowland girls although the proportions of overweight and obesity were higher in the low activity group and lowest in the moderate activity group . this may be because this population of girls was homogenous with regard to physical activity , which had little or no apparent effect on overweight and obesity in individuals . it is obvious that childhood overweight and obesity are a serious public problem with adverse effects on health and longevity . in view of the significant association between physical activity and overweight and obesity , at least in high and lowland boys , health education programs that encourage physical activity as means of preventing overweight and obesity and as strategy for weight reduction in overweight and obese children

objective : to assess the relationship between overweight and obesity and physical activity in saudi children born and permanently domiciled at high and low altitudes in southwestern saudi arabia.subjects and methods : a cross - sectional study of 145 healthy saudi children aged 1015 years who were born and lived permanently at high altitude ( 30003100 m ) and 154 healthy saudi children of comparable age who were born and lived permanently at a relatively low altitude ( 500 m ) was conducted . for each subject selected , body weight and body height were measured using an avery beam weighing scale and a stadiometer , respectively . body mass index ( bmi ) was calculated using the equation bmi = ( weight [ kg]/height [ m2 ] ) . physical activity scores were determined using international physical activity questionnaire - short form - a . resting radial pulse rate ( beat / minutes ) was determined clinically.results:physical activity was significantly and inversely associated with overweight and obesity in boys at both high ( 2 = 15.8 , p < 0.001 ) and low ( 2 = 14.7 , p < 0.001 ) altitudes , but there was no clear trend for girls at either altitude . the lack of association between physical activity and overweight and obesity in girls was attributed to the low and homogeneous level of physical activity.conclusion:physical activity should be encouraged as a strategy for weight reduction in the overweight and the obese and the prevention of overweight and obesity in saudi children at high and low altitudes .

INTRODUCTION SUBJECTS AND METHODS Statistical analysis RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

PMC3976005

when performing exercise , humans generally assume a basic , erect , bipedal posture . in this posture , we engage in different movements at varying speeds , such as walking , running , kicking , and jumping . the feet are an important body component , as they constantly function as shock eliminators , weight support structures , and locomotive organs at any given time1,2,3,4,5,6,7,8,9 . in recent years , many reports have indicated that toe flexor strength and grip force training leads to significant changes in walking speed or pace and improved dynamic balance and prevents falls10 , 11 . however , the common training methods for foot - related research , treatment , and prevention have typically included flat - footed exercises such as towel gathering or toe exercises . these exercises use extrinsic and intrinsic muscles without distinction , and many points regarding their frequency , load , implementation time , implementation period , and effects remain unclear . furthermore , previous studies , including one conducted by our research team , have reported the importance of the intrinsic foot muscles , but the effects of such training have not been clarified12 . if a training effect is acceptable , improvements in athletic performance , treatment of foot disease , long - term foot function maintenance , obstacle prevention , enhancements in the standing and walking positions , and further exercise of a flat feet loads are expected . the purpose of the present study was to focus on the intrinsic foot flexor muscles , and to verify the effects of a strength training method with fixed repetitions and loads on ( 1 ) muscle strength , ( 2 ) foot arch formation , and ( 3 ) dynamic test items ( 1-legged long jumping , vertical jumping , and 50-m dash time ) . the subjects were 12 healthy males ( 12 left and 12 right feet ) without motor system diseases or history of foot - related conditions such as flat feet ( low arches ) . the mean ( standard deviation ) age , height , and weight were 29 5 years , 172.5 7.3 cm , and 64.9 12.8 kg , respectively . characteristics of the subjectssubject being testedage ( years)height ( cm)height ( cm)height ( cm)a3218210030.2b271646524.2c291806419.8d2617059.820.7e291746120.1f301685619.8g311707325.3h36174.56320.7i221655118.7j221746220.5k321867220.8l361635319.9bmi : body mass index . there is no sport history which continued the subject being tested in the period before and behind training , and it is not carrying out except this training . the test items were measured before ( pre - training ) and after 8 weeks of training ( post - training ) , and the values were compared using statistical analysis . a digital grip dynamometer ( model # 5101 , takei scientific instruments co. , ltd . , niigata , japan ) was used to measure intrinsic foot flexor strength and could display values 5 kg in increments of 0.1 kg . a wooden box was constructed for the dynamometer , measuring 50 14 60 cm ( length width height ) , which stabilized the device and lower limbs during the measurements . the limbs were fixed with the subject in the supine position , with their knees in extension and the ankles in maximum plantar flexion . next , after correcting any distal interphalangeal ( dip ) joint curvature caused by extrinsic muscle contraction resulting from passively flexing the toe , a finger sling ( c7791 , sakai medical co. , ltd . , tokyo , japan ) was used to flex each toe , with the dynamometer fixed at a 5-kg load . the intrinsic foot flexor strength was quantified for all toes by measuring the flexion - gripping force in the interphalangeal ( ip ) and metatarsophalangeal ( mp ) joints . these measurements were performed with the subjects barefoot , and 3 measurements were performed for the left and right feet , which were then averaged for each foot . the muscle contraction time for each measurement was 3 s. the measurements were collected on a total of 3 days with the same testers , and were performed at a 23-day interval . the interobserver correlation ( interclass correlation coefficient , 1.3 ) indicated a high reliability of 0.98 between testers for muscle strength measurement values . therefore , these measurements were collected three times and the mean values of these 3 measurements were used . for arch measurement , the longitudinal and horizontal arch lengths were recorded for the left and right feet to create a static standing position using a berkemann footprint . the longitudinal arch length was defined as the distance from the head of the first metatarsal bone to the calcaneal tuberosity . the horizontal arch length was defined as the distance encompassing the head of the first metatarsal bone to the head of the fifth metatarsal bone . the dynamic test items were 1-legged long jumping , vertical jumping , and 50-m dash time . one - legged long jumping was evaluated on the basis of the measurement method used for the standing broad jump in the new physical strength test used by the ministry of education , culture , sports , science and technology , japan . in this test , a tape measure was used to measure the distance from the toes to the heel of the measured lower limb . the measurements were collected twice for the left and right lower limbs , and the mean values for these measures were used . vertical jumping measurement values were displayed numerically on a jump md measurement device ( takei scientific instruments co. , ltd . , niigata , japan ) , which can display measurements in 1- cm increments . vertical jump measurements were collected twice , and their mean value was used . for measuring the 50-m dash time , a start signal was used by the tester who announced the start time and bent their arm at the start line . subjects started the 50-m dash from a standing position , and the tester used a stopwatch to time how long it took for the subject s torso to cross the finish line . subjects were positioned so that their hip joints were flexed at 90 angles . then , similar to the other methods for measuring muscle strength , a barefoot toe ip and mp joint - flexing exercise was performed simultaneously with the left and right feet . this exercise was performed for 200 repetitions , once per day , three times per week with a 3-kg load that was set using scales with a maximum capacity of 10 kg ( sanko seikohjyo co. , ltd . , a metronome indicated the desired exercise rhythm until the subjects could independently maintain the rhythm . the subjects flexed their toes for 1 s before extending them to their resting position for 1 s , and this movement was performed repeatedly , thereby creating a rhythm of 1 repetition per 2 s. the pre- and post - training measurement values are presented as mean standard deviation values ( mean sd ) . a wilcoxon signed - rank test was used to examine differences in the test results . the ethics committee of juntendo university graduate school of health and sports science approved this study ( no . 21 - 34 ) . all subjects were given an explanation of the objectives and content of the study before they provided written consent . the left and right intrinsic foot flexor strengths significantly increased ( p < 0.01 ) , and the left and right longitudinal arch lengths ( p < 0.01 ) and horizontal arch lengths , measured from a static standing position ( p < 0.01 ) , significantly decreased after training compared with those before training ( table 2table 2 . test item resultsinspectedpre - trainingpost - trainingtoe grip ( kg)l9.3 ( 5.611)14.4 ( 11.915.5)**r9.4 ( 6.212.7)14.2 ( 12.115.7)**arch length ( cm)l18.6 ( 17.320.1)17.7 ( 17.219.4)**r18.7 ( 17.919.6 ) 18.1 ( 16.719)**transverse arch length ( cm)l10.4 ( 9.711.3)10.1 ( 9.510.7)**r10.3 ( 1011.1 ) 10.1 ( 9.811)**long jump with one leg standing ( cm)l178.8 ( 163.5217)198.3 ( 177226.5)**r179 ( 151.5206)189.5 ( 166.5227)**vertical jump ( cm)54 ( 43.571.5)55.5 ( 49.573)*50-meter dash time ( s)7.34 ( 6.798.58)7.05 ( 6.58.22)****p<0.01 ; * p<0.05 . ) . additionally , the left and right 1-legged long jumping distances ( p < 0.01 ) and the vertical jumping height ( p < 0.05 ) significantly increased , and the 50-m dash time significantly decreased ( p < 0.01 ) after training compared with those before training ( table 2 ) . in contrast to previous studies , we implemented a strength training program that focused on intrinsic foot muscle strength and excluded extrinsic foot muscle function as much as possible . with the ankle in plantar flexion , the extrinsic foot muscle origin and insertion points draw closer to each other , thereby lowering the flexion action and becoming more dependent on the intrinsic foot muscles . this led hayashi et al.12 to establish a dip joint flexion measurement method , so that the intrinsic foot muscle strength contributed to almost 100% of the proximal ip ( pip ) and mp joint flexion . the present study implemented training and muscle strength measurements specialized for the intrinsic foot flexor muscles with the gripping force of all of the toes . in order to measure intrinsic foot flexor strength , we created an original strength measurement device because no commercially available device exists . as the test retest method indicated reproducibility without observable differences and high interobserver correlation , the measurement device used in this study is sufficiently reliable . statistically significant differences were found for each measurement test item when the pre- and post - training scores were compared . that is , we observed an increased intrinsic foot flexor strength , shorter longitudinal and horizontal foot arches , greater 1-legged long jumping distances , greater vertical jumping heights , and shorter 50-m dash times . increased intrinsic foot flexor strength values were observed after training . toe gripping force training has been reported to elicit positive results that can be observed after 3 weeks according to fukuda et al.10 or 4 weeks according to usaba and ihara13 , 14 . because the results of the present study indicated that the training load was approximately 30% of the mean maximum strength for all subjects and increased strength scores were recorded for all subjects , it appears that these results were due to neurological rather than muscle adaptations ( i.e. , hypertrophy ) , as was indicated by previous studies . foot - arch measurements revealed that the longitudinal and horizontal foot arches were shortened after training , which suggests arch formation . this finding also suggests that arch maintenance function improved along with increased intrinsic foot flexor strength scores . ukai et al.15 reported that forefoot arch maintenance is dependent on the intrinsic foot muscles , whereas umeki et al.16 reported that the arches are shortened by intrinsic and extrinsic foot muscle contraction during 1-legged standing , which protects the plantar support ligaments and joint tissue . we believe the shortened arches observed in the present study were due to an increased intrinsic foot flexor strength , which made arch formation possible through muscle contraction . performance of all dynamic test items improved in the post - training period , with increased 1-legged long jump distances , greater vertical jumping heights , and shorter 50-m dash times . rabita et al.17 reported that the elements that improve jumping function are muscle tendon structure and intrinsic muscle hardness , although it is also important to take a neuromuscular function approach to training . this suggests that we were able to achieve increased foot hardness that was accompanied by an improved intrinsic foot flexor toe gripping strength and arch formation . mann et al.18 reported that when running at top speed , the intrinsic muscles are continuously bearing weight . thus , we inferred that the volume of intrinsic foot flexor activity would improve under high - load conditions . in addition , running and jumping movements involve high loads and require plantar flexion position movements . the present training position was a toe flexion movement with the ankle positioned in maximum plantar flexion , and the necessary ankle plantar flexion position stability in the final exercise area , along with pip and mp joint ejection power , was exhibited . we believe that this resulted in greater anterior propulsive force and jumping power , leading to improved performance . although it can not be ignored that other factors relating to the joints and muscles , such as quadriceps activity , may have affected the results of the present study , we believe that reinforcement of the intrinsic foot flexor muscles should receive sufficient focus . furthermore , we believe that intrinsic foot flexor strength training is useful for improving standing and walking performance , in addition to improving the performance of sports athletes engaged in activities involving greater exercise loads .

[ purpose ] the purpose of the present study was to verify the effects of intrinsic foot flexor strength training . [ subjects ] the subjects were 12 healthy males without motor system disease . [ methods ] a training method that involved flexion of all toe interphalangeal and metatarsophalangeal joints against a 3-kg load was implemented and was performed for 200 repetitions once per day , three times per week , for a period of eight weeks . [ results ] significant changes were observed for intrinsic foot flexor strength scores , foot arches , vertical jumping , 1-legged long jumping , and 50-m dash time . [ conclusion ] this muscle strength training method significantly improved muscle strength scores , foot arch shape , and movement performance .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4756027

the achilles tendon attaches the plantaris , gastrocnemius , and soleus muscles to the heel1 . excessive stress or tight or fatigued gastrocnemius and soleus muscles can result in microtrauma , degeneration , or inflammation of the tendon a condition called achilles tendonitis / tendinosis1 . this condition can occur in the general population2 and is exacerbated by prolonged walking , excessive running , jumping , or walking hills . mountaineering and mountain - related activities are associated with significant risk of injury in the ankle joint and achilles tendon3 . the nomenclature for disorders of the achilles tendon is important , as the treatment is dependent on the underlying tendon pathology4 achilles tendon conditions such as tendinosis are often chronic and non - inflammatory , although the area can be painful to touch . based on the etiology , achilles tendinosis can be caused by a combination of factors such as failure of the normal healing mechanisms and repeated trauma5 . the achilles tendon serves to attach the plantaris , gastrocnemius , and soleus muscles to the calcaneus ( heel ) bone1.this study developed heel support banding ( hsb ) using an elastic band for flexible heel support and investigated its effect on chronic achilles tendon pain of a mountaineer . a 40-year - old male mountaineer with chronic achilles tendon pain participated in this study . the purpose and methods of the study were explained to the participant and informed consent was obtained according to the principles of the declaration of helsinki . the patient complained of chronic pain in the achilles tendon of the right leg . he developed severe pain during active plantar flexion and palpation of the pre - insertion area of the achilles tendon after mountain climbing , which he does three times per week . active dorsiflexion and active plantar flexion , measured with a goniometer , were 13 ( population normal angle , 20 ) and 28 ( population normal angle , 45 ) , respectively , due to pain . the visa - a questionnaire is a reliable and valid instrument for the evaluation of pain intensity and levels of function in patients with achilles tendon disorders . the visa - a questionnaire score was 56 ( normal population range > 95 ) , and the load - induced pain , assessed on a numeric rating scale from 0 to 10 , was 7 . the pressure - pain threshold , assessed with an algometer ( pain test - model fpk ; wagner instruments , greenwich , ct ) , was 2.0 kg . rompe et al.6 defined tenderness using a numeric rating scale following the application of 3 kg of pressure to the most tender area of the achilles tendon . the tenderness at 3 kg , assessed on a numeric rating scale from 0 to 10 , was 8 . hsb is an elastic band ( about 250 cm long ) , divided into four sections with the center piece binding together to form a figure x. to apply the band , the center of the band was placed above the knee approximately 5 cm from the patella . second , the other two pieces were placed longitudinally along the lower leg , while the third section of the band wraps around the lower leg , approximately 5 to 7 cm below the knee , and was bound to itself laterally . the third and fourth pieces of the band crossed each other at half of the distance between the foot and patella . finally , these pieces of the band were placed around the foot and support the heel and foot arch . an elastic band of proper strength for the patient was selected by a therapist for maximum support of the heel and foot arch . the range of elastic bands includes black , silver , blue , green , red , and yellow . the patient in the study applied a level silver band prior to mountain climbing and proceeded to climb three times a week for one month . after one month of applying the hsb , the dorsiflexion and plantar flexion angles increased to 18 and 37 , respectively , compared to the initial 13 and 28. the visa - a questionnaire score increased to 79 compared to the initial 56 . the load - induced pain assessment decreased to 4 compared to the initial 7 . the pain threshold increased to 5.2 kg compared to the initial 2.0 kg and tenderness at 3 kg , assessed on a numeric rating scale , decreased to 3 compared to the initial 8 . the function of this musculotendinous unit is to serve as the chief plantar flexor of the ankle joint during walking and running , providing the primary dynamic force for locomotion and jumping5 , 7 . based on the etiology , the achilles tendinosis was caused by a combination of failure of the normal healing mechanisms and repeated trauma5 . the severe pain may have occurred gradually as repetitive microtrauma was imposed on the achilles tendon . for this , this study developed hsb using an elastic band and investigated its effect on chronic achilles tendon pain of a mountaineer . the result showed that after one month after applying hsb , the dorsiflexion and plantar flexion angles increased , the visa - a questionnaire increased , the load - induced pain assessment decreased , the pain threshold increased and tenderness at 3 kg , assessed on a numeric rating scale , decreased . the tension of hsb created by increased stimulation during active ankle movement may have also created tension in the soft tissue structures . increased tension may facilitate a pain inhibitory mechanism by providing an afferent stimulus to large - diameter nerve fibers to mitigate nociception . as the patient s pain level reduced , the fear of pain during ankle movement might have reduced by proper sensory feedback , thus improving ankle range of motion . a thera - band , which provides varied resistance through the range of movement , has been used for rehabilitation in combination with therapeutic exercise8 . it is light and portable , has low resistance , and can be adjusted to accommodate various situations9 . in conclusion , one month of hsb for flexible heel support improved range of motion in the ankle and reduced pain . therefore , the hsb could provide a new approach for effective intervention and management of chronic achilles tendon pain .

[ purpose ] this study developed heel support banding ( hsb ) using an elastic band for flexible heel support and investigated its effect on chronic achilles tendon pain of a mountaineer . [ subject ] a 40-year - old male mountaineer with chronic achilles tendon pain [ methods ] ankle dorsiflexion and plantar flexion angles , visa - a questionnaire , load - induced pain , total pain threshold and tenderness at 3 kg of pressure were measured before and after applying hsb . [ results ] after one month of applying hsb , the dorsiflexion and plantar flexion angles increased ; the visa - a questionnaire score increased ; the load - induced pain assessment score decreased ; the pain threshold increased ; and tenderness at 3 kg decreased . [ conclusion ] these results indicate that hsb use improves ankle range of motion , decreases pressure and pain , and could provide a new approach for effective intervention and management of chronic achilles tendon pain .

INTRODUCTION SUBJECT AND METHODS RESULTS DISCUSSION

PMC1852384

approximately 5 to 8% have clinically severe or morbid obesity and are candidates for bariatric surgery . this obesity epidemic has been accompanied by a geometric rise in the number of bariatric surgical procedures . the american society for bariatric surgery ( asbs ) estimates that the number of bariatric surgery procedures has increased from approximately 20,000 in 1996 to over 140,000 in 2004 ( http://www.asbs.org ) . during this period , membership in the asbs the most common bariatric procedure performed in the united states is roux - en - y gastric bypass ( rygb).1 the explosive growth of bariatric surgery has garnered much attention in the media , with much speculation about the risks , both short- and long - term , of rygb . unfortunately , to date , little information from large series is available concerning the operative mortality of rygb and those factors that predict mortality . in 2002 , livingston et al.2 reported an operative mortality of 1.3% in 1,067 patients after open rygb and found that only age over 55 years correlated with perioperative mortality . more recently , fernandez et al.3 analyzed their patient cohort of 1,431 patients having open rygb and found a 1.9% mortality , which was associated with age , weight , longer limb gastric bypass , and the occurrence of a leak or pulmonary embolism . this report is a multivariate analysis of preoperative mortality and morbidity in 1,000 consecutive open rygbs performed over a 5-year period by a single surgeon ( lf ) in at a single institution . roosevelt hospital center in new york city was initiated in april 1999 and the first operation performed in june 1999 . the basis of this report consists of 1,000 consecutive open rygbs ( primary cases and revisions ) performed by a single surgeon , lf , between june 1999 and june 2004 . clinical protocol and surgical technique all patients were evaluated preoperatively and met generally accepted criteria outlined by the nih at its consensus development conference on gastrointestinal surgery for severe obesity.4 in addition , all patients were routinely evaluated by a registered dietitian experienced in the treatment of obesity . mental health and other specialty consultations were only obtained if they were felt to be clinically indicated or were required by an insurance carrier . all patients were evaluated by an attending anesthesiologist before surgery.routine preoperative studies included : electrocardiogram , chest x - ray , gallbladder ultrasonography , serum electrolytes , glucose , hba1c , calcium , albumin , lipid profile , liver function tests , complete blood count , platelets , prothrombin time , partial thromboplastin time , inr , and urinalysis . over time , additional preoperative studies , including serum insulin , iron , ferritin , vitamin b12 , 25-oh vitamin d , and thiamine ( vitamin b1 ) were added.rygb was performed in a standard fashion with the following common elements : ( 1 ) open technique ; ( 2 ) 2030 ml pouch , nondivided stomach , ta 90b ( us surgical , norwalk , ct , usa ) applied twice ; ( 3 ) hand - sewn two - layer retrocolic , antegastric gastrojejunostomy , 12 mm in length , tested with methylene blue under pressure intraoperatively ; ( 4 ) side - to - side , functional end - to - end jejunojejunostomy with dispsable gia ( us surgical ) or lc ( ethicon endosurgery , sommerville , nj , usa ) 75 mm staplers , and ta55 ( us surgical ) or tx 60 ( ethicon endosurgery ) staplers and routinely oversewn with 30 silk lembert sutures . the biliopancreatic limb - length measured 75 cm along the antimesenteric border for patients with bmi less than 50 and 150 cm for patients with bmi greater than or equal to 50 . the roux or alimentary limb - length was 150 cm in all patients . this technique was abandoned and switched to the nondivided stomach after a stapler malfunction led to a leak.closed suction drains were placed in all revisions , patients with bmi > 55 , or if clinically indicated ( e.g. , identification of an intraoperative leak , technically difficult anastomosis ) . fascia was closed with a running looped # 1 pds ( ethicon , sommerville ) and infiltrated with 0.25% bupivacaine . the subcutaneous space was drained with a # 10 jackson pratt drain and the skin was closed with a running subcuticular stitch.the gallbladder was removed if gallstones were documented by preoperative ultrasound . incisional or umbilical hernias were primarily repaired if encountered , oftentimes through a separate periumbilical incision.invasive monitoring was not used routinely and foley catheters were only placed if patients required invasive hemodynamic monitoring or in the case of revisions or when patients had multiple prior abdominal operations . nasogastric tubes were left in placed as clinically indicated ( intraoperative leak identified ) or in patients with bmi > 55 . all patients received perioperative antibiotics for 24 h , cefazolin ( 2 g intravenously every 8 h for three total doses ) or clindamycin ( 900 mg intravenously every 6 h for four doses ) . prophylaxis against deep vein thrombosis consisted of 5,000 units of unfractionated heparin administered subcutaneously every 8 h and pneumatic compression stockings until ambulatory . all patients were given a pca pump ( patient - controlled analgesia ) for pain . patients were routinely cared for on a regular surgical floor , equipped for severely obese patients . patients with significant sa or documented cad were usually observed in the recovery room overnight , and only rarely admitted to the intensive care unit . on the morning of postoperative day 1 , patients were routinely studied with gastrograffin upper - gi studies . if no leak was identified , they were given liquids for lunch and advanced to soft food ( yogurt , apple sauce , and cottage cheese ) for dinner and switched to oral pain medications . patients were discharged on pod # 2 or # 3 as indicated with the following medications : a codeine - derivative for pain , prenatal vitamins , iron polysaccharide , calcium citrate , ursodeoxycholic acid , if the gallbladder was present.after hospital discharge , patients were scheduled to be seen at 2 and 8 weeks , 6 , 12 , 18 , 24 months , and yearly thereafter . all routine follow - up appointments included nutritional counseling and those after 2 weeks included laboratory studies [ serum electrolytes , glucose , hba1c , calcium , albumin , lipid profile , liver function tests , complete blood count , platelets , serum insulin , iron , ferritin , vitamin b12 , 25-oh vitamin d , and thiamine ( vitamin b1 ) ] . follow - up was 74% at 1 year , 68% at 2 years , 59% at 3 years , 53% at 4 years , and 48% at 5 years . clinical data and data analysis clinical and laboratory data were pc - based database prospectively maintained since the program s inception in 1999 . data collected included : age , sex , height weight , bmi , race / ethnicity , payer status , obesity - related comorbidities , operative procedure , duration of stay , mortality , major complications , and death . complications were classified as systemic ( prolonged intubation , deep venous thrombosis , pulmonary embolism , and myocardial infarction / fatal arrhythmia ) or technical ( incisional hernia , intestinal obstruction , leak / perforation , dehiscence , gi bleeding , anastomotic stricture , and anastomotic ulcer ) . deaths were analyzed with respect to bmi , demographics , comorbidities , and complications.superficial wound infections were not included ; the incidence of urinary tract infections was not tracked . nutritional complications were not evaluated because all patients were routinely maintained on vitamins , iron , and calcium supplements postoperatively ; it would be impossible to determine the true incidence of any of these nutritional deficiencies.univariate analyses and logistic regression with spss 11.0 were used to determine significance . the prevalence of obesity - related comorbid conditions is shown in table 2 . table 1demographic characteristics by sex and race womenmentotalp - valueage ( years)38 1.0 ( 1573)40 11.9 ( 1565)38 11.17 ( 1573)0.064weight ( kg)134 27 ( 84263)170 43 ( 81345)139 33 ( 82345)<0.01bmi ( kg / m)51 10 ( 351,000)55 13 ( 24116)51 10 ( 24116)<0.01caucasian(28% ) 238/853(53% ) 78/14732%<0.01african american(30% ) 253/853(20% ) 29/14728%0.01hispanic(42%)358/853(27%)40/14740%<0.01other(0.5% ) 4/853(0% ) 0/1470.4%0.41table 2prevalence of obesity - related comorbid conditions by sex women ( n = 853 ) ( % ) men ( n = 147 ) ( % ) total ( % ) p - valuetype ii diabetes mellitus177 ( 21)54 ( 37)23<0.01hypertension310 ( 36)79 ( 54)39<0.01cad / chf29 ( 3)26 ( 18)6<0.01dyslipidemia376 ( 44)80 ( 54)470.02sleep apnea172 ( 20)63 ( 43)24<0.01asthma135 ( 16)16 ( 11)150.12dyspnea on exertion811 ( 95)127 ( 86)48<0.01gerd513 ( 60)77 ( 52)590.08osteoarthritis791 ( 93)126 ( 87)92<0.01urinary stress incontinence430/(50)5 ( 3)44<0.01irregular menses276 ( 32)na32nacad / chf coronary artery disease / congestive heart failure , gerd gastroesophageal reflux disease demographic characteristics by sex and race prevalence of obesity - related comorbid conditions by sex cad / chf coronary artery disease / congestive heart failure , gerd gastroesophageal reflux disease the most common comorbidites encountered were dyspnea on exertion ( 94% ) , joint pain / arthritis ( 92% ) , and gastroesophageal reflux disease ( 59% ) . the comorbidites typically associated with systemic disease included hypertension ( htn , 39% ) , obstructive sleep apnea ( sa , 24% ) , dyslipidemia ( 46% ) , and asthma ( 15% ) . approximately 23% of the patient population suffered from type ii diabetes mellitus ( dm ) . at time of initial evaluation , 13.0% of this diabetic patient subset had a prior history of insulin - dependent dm , 57.6% had noninsulin dependent dm , and 23% had a previous diagnosis of dm intermittently controlled on diet or were newly diagnosed with dm during their preoperative evaluation . six percent of the patient population had angiographically documented histories of coronary artery disease ( cad ) but were deemed suitable risk by their respective specialists . procedures and duration of hospital stay there were 966 primary rygb and 34 revisions of failed bariatric procedures ( 21vbg , 5rygb , 8other ; all performed at outside institutions ) to rygb . the median length of stay for primary procedures was 2.4 days compared to 3.7 days for revisions . average ( los ) for all patients having primary rygb was 3.8 days with 87% of the group leaving in 3 days or less . complications overall , 91% of the procedures were without systemic or technical complications.the incidence of complications in relation to bmi is summarized in table 3 . the most common technical complications were incisional hernia ( 3.5% ) , intestinal obstruction ( 1.9% ) , and leak / perforation ( 1.6% ) . table 3incidence of complications after rygbcomplicationbmi < 50 ( % ) ( n = 481)bmi > 50 ( % ) ( n = 515)total ( % ) p - valuesystemic complicationsprolonged intubation3 ( 0.6)8 ( 1.5)1.10.16 ( ns)deep venous thrombosis0 ( 0)2 ( 0.4)0.2nspulmonary embolism0 ( 0)3 ( 0.6)0.3nsmi / fatal arrhythmia1 ( 0.2)1 ( 0.2)0.2nstechnical complicationsincisional hernia10 ( 2.1)25 ( 4.8)3.50.019intestinal obstruction10 ( 2.1)9 ( 1.7)1.9nsleak7 ( 1.5)9 ( 1.9)1.6nsdehiscence2 ( 0.4)2 ( 0.4)0.4nsgi bleeding requiring transfusion3 ( 0.6)6 ( 1.2)0.9nsanastomotic ulcer2 ( 0.4)0 ( 0)0.2nsanastomotic stricture6 ( 1.2)2 ( 0.4)0.8nsdeath4 ( 0.8)11 ( 2.1)1.5.03thirty - one patients ( 3.1% ) required reoperation within 30 days of the original procedure . the indications for reoperation within 30 days were leak / perforation ( 11 ) , intestinal obstruction ( 9 ) , bleeding ( 4 ) , rule - out leak ( 2 ) , dehiscence ( 4 ) , and subphrenic abscess ( 1).indications for late operations ( > 30 days postoperatively ) included incisional hernia repair in 35 patients , intestinal obstruction in 10 patients , and repair of leaks / perforations in five patients who were experiencing ongoing postoperative complications . one patient developed a gastrogastric fistula after repair of an early leak , which was treated expectantly since it was clinically insignificant . patients with late deaths due to unrelated events , such as motor vehicle accidents ( n = 2 ) or drug overdoses ( n = 1 ) , were excluded and not classified as mortalities in the analysis . mortality correlated with bmi , with four ( 0.8% ) patients having a bmi < 50 dying compared to 11 ( 2.1% ) patients with bmi 50 ( p = 0.03 ) ( table 3).causes of death after rygb , along with their timing and relationship to bmi are shown in table 4 . both were males , ages 48 and 54 , with bmis > 50 , dm , cad , htn , and sa . one patient , a 43-year - old woman with a bmi 59 , died of a pulmonary embolism ( identified at autopsy ) at home after 2 weeks . table 4causes of early and late deaths related to bmicause of deathearly ( < 30 days ) ( n = 11)late ( > 30 days ) ( n = 4)bmi < 50bmi 50bmi < 50bmi 50mi / arrhythmia0200pulmonary / pe0100msof 2 leak1212msof 2 bowel obstruction0100msof cause unknown1100bleeding complications1200pe pulmonary embolism , msof multisystem organ failuresix patients died from msof after postoperative leaks , three from the gastrojejunostomy and three from perforations of the distal small bowel within the common channel . of these , four occurred and were diagnosed within 48 h of the initial rygb and all were emergently explored . all four developed msof and expired between 14 and 211 days postoperatively . of the remaining two deaths , one patient , 46-year - old woman with a bmi 79 and a history of chronic ventilator dependence due to a paralyzed left hemidiaphragm on chronic steroid therapy developed a late leak and multiple intestinal fistulae and eventually succumbed to msof . the other patient , a 43-year - old woman , bmi 43 , had undergone a laparoscopic rygb at another institution that was complicated by a strangulated internal hernia , massive intestinal gangrene , and short - bowel syndrome . she underwent a reversal of her rygb with reconstruction of her gi tract , but developed multiple small bowel fistulae 2 weeks postoperatively and died of msof several months later.three patients died from a severe systemic inflammatory response syndrome ( sirs ) with msof accompanied by extremely high fevers , without any identifiable source . one was a 70-year - old woman with bmi 68 underwent reexploration for an early postoperative small bowel obstruction . the others were a 54-year - old woman with a bmi 47 , dm , htn , and sa and a 37-year - old male with bmi 94 and severe sa and htn . no intraabdominal or other sources were identified despite numerous cultures and radiologic studies.three patients died of bleeding complications . a 43-year - old woman with these symptoms were initially addressed with rehydration allowing the patient s hematocrit to fall to a level where hypovolemic shock and resulting coagulopathy obscured efforts to surgically control or identify a single source . a 31-year - old woman with a bmi 50 was returned to the or 4 h postoperatively for control of bleeding from the small bowel mesentery . after this second procedure , she developed sirs , temperatures of 106f , and msof , with no identifiable source of sepsis . a 54-year - old male with a bmi 56 and a history of htn , cad , and chronic atrial fibrillation suffered a postoperative myocardial infarction and developed a coagulopathy complicated by a massive intraspenic hematoma after restarting coumadin , which rapidly progressed to anuria and msof . attempts to reverse his anticoagulation and control the bleeding angiographically were unsuccessful.the necessity for reoperation within 30 days of the original procedure was particularly ominous . overall , the incidence of death after a second operative procedure within 30 days was 9/31 ( 29% ) . two of the 16 patients with a bmi < 50 who required reoperation within 30 days died ( 12.5% ) compared to 7 out of 15 patients with bmi 50 ( 47% p = 0.03 ) . this is similar to the mortality for the entire series where 0.8% for patients with a bmi < 50 died compared to 2.1% for patients with bmi 50 ( p = 0.03 ) . however , among those 15 patients classified as operative deaths for the entire series , 9 ( 60% ) died after their second operative procedure.logistic regression demonstrated that cad [ lr 7.5 p < 0.01 ( 95% ci 2.2 to 25.3 ) ] , and sa [ lr 3.3 p = 0.03 ( 95% ci 1.1 to 10.1 ) ] , followed by age [ lr 1.06 p = 0.042 ( 95% ci 1.00 to 1.12 ) ] , were risk factors for death in all patients ( table 5 ) . although a small sample set , 12.7% of patients with cad died ( 7/55 ) and 29% of patients with bmi > 50 and cad and sa died . table 5logistic regression evaluation of patient comorbidity as predictors of mortality p - valuerelative risk95% confidence intervalage0.0421.0591.0021.120bmi0.1301.0250.9931.058female0.6120.7290.2152.474dm0.8520.8890.2593.054htn0.2572.0850.5857.438cad0.0017.4462.19525.258dyslipidemia0.0990.3590.1061.211asthma0.0703.0650.91310.293sa0.0333.3421.10410.115sob0.6930.6440.0735.725although the average bmi of males was slightly greater than that of females ( 55.2 vs 51.2 kg / m , p < 0.01 ) , the two populations also differed in characteristics other than bmi . the male population had a significantly significant greater prevalence of dm , htn , cad , dyslipidemia , and sa . females had a greater prevalence of pulmonary comorbidities , including asthma and dyspnea on exertion ( table 2).when logistic regression was performed specific to the subsets of sex , males with angiographically demonstrated cad were 30 times more likely to die [ lr 30.1 p = 0.028 ( 95% ci 1.4 to 631.4 ) ] . logistic regression did not identify cad as a significant predictor when the analysis was limited to women . predictors of death for women include age [ lr 1.07 p = 0.033 ( 95 % ci 1.0 to 1.14 ) and sa [ lr 4.1 p = 0.040 ( 95% ci 1.07 to 16.2)].logistic regression was repeated specific to race . when limited to caucasian patients only , caucasians with cad were 58 times more likely to die [ lr 58.8 p < 0.01 ( 95% ci 4.8 to 716.9 ) ] than those caucasians without cad . [ lr 1.08 p = 0.02 ( 95% ci 1.01 to 1.16 ) ] . evaluation of african american patients demonstrated that only sa was significant [ lr 19.1 p = 0.03 ( 95% ci 1.29 to 282.8 ) ] . one - way anova did not demonstrate any significant differences in prevalence of death or cad between the three racial groups for the entire population or specific to sex.hispanic patients had significantly less sa than either the caucasian or african american patients ( 18.1 vs 25.6 and 27.4% , respectively ( p = 0.01 ) ) . when examined specifically in relation to sex , there were no differences among prevalence of sa in the males . african american women , however , had the greatest prevalence of sa ( 26.4% ) compared to caucasian ( 18.9% ) and hispanic ( 15.7% ) women ( p < 0.01 ) . causes of early and late deaths related to bmi pe pulmonary embolism , msof multisystem organ failure logistic regression evaluation of patient comorbidity as predictors of mortality the tremendous growth of bariatric surgery over the past several years has spawned much interest in its complications and mortality , first in the media , but most recently in the public health arena as well . health and malpractice insurance carriers as well as governmental agencies and professional societies are evaluating the risks of bariatric surgery and the surgeons that perform it . in several states , insurance companies have stopped covering bariatric surgery at the same time that the centers for medicare and medicaid services have approved coverage for them . several malpractice carriers have stopped issuing policies for surgeons performing bariatric procedures while others are categorizing bariatric surgery as a high - risk subspecialty area , similar to obstetrics and neurosurgery , and increasing premiums accordingly . in each of these instances , the overriding fear or consideration appears to be that the risks associated with bariatric surgery are excessively high . much of the information being utilized in this regard has come form series utilizing pooled data from multiple smaller series or government databases.1,59 buchwald et al.5 performed a metaanalysis of 16,944 patients which included 7,074 patients that underwent gastric bypass ( open and laparoscopic ) in 44 studies with a 30-day mortality rate of 0.5% . several authors have reported that complication rates of bariatric surgery were inversely correlated with case loads , reporting mortality rates of in the range of 0.10.5% for surgeons more than 100 or 150 cases per year.68 flum et al.9 recently reported 30- and 90-day mortality rates of 2.0 and 2.8% , respectively in medicare beneficiaries having bariatric surgery , with men having higher rates than women and those over 65 years of age having higher rates than those younger than 65 . many of these series lack data concerning bmi and comorbidities , making risk assessment difficult or impossible . in one large series from a single - center , christou et al.10 reported a 30-day mortality of 0.4% in 1,035 patients undergoing rygb , of whom 820 had open rygb . the results of these pooled series differ from those in several large series of open gastric bypasses2,3,11 . livingston et al.2 reported an operative mortality of 1.3% in 1,067 patients after open rygb . in his series , mean bmi was 53.6 kg / m , mean age 42.3 years , and the incidence of comorbidities was diabetes ( 23% ) , hypertension ( 48% ) , and sleep apnea ( 39% ) . only male sex was predictive of severe life - threatening complications ; mortality in patients over 55 years was significantly greater than in patients under 55 ( 3.5 vs 1.1% , p < 0.05 ) . more recently , fernandez et al.3 analyzed their patient cohort of 1,431 patients having open rygb and found a 1.9% mortality , which was associated with age , weight , longer limb gastric bypass , and the occurrence of a leak or pulmonary embolism . in that series , the mean bmi was 53.3 , mean age 40.7 years , and incidence of serious comorbidities was diabetes ( 19.5% ) , hypertension ( 51% ) , and sleep apnea ( 33% ) . in both of these series , the bmi was higher , the patients were older and the incidence of serious comorbidities , such as diabetes , hypertension , dyslipidemia , and sleep apnea , higher . pories et al.11 reported a 1.5% perioperative mortality rate , with 0.8% dying of sepsis , 0.5% dying of pulmonary embolism , and 0.2% of an unknown cause . additional information regarding the incidence of leaks , bowel obstructions and other complications was not reported as these were not the focus of the original paper . similarly , in the present series , the 30-day mortality rate was 1.2% in 1,000 patients with a mean bmi of 52 kg / m . the prevalence of preoperative comorbidities was comparable to the larger series ( diabetes23% , hypertension39% , dyslipidemia46% , coronary artery disease / congestive heart failure5.5% , sleep apnea23.5% , and asthma15% ) and generally higher than in the pooled series . the incidence of leaks and postoperative small bowel obstruction in our series was comparable to the other series . the incidence of pulmonary embolism was less than those reported , perhaps related to the use of both pneumatic compression stockings and subcutaneous heparin and early ambulation with a shorter length of stay . although the incidence of incisional hernia in our series was low ( 3.5% ) , this may well be affected by our suboptimal follow - up . multisystem organ failure accounted for 11 deaths in our series ( 73% ) . in six patients , this resulted from leaks or perforations ; in one it followed a bowel obstruction , in two it followed postoperative hemorrhage and in two patients , the cause was never determined . in each of these instances , the complication was identified early and appropriate treatment and supportive care instituted . in four of the patients , the clinical course of sirs and msof was characterized by extremely high temperatures ( > 105f ) , with no apparent source ever identified . to our knowledge , this syndrome has not been reported , but may be due to the fact that the enormous adipose tissue stores in these patients may act as a metabolic sink , releasing cytokines and other mediators and perpetuating this extreme systemic inflammatory response . two of the deaths were due to fatal arrhythmias , both in patients with known cad , who were extensively evaluated preoperatively . the death due to pulmonary embolism occurred after discharge , even though the patient received prophylaxis with both heparin and pneumatic compression stockings in the hospital . although males were significantly heavier and had higher bmis than women , sex was not an independent predictor of morbidity . however , the presence of angiographically documented coronary artery disease was particularly ominous in men . males with angiographically demonstrated cad were 30 times more likely to die . in women , with respect to race , caucasian males with bmi > 50 and cad were most likely to die , whereas sa was a predictor in african american men . there were no predictors in hispanics . despite the increased mortality in caucasian males with cad , these patients were not candidates for cardiac revascularization and extreme weight loss was the only intervention thought to make a beneficial health impact . based upon our data and those of others2,3,11 , it appears that the risk of open rygb is in the range of 12% . risk appears to be adversely affected by increasing bmi and those factors with which it is often associated namely male sex and coronary artery disease . how this compares with the perioperative mortality after laparoscopic rygb is still unclear because many series of patients having laparoscopic rygb do not include patients with the highest bmis , above 60 or 70 kg / m , or patients having revisional surgery . a perioperative mortality rate of 1.2% after rygb compares favorably with that after other common surgical procedures . for example , perioperative mortality after elective surgery for abdominal aortic aneurysms has been reported at 3.14.7% overall , 1.02.7% in patients under 65 years of age , and 3.55.2% in patients over 65 years of age.12 using medicare data adjusted for high volume surgeons , birkmeyer et al.13 reported perioperative mortality rates of 4.5% for colectomy , 8.6% for gastrectomy , 8.4% for esophagectomy , 3.8% for pancreatic resection , 4.0% for pulmonary lobectomy , and 10.7% for pneumonectomy . while it is true that all of these patients were over 65 years of age , the fact still remains that these perioperative mortality rates are all substantially greater than that after rygb ( even in medicare recipients , as recently reported9 ) and neither the public , the press , the insurance industry , or various state departments of health are appalled or alarmed , or calling for a moratorium on those procedures . this is not meant to suggest that every effort should not be made to lessen the risks of bariatric surgery and to improve operative mortality , but rather to inject some proportionality into the discussion . the importance of such careful analysis of bariatric surgical data , including its limitations , and the need to continue to offer bariatric surgery to those patients for whom it constitutes the best available treatment has recently been emphasized.14 in his 2004 perspective , surgery for severe obesity , steinbrook15 quotes robert brolin , md , cautioning physicians and the public ... to reconcile the fact that the operation has a real mortality and it will continue to have a real mortality under the best of circumstances . some of these patients are just profound operative risks for any kind of surgical intervention ... the sickest ones are the ones who benefit the most , but they are also the highest risk . rygb can be performed with acceptable perioperative morbidity in patients over a wide range of bmis . patients with bmi 50 have a higher morality for both initial operations and after reexploration . these three comorbidities were more prevalent in these patients and may contribute to this finding .

introductiondeterminants of perioperative risk for rygb are not well defined.methodsretrospective analysis of comorbidities was used to evaluate predictors of perioperative risk in 1,000 consecutive patients having open rygb by univariate analyses and logistic regression.resultsone hundred forty - six men , 854 women ; average age 38.3 11.2 years ; mean bmi 51.8 10.5 ( range 24116 ) were evaluated . average hospital stay ( los ) was 3.8 days ; 87% <3 days . 91.3% of procedures were without major complication . the most common complications were incisional hernia 3.5% , intestinal obstruction 1.9% , and leak 1.6% . 31 patients required reoperation within 30 days ( 3.1% ) . a 30-day mortality was 1.2% . logistic regression evaluating predictors of operative mortality correlated strongly with coronary artery disease ( cad ) ( p < 0.01 ) , sleep apnea ( p = 0.03 ) , and age ( p = 0.042 ) . bmi > 50 ( 0.6 vs 2.3% , p = 0.03 ) and male sex were associated with increased mortality ( 1.3 vs. 4.0% , p = 0.02 ) . sex - specific logistic regression demonstrated males with angiographically proven cad were more likely to die ( p = 0.028 ) than matched cohorts . age ( p = 0.033 ) and sleep apnea ( p = 0.040 ) were significant predictors of death for women.conclusionperioperative mortality after rygb appears to be affected by sex , bmi , age , cad , and sleep apnea . strategies employing risk stratification should be developed for bariatric surgery .

Introduction Materials and Methods Results Discussion Conclusion

PMC2868156

pneumonectomy is a lung resection technique that has been used to remove all lung lobes in humans and dogs when bilobectomy or lobectomy techniques are inadequate to remove the pathology in the hemi - thorax . in dogs , pneumonectomy has been performed in some pathological conditions , such as lung tumors , congenital lung anomalies , chronic lung collapse , chronic progressive lung inflammation , post - traumatic diffuse parenchymal laceration , and bronchial rupture . the reported morbidity and mortality rate after pneumonectomy is extremely high in humans suffering from respiratory , cardiac and gastrointestinal system problems , acute respiratory distress , pneumonia , pulmonary edema , pulmonary thromboembolism , bronchopleural fistula ( bpf ) , pyothorax , esophagopleural fistula , cardiac herniation , lung lobe torsion , hemathorax and chylothorax . the most commonly encountered complication of pneumonectomy in humans is bpf , which is described as a pathologic connection between the bronchus and pleural space [ 2,3,18,19,21,31 - 33,36 ] . closure failure of the bronchial stump ( bs ) after partial or complete lung resection is the primary cause of bpf in humans , leading to a prolonged hospitalization period and the need for multiple operations [ 1,11,16 - 18,19,21,31 - 34,36 ] . some authors have reported of the incidence of bpf to be in the range of 0 - 28% , while others have reported the incidence to be between 0% and 12% . the mortality rate of bpf has been reported to be between 15% and 70% [ 8,10,14,16,18 - 20,37 ] . pneumonia , pleural infections and failure in bs closure are the primary risk factors for bpf . the proper bs closure technique to prevent bs dehiscence after pneumonectomy has been long debated in the field of human thoracic surgery . therefore , the stapler technique has been used to prevent bpf in humans and dogs [ 1 - 3,17,22 ] . there seems to be no consensus between researchers regarding the optimal method for bs closure because several authors have made different suggestions for this procedure . the surgical procedures for bs closure are focused on suture line ( transversal versus longitudinal ) , suture technique ( manual versus mechanical ) , clamping technique ( open stump technique versus closed stump technique ) and whether or not to wrap the bs ( e.g. pleuralization , flap ) . both manual ( by running or interrupted suture ) and mechanical sutures have been routinely used in humans and dogs . the superiority of both methods has been clearly demonstrated by incidence of bpf complications in humans . in recent years , the autogenic tissue wrapping of the bs after pneumonectomy has been reported to decrease the incidence of bpf . therefore , in this technique the intercostal muscle flaps are primarily used to support the vascularity of the bs and the line of anastomosis in the trachea . in addition , the popularity of intercostal muscle flap usage has been increased in human thoracic surgery due to their thickness , autogenous and self - vascularization properties . in the present study , this literature data was compared with the histologic healing and bpf complications encountered following three different bs closure techniques ( manual suture , stapler and manual suture plus tissue flab ) after pneumonectomy in dogs were evaluated for a period of one month . a total of 27 hybrid dogs ( two years old , 20 kg in weight and sex was not considered ) were used in this study . histological healing was evaluated in 18 dogs ( n = 18 ) ; nine dogs ( n = 9 ) were reviewed separately due to postoperative complications . the 18 dogs were separated into two groups : group i ( gi ) ( n = 9 ) and group ii ( gii ) ( n = 9 ) . right and left pneumonectomies were performed in the dogs in gi and gii , respectively . the dogs in gi and gii were further divided into three subgroups [ subgroup i ( sgi ) ( n = 3 ) , subgroup ii ( sgii ) ( n = 3 ) and subgroup iii ( sgiii ) ( n = 3 ) ] according to the bs closure technique performed . the main stem bronchus was closed with a manual suture in subgroup i ( sgi ) ( n = 3 ) , with a stapler in subgroup ii ( sgii ) ( n = 3 ) , and with a manual suture plus tissue flab ( pedicled intercostal externus - internus muscle ) in subgroup iii ( sgiii ) ( n = 3 ) following pneumonectomy ( table 1 ) . the dogs were anesthetized and continued with 2% isofloran after the administration of xylasine hcl ( 1 mg / kg i m ) and thiopental na ( 15 mg / kg iv ) . respiration was ensured by mechanical ventilation ( 15 ml / kg tidal volume , respiration rate 15/min and 25 cm h2o alveolar pressure ) . thoracotomy incision was made on the 4th intercostal space according to the standard procedure in the sgi and sgii groups , but the 4th intercostal externus - internus muscle prepared as a pedicled flap in the sgiii group . pneumonectomy was carried out using the open bronchi resection technique in groups sgi and sgiii . in sgii , however , resection was performed after stapler closure . subgroup i ( manual suture ) : vertical mattress sutures were applied over the excision line , and the end of the stump was sutured with a simple interrupted suture pattern using 2 - 0 vicryl ( ethicon , uk ) ( fig . subgroup ii ( stapler ) : a ta-30 stapler ( ethicon , uk ) ( 4.8 mm ) was initially applied to the main stem bronchus , and the bronchus was then excised ( fig . subgroup iii ( manual suture plus tissue flab ) : pedicled intercostal externus - internus muscle was extended towards the bs and then wrapped and sutured using a simple continuous suture pattern with 2 - 0 vicryl after manual suturing of the bs with 2 - 0 vicryl ( fig . the pleural space was filled with warm sterile saline , and then 50 cm h2o endobronchial pressure was applied to determine the bronchial air leakage . extra vertical mattress suture(s ) was / were applied over the excision side of the bs in cases where air bubbles occurred due to the bs . saline was aspirated , and a 28-french thoracostomy tube ( argyle , usa ) was inserted into the pleural cavity ventro - cranially . the tube was fixed to the skin with a ' chinese finger trap ' suture pattern , and a heimlich flutter valve was connected to the tube for pleural drainage . postoperative care was carried out in compliance with the rules of the national society of medical research principles of laboratory animal care for a period of one month . the dogs were relieved with carprofen ( 5 mg / kg / d sc ) . cefazolin na ( 20 mg / kg iv tid ) was used as an antibiotic agent for 5 days . hematologic parameters and blood gas analysis of the dogs were controlled routinely , and the dogs were closely monitored for suspected cardiac problems and respiratory failure after pneumonectomy . the thoracostomy tube and skin sutures were removed on days 5 and 7 after surgery , respectively . at the end of the postoperative first month , the dogs in gi and gii were sacrificed with high - dose thiopental na . the bs was removed from each animal , fixed in 10% neutral - buffered formalin and then embedded in paraffin . five micrometer thick sections from these samples were placed on slides and stained with hematoxylin and eosin for microscopic examination . statistical analysis was carried out using fisher 's exact test and pearson 's chi - square test to determine the differences between subgroups in terms of histological healing and bpf complications , respectively . a total of 27 hybrid dogs ( two years old , 20 kg in weight and sex was not considered ) were used in this study . histological healing was evaluated in 18 dogs ( n = 18 ) ; nine dogs ( n = 9 ) were reviewed separately due to postoperative complications . the 18 dogs were separated into two groups : group i ( gi ) ( n = 9 ) and group ii ( gii ) ( n = 9 ) . right and left pneumonectomies were performed in the dogs in gi and gii , respectively . the dogs in gi and gii were further divided into three subgroups [ subgroup i ( sgi ) ( n = 3 ) , subgroup ii ( sgii ) ( n = 3 ) and subgroup iii ( sgiii ) ( n = 3 ) ] according to the bs closure technique performed . the main stem bronchus was closed with a manual suture in subgroup i ( sgi ) ( n = 3 ) , with a stapler in subgroup ii ( sgii ) ( n = 3 ) , and with a manual suture plus tissue flab ( pedicled intercostal externus - internus muscle ) in subgroup iii ( sgiii ) ( n = 3 ) following pneumonectomy ( table 1 ) . the dogs were anesthetized and continued with 2% isofloran after the administration of xylasine hcl ( 1 mg / kg i m ) and thiopental na ( 15 mg / kg iv ) . respiration was ensured by mechanical ventilation ( 15 ml / kg tidal volume , respiration rate 15/min and 25 cm h2o alveolar pressure ) . thoracotomy incision was made on the 4th intercostal space according to the standard procedure in the sgi and sgii groups , but the 4th intercostal externus - internus muscle prepared as a pedicled flap in the sgiii group . pneumonectomy was carried out using the open bronchi resection technique in groups sgi and sgiii . in sgii , however , resection was performed after stapler closure . subgroup i ( manual suture ) : vertical mattress sutures were applied over the excision line , and the end of the stump was sutured with a simple interrupted suture pattern using 2 - 0 vicryl ( ethicon , uk ) ( fig . subgroup ii ( stapler ) : a ta-30 stapler ( ethicon , uk ) ( 4.8 mm ) was initially applied to the main stem bronchus , and the bronchus was then excised ( fig . subgroup iii ( manual suture plus tissue flab ) : pedicled intercostal externus - internus muscle was extended towards the bs and then wrapped and sutured using a simple continuous suture pattern with 2 - 0 vicryl after manual suturing of the bs with 2 - 0 vicryl ( fig . the pleural space was filled with warm sterile saline , and then 50 cm h2o endobronchial pressure was applied to determine the bronchial air leakage . extra vertical mattress suture(s ) was / were applied over the excision side of the bs in cases where air bubbles occurred due to the bs . saline was aspirated , and a 28-french thoracostomy tube ( argyle , usa ) was inserted into the pleural cavity ventro - cranially . the tube was fixed to the skin with a ' chinese finger trap ' suture pattern , and a heimlich flutter valve was connected to the tube for pleural drainage . postoperative care was carried out in compliance with the rules of the national society of medical research principles of laboratory animal care for a period of one month . the dogs were relieved with carprofen ( 5 mg / kg / d sc ) . cefazolin na ( 20 mg / kg iv tid ) was used as an antibiotic agent for 5 days . lactate ringer and hematologic parameters and blood gas analysis of the dogs were controlled routinely , and the dogs were closely monitored for suspected cardiac problems and respiratory failure after pneumonectomy . the thoracostomy tube and skin sutures were removed on days 5 and 7 after surgery , respectively . at the end of the postoperative first month , the dogs in gi and gii were sacrificed with high - dose thiopental na . the bs was removed from each animal , fixed in 10% neutral - buffered formalin and then embedded in paraffin . five micrometer thick sections from these samples were placed on slides and stained with hematoxylin and eosin for microscopic examination . statistical analysis was carried out using fisher 's exact test and pearson 's chi - square test to determine the differences between subgroups in terms of histological healing and bpf complications , respectively . postoperative ventrodorsal and lateral radiographs showed no signs of intra - thoracic pathology , such as pneumothorax or hemathorax , in the dogs during a one - month period following pneumonectomy . 2a ) ; however , no abnormal clinical condition due to mediastinal shift was observed . complications were encountered in 9 of the 27 dogs ( 10 dogs in sgi , 7 dogs in sgii and 10 dogs in sgiii ) and included two bpfs ( on the 5th and 10th day ) , one postoperative cardiac failure ( 15th day ) and one postoperative respiratory arrest ( 1st h after surgery ) in the sgi group ; one bpf ( 5th day ) in the sgii group ; and two bpfs ( on the 1st week and 15th day ) , one cardio - pulmonary failure ( on 3rd week ) and one peri - operative cardiac arrest in the sgiii group ( table 2 ) . respiratory arrest and sudden onset of death tension pneumothorax and subcutaneous emphysema were observed in the postmortem radiographs of these animals ( fig . necropsy on the dog that died on the 15th day revealed myocardial petechial hemorrhagic spots related to the cardiac failure . in a different dog in sgi , the cause of the postoperative respiratory arrest ( 1 h after surgery ) was insufficient lung volume , which caused the postoperative spo2 value of the dog to decrease . in sgiii , alveolar emphysema , pneumonia and petechial myocardial hemorrhage were seen macroscopically in the dog with cardiopulmonary failure that died during the 3rd week after surgery . peri - operative cardiac arrest after left pneumonectomy was seen as a complication in one of the dogs in sgiii . this was related to an underlying cardiac problem , which was determined to be an atrioventricular blockage and sinus pause after pneumonectomy based on the results of ecg . subgroup i ( manual suture ) : histological examinations revealed complete healing in 4 out of 6 bronchial stumps . granulation tissue formation with new vessel formations as well as continued phagocytosis of the suture material was seen around these 4 bronchial stumps ( fig . one had severe neutrophil infiltration , and the other had severe neutrophil infiltration along with purulent bronchitis . subgroup ii ( stapler ) : in the histological examination , complete healing was seen in 4 of these 6 bronchial stumps . new granulation tissues were seen around the bs and stapler particles were surrounded by macrophages and were being phagocytosed ( fig . the other two samples had diffuse and severe hemorrhages in the regeneration area , and their healing was considered to be incomplete . subgroup iii ( manual suture plus tissue flab ) : complete healing was observed in 3 out of 6 bronchial stumps . the muscle flap used for bs closure was degenerated and necrotized to a great extent , and a granulation tissue forming around the bs covered the area ( fig . in 2 of the 3 incomplete bronchial stumps , severe neutrophil infiltrations and severe hemorrhage were observed . the numbers of complete and incomplete histological healings and bpf complications within the subgroups are presented in table 3 . there were no statistically significant differences in histological healing between the sgi and sgiii groups ( p = 1.00 ; p > 0.05 ) , nor between the sgii and sgiii groups ( p = 1.00 ; p > 0.05 ) . similarly , there were no significant differences in bpf complications between the sgi , sgii and sgiii groups ( p = 0.945 ; p > 0.05 ) . postoperative ventrodorsal and lateral radiographs showed no signs of intra - thoracic pathology , such as pneumothorax or hemathorax , in the dogs during a one - month period following pneumonectomy . 2a ) ; however , no abnormal clinical condition due to mediastinal shift was observed . complications were encountered in 9 of the 27 dogs ( 10 dogs in sgi , 7 dogs in sgii and 10 dogs in sgiii ) and included two bpfs ( on the 5th and 10th day ) , one postoperative cardiac failure ( 15th day ) and one postoperative respiratory arrest ( 1st h after surgery ) in the sgi group ; one bpf ( 5th day ) in the sgii group ; and two bpfs ( on the 1st week and 15th day ) , one cardio - pulmonary failure ( on 3rd week ) and one peri - operative cardiac arrest in the sgiii group ( table 2 ) . respiratory arrest and sudden onset of death tension pneumothorax and subcutaneous emphysema were observed in the postmortem radiographs of these animals ( fig . necropsy on the dog that died on the 15th day revealed myocardial petechial hemorrhagic spots related to the cardiac failure . in a different dog in sgi , the cause of the postoperative respiratory arrest ( 1 h after surgery ) was insufficient lung volume , which caused the postoperative spo2 value of the dog to decrease . in sgiii , alveolar emphysema , pneumonia and petechial myocardial hemorrhage were seen macroscopically in the dog with cardiopulmonary failure that died during the 3rd week after surgery . peri - operative cardiac arrest after left pneumonectomy was seen as a complication in one of the dogs in sgiii . this was related to an underlying cardiac problem , which was determined to be an atrioventricular blockage and sinus pause after pneumonectomy based on the results of ecg . subgroup i ( manual suture ) : histological examinations revealed complete healing in 4 out of 6 bronchial stumps . granulation tissue formation with new vessel formations as well as continued phagocytosis of the suture material was seen around these 4 bronchial stumps ( fig . one had severe neutrophil infiltration , and the other had severe neutrophil infiltration along with purulent bronchitis . subgroup ii ( stapler ) : in the histological examination , complete healing was seen in 4 of these 6 bronchial stumps . new granulation tissues were seen around the bs and stapler particles were surrounded by macrophages and were being phagocytosed ( fig . the other two samples had diffuse and severe hemorrhages in the regeneration area , and their healing was considered to be incomplete . subgroup iii ( manual suture plus tissue flab ) : complete healing was observed in 3 out of 6 bronchial stumps . the muscle flap used for bs closure was degenerated and necrotized to a great extent , and a granulation tissue forming around the bs covered the area ( fig . in 2 of the 3 incomplete bronchial stumps , severe neutrophil infiltrations and severe hemorrhage were observed . the numbers of complete and incomplete histological healings and bpf complications within the subgroups are presented in table 3 . there were no statistically significant differences in histological healing between the sgi and sgiii groups ( p = 1.00 ; p > 0.05 ) , nor between the sgii and sgiii groups ( p = 1.00 ; p > 0.05 ) . similarly , there were no significant differences in bpf complications between the sgi , sgii and sgiii groups ( p = 0.945 ; p > 0.05 ) . a considerable number of retrospective studies have been performed on bronchial closure techniques in human medicine , and a few experimental studies have been carried out on the basis of these retrospective studies . however , bs closure techniques , their histological healing patterns and possible cardiac , respiratory and gastrointestinal system complications after pneumonectomy have not been compared experimentally and have only been reviewed in dogs . , manual suture has been shown to be a safe and cost - effective technique . the superiority of stapler suture to manual suture remains questionable , and several recent studies have compared the stapler with manual suture techniques . therefore , in the present study , manual and stapler suture techniques were used in the sgi and sgii groups , respectively . manual suture , stapler and bs covering with flaps after manual suture closure are still in use today . pleural flaps , patch with pedicle , pericardial grafts , pericardial fat pad grafts , diaphragm , azygous vein , pericardiophrenic pedicles , mediastinum and transposition of extra - thoracic muscles ( intercostal , serratus and latissimus dorsi ) have been used as flaps in human thoracic surgery . intercostal muscle flaps are superior to other flaps due to their flexibility , thickness , vascularity and autogenous features , and the usage of these flaps has been recommended by a number of thoracic surgeons . thus , in this study , pedicled intercostal externus - internus muscle was used to buttress the manual suture line of the bs closure in the sgiii group . advised the use of simple interrupted and over - to - over suture patterns for bs closure in humans . in veterinary literature , interrupted horizontal and continuous suture patterns have been used for bs closure . in this study , hermetically horizontal mattress sutures were performed over the excision site , without affecting the vascularity of the main stem bronchus , and the end of the bs was manually sutured with a simple interrupted suture pattern in sgi . in humans and dogs , ta-55 and ta-30 staplers have been reported during pneumonectomy by the parallel approximation of the mucosal membranous and cartilaginous portions of the bronchus and applying the staple transversally to the bronchus . if the bs is to be closed with a suture , it is important that the same surgical team perform the procedure . therefore , in the present study , all bs closures were carried out by the same surgical team . current literatures indicate that the application of 20 - 40 cm h2o intra - bronchial pressure is sufficient to determine the amount of bronchial air leakage before thoracic closure . similarly , the intra - bronchial pressure increases by up to 200 mmhg in humans at the time of coughing . considering these findings and the possible postoperative coughing and barking of the dogs , 50 cm h2o pressure was applied intra - bronchially to test for air leakage . in cases of air bubbles occurring from the bs , extra vertical mattress suture(s ) pulmonary resection is associated with a considerable risk of infection ; therefore , antibiotic prophylaxis has become a routine procedure during and after pulmonary operations . in addition , only one dose of prophylactic cephalosporin has been suggested to be an effective dosage following pneumonectomy . in this study , cefazolin na was administered during the postoperative period , and no infection - related complications were encountered . radiologically , the shifted mediastinum was observed in all dogs , and no complications from the mediastinal shift were seen for one month after the operation . in humans , a history of fever and sudden onset of continuous coughing after lung resection raises the suspicion of bpf . in this study , in contrast to the results in humans , the clinical findings in the dogs with bpf were respiratory arrest and sudden onset of death . tension pneumothorax and subcutaneous emphysema were seen in the postmortem radiographs , and necropsy of the dogs revealed the occurrence of bronchial dehiscence and confirmed the bpf . possible cardiac , respiratory and gastrointestinal system complications after pneumonectomy have been reported in humans and dogs . in this study , five bpfs , one cardiac failure , one postoperative respiratory arrest , one cardio - pulmonary failure and one peri - operative cardiac arrest were seen in the dogs ( table 2 ) . necropsy findings , postoperative spo2 values and ecg findings revealed these complications in this study . it is our opinion that further studies can be planned to demonstrate the complications after pneumonectomy in dogs . in addition , the fifth postoperative day is the most critical day for bronchial dehiscence , and the incidence of early bpf in the suture technique is 8.6% , although its incidence is 1% in the stapler technique . the incidence of bpf following manual suture plus autogenic tissue coverage is 3.9% in humans . the main focus of this study was not to determine the incidence of bpf in dogs , but we do report that we encountered five bpf complications in 27 dogs . information on the inflammatory reactions between the suture material and bronchi are important to the prevention of postoperative complications . in the present study , neutrophil reactions with different intensities were seen after manual suture closure . in the present study , the stapler closure of the bronchi proved to be the preferred alternative within the three closure techniques used . however , it is unclear which technique is superior to the others from a histological healing point of view . it is important to note that avascular necrosis and inflammation of the bs after stapler closure have not been well explained in the literature . in the present study , the stapler technique was better than the other techniques and resulted in less severe inflammation and a faster healing process . current literatures indicate that vascular tissue with pedicle is required for the early healing of the bs . in addition , pedicle flaps warranted the bs healing in the critical revascularization period continuing for 3 - 4 weeks , and decreased the risk of infection . self - vascularized flaps are reported to enhance the healing of the bs . according to algar et al . although yamamoto et al . indicated that intercostal flaps prevented bpf complications , demos reported that these flaps could cause fibrosis , ossification and calcification . in our study , a severe inflammatory reaction was observed against the degeneration and necrosis of the flap and suture material . there were no statistically significant differences in histological healing among the subgroups . in the present study , we emphasize that the use of a thinner muscle flap can potentially reduce the inflammatory reaction , thus resulting in faster healing . in conclusion , the usage of a stapler for bs closure is the best choice according to our histological data , although it can not be considered to be a very economic technique . manual suture plus intercostal externus- internus muscle flap technique has advantage for buttressing to bs . there were no statistically significant differences among the subgroups with regard to bpf complications . according to the histological healing results , the manual suture technique did not have an apparent advantage over the other techniques , but stapler closure seemed to be more acceptable than the other methods of closure . the use of manual suture plus tissue flap technique could not be recommended due to the observation of intense inflammatory reactions following this procedure . statistical data indicated that none of the techniques seemed to have any superiority over the others with regard to histological healing . taking the results of the statistical analysis into consideration , all three techniques ( suture , stapler and suture plus tissue flab ) can be used for bs closure .

the comparison of the histologic healing and bronchopleural fistula ( bpf ) complications encountered with three different bs closure techniques ( manual suture , stapler and manual suture plus tissue flab ) after pneumonectomy in dogs was investigated for a one - month period . the dogs were separated into two groups : group i ( gi ) ( n = 9 ) and group ii ( gii ) ( n = 9 ) . right and left pneumonectomies were performed on the animals in gi and gii , respectively . each group was further divided into three subgroups according to bs closure technique : subgroup i ( sgi ) ( n = 3 ) , manual suture ; subgroup ii ( sgii ) ( n = 3 ) , stapler ; and subgroup iii ( sgiii ) ( n = 3 ) , manual suture plus tissue flab . the dogs were sacrificed after one month of observation , and the bronchial stumps were removed for histological examination . the complications observed during a one - month period following pneumonectomy in nine dogs ( n = 9 ) were : bpf ( n = 5 ) , peri - operative cardiac arrest ( n = 1 ) , post - operative respiratory arrest ( n = 1 ) , post - operative cardiac failure ( n = 1 ) and cardio - pulmonary failure ( n = 1 ) . histological healing was classified as complete or incomplete healing . histological healing and bpf complications in the subgroups were analyzed statistically . there was no significant difference in histological healing between sgi and sgiii ( p = 1.00 ; p > 0.05 ) , nor between sgii and sgiii ( p = 1.00 ; p > 0.05 ) . similarly , no significant difference was observed between the subgroups in terms of bpf ( p = 0.945 ; p > 0.05 ) . the results of the statistical analysis indicated that manual suture , stapler or manual suture plus tissue flab could be alternative methods for bs closure following pneumonectomy in dogs .

Introduction Materials and Methods Experimental group Bronchial closure Results Bronchial closure Histological findings Discussion

PMC1469784

there are many distinct differences ( morphologic , physiologic , and mechanical ) between the bird 's lung - air - sac respiratory system and the mammalian bronchoalveolar lung . in this paper , we review the physiology of the avian respiratory system with attention to those mechanisms that may lead to significantly different results , relative to those in mammals , following exposure to toxic gases and airborne particulates . we suggest that these differences can be productively exploited to further our understanding of the basic mechanisms of inhalant toxicology ( gases and particulates ) . the large mass - specific gas uptake by the avian respiratory system , at rest and especially during exercise , could be exploited as a sensitive monitor of air quality . birds have much to offer in our understanding of respiratory toxicology , but that expectation can only be realized by investigating , in a wide variety of avian taxa , the pathophysiologic interactions of a broad range of inhaled toxicants on the bird 's unique respiratory system.imagesp188-afigure 1.figure 2.figure 3.figure 4.figure 5 . afigure 5 . bfigure 5 . cfigure 6.figure 7.figure 8 .

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PMC3198235

traditionally , open dismembered pyeloplasty has been the standard treatment for ureteropelvic junction obstruction ( upjo ) . in 1993 , schuessler and associates described the first laparoscopic pyeloplasty , and since then it has been shown to result in similar outcomes to open pyeloplasty with a lower morbidity . to decrease the morbidity and improve cosmetic outcomes from conventional laparoscopy , efforts have been made to develop new techniques with multichannel single - access ports and articulating instruments that allow the laparoscopic procedure to be performed through a single skin incision that is often hidden within the umbilicus , otherwise referred to as laparoendoscopic single - site surgery ( less ) . less has been used for diverse urological diseases and initial studies have shown that less is safe and feasible in comparison to conventional laparoscopic surgery [ 4 - 8 ] . in particular , after the report of desai et al on the first less pyeloplasty ( less - p ) , follow - up studies showed similar outcomes with conventional laparoscopic pyeloplasty ( cl - p ) . however , less is still a technically challenging procedure for beginners because of the difficulty in suturing . here , we report on our techniques and initial experience with less - p using additional needlescopic instruments and compare the results with cl - p . we analyzed 9 consecutive patients who underwent less - p between july 2009 and april 2010 . the clinical data were compared with data from 18 patients out of 31 consecutive patients who underwent cl - p from november 2003 to april 2009 . the 9 less - p patients were matched 1:2 with the 18 cl - p patients on the basis of age and side of surgery . the choice of operation was based on the presence of obstruction on the diuretic renogram with either symptoms ( urinary tract infection , recurrent pain , or stone formation ) or functional impairment of the kidney ( relative uptake 40% ) . all less - p procedures were performed by a single surgeon ( d.h.h . ) , whereas the cl - p procedures were performed by two surgeons ( 17 cases by d.h.h , 1 case by s.s.j . ) in both groups , the operating procedures were performed similarly , except for the number of access points . a ureteral stent was placed antegradely , except in cases where the stent had been placed before surgery . to perform less - p , we used a wound retractor ( alexis ; applied medical , rancho santa margarita , ca , usa ) and a surgical glove as the homemade single - port device as described in our previous series . to make a single port similar to that of a commercial multichannel trocar we firmly fastened the first , third , and fifth glove finger tips to the end of the three trocars ( two 12 mm trocars and one 5 mm trocar ) with a tie or rubber band . pneumoperitoneum was made by co2 gas insufflation to 14 mm hg , and a 10 mm rigid laparoscope angled at 30 degrees was inserted . the operation was performed by using conventional laparoscopic straight working instruments and 5 mm articulating instruments ( autonomy laparo - angle ; cambridge endo , framingham ma , usa ) to overcome the lack of triangulation that occurs with a single port . we also used additional needlescopic instruments with a 2 mm trocar inserted at the subcostal area to create an environment similar to that for conventional laparoscopic pyeloplasty with ureteropelvic anastomosis ( fig . 1 , 2 ) . the 2 mm grasper was used to assist with suturing by manipulating the renal pelvis or ureter and adjusting the needle direction ( fig . the anastomosis was performed by means of a continuously running suture with 4 - 0 polygalactin suture . a ureteral stent was inserted antegradely through the 2 mm trocar by using a 5 fr . the 2 mm trocar site did not need to be closed after the operation . in the cl - p group all patients underwent placement of a jackson - pratt drain through a 5 mm trocar incision in all cl - p cases and through the margin of the umbilical incision in all less - p cases . prophylactic antibiotics were routinely prescribed . for pain control , we prescribed intravenous ( iv ) patient - controlled analgesia ( pca ) . infusion of 1,500 g of fentanyl in 100 ml of normal saline was started at a basal infusion rate of 1.0 ml / hr with an iv infusion pump ( automed3200 ; ace medical , seoul , korea ) . the analgesic bolus of pca ( using a 1.0 ml bolus and a lockout time of 15 minutes in the iv pca group ) was started at the post - anesthesia care unit by the patient after awakening . additional mso4 was applied when the iv pca was ineffective at reducing or eliminating the pain . the closed suction drain was subsequently removed if the drainage output did not increase and was less than 100 ml in 24 hours after foley catheter removal . the perioperative parameters including operative time , intraoperative estimated blood loss , analgesic use , postoperative hospital stay , complications , and success rates were compared between the two groups . the operative time was recorded from the time of the initial skin incision to the final skin suture . to compare postoperative pain , we used a numerical rating scale ( nrs ) from 0 ( no pain ) to 10 ( worst possible pain ) , and we looked at the additional mso4 requirement on postoperative day 1 and the discharge day . the assessment of success was based on improvement in the diuretic renogram , including resolution of clinical symptoms . all statistical analyses were performed with the spss ver . 17.0 ( spss inc . , numerical data from a normal distribution were expressed as a group mean with the standard deviation and were compared by using the student 's t - test . numerical data from a non - normal distribution were expressed as a group median evaluated by using the mann - whitney rank sum test . we analyzed 9 consecutive patients who underwent less - p between july 2009 and april 2010 . the clinical data were compared with data from 18 patients out of 31 consecutive patients who underwent cl - p from november 2003 to april 2009 . the 9 less - p patients were matched 1:2 with the 18 cl - p patients on the basis of age and side of surgery . the choice of operation was based on the presence of obstruction on the diuretic renogram with either symptoms ( urinary tract infection , recurrent pain , or stone formation ) or functional impairment of the kidney ( relative uptake 40% ) . all less - p procedures were performed by a single surgeon ( d.h.h . ) , whereas the cl - p procedures were performed by two surgeons ( 17 cases by d.h.h , 1 case by s.s.j . ) in both groups , the operating procedures were performed similarly , except for the number of access points . a ureteral stent was placed antegradely , except in cases where the stent had been placed before surgery . to perform less - p , we used a wound retractor ( alexis ; applied medical , rancho santa margarita , ca , usa ) and a surgical glove as the homemade single - port device as described in our previous series . to make a single port similar to that of a commercial multichannel trocar , we firmly fastened the first , third , and fifth glove finger tips to the end of the three trocars ( two 12 mm trocars and one 5 mm trocar ) with a tie or rubber band . pneumoperitoneum was made by co2 gas insufflation to 14 mm hg , and a 10 mm rigid laparoscope angled at 30 degrees was inserted . the operation was performed by using conventional laparoscopic straight working instruments and 5 mm articulating instruments ( autonomy laparo - angle ; cambridge endo , framingham ma , usa ) to overcome the lack of triangulation that occurs with a single port . we also used additional needlescopic instruments with a 2 mm trocar inserted at the subcostal area to create an environment similar to that for conventional laparoscopic pyeloplasty with ureteropelvic anastomosis ( fig . 1 , 2 ) . the 2 mm grasper was used to assist with suturing by manipulating the renal pelvis or ureter and adjusting the needle direction ( fig . the anastomosis was performed by means of a continuously running suture with 4 - 0 polygalactin suture . a ureteral stent was inserted antegradely through the 2 mm trocar by using a 5 fr . the 2 mm trocar site did not need to be closed after the operation . in the cl - p group all patients underwent placement of a jackson - pratt drain through a 5 mm trocar incision in all cl - p cases and through the margin of the umbilical incision in all less - p cases . prophylactic antibiotics were routinely prescribed . for pain control , we prescribed intravenous ( iv ) patient - controlled analgesia ( pca ) . infusion of 1,500 g of fentanyl in 100 ml of normal saline was started at a basal infusion rate of 1.0 ml / hr with an iv infusion pump ( automed3200 ; ace medical , seoul , korea ) . the analgesic bolus of pca ( using a 1.0 ml bolus and a lockout time of 15 minutes in the iv pca group ) was started at the post - anesthesia care unit by the patient after awakening . additional mso4 was applied when the iv pca was ineffective at reducing or eliminating the pain . the closed suction drain was subsequently removed if the drainage output did not increase and was less than 100 ml in 24 hours after foley catheter removal . the perioperative parameters including operative time , intraoperative estimated blood loss , analgesic use , postoperative hospital stay , complications , and success rates were compared between the two groups . the operative time was recorded from the time of the initial skin incision to the final skin suture . to compare postoperative pain , we used a numerical rating scale ( nrs ) from 0 ( no pain ) to 10 ( worst possible pain ) , and we looked at the additional mso4 requirement on postoperative day 1 and the discharge day . the assessment of success was based on improvement in the diuretic renogram , including resolution of clinical symptoms . numerical data from a normal distribution were expressed as a group mean with the standard deviation and were compared by using the student 's t - test . numerical data from a non - normal distribution were expressed as a group median evaluated by using the mann - whitney rank sum test . the demographics of the patients in the cl - p and open less - p group are shown in table 1 . there were no significant differences between the 2 groups in terms of age , operation side , sex , body mass index , and crossing vessel . the number of attempts at previous endoscopic management was significantly higher in the less - p ( p=0.029 ) group . all cl - p procedures were completed successfully with no conversions to open surgery and all open procedures were performed as planned . one conversion to cl - p was present in the less - p group because of severe adhesion resulting from previous endoscopic management and intraperitoneal operation . there was no statistically significant difference in the conversion rate to other operations , however . there was also no mortality in either the cl - p or the less - p group . less - p was associated with a shorter operative time ( 252.2 vs. 309.7 minutes , p=0.044 ) and a lower nrs on postoperative day 1 ( 3.7 vs. 5.6 , p=0.024 ) . however , there were no significant differences between the two groups in terms of length of stay , estimated blood loss volume , double j indwelling time , foley catheter indwelling time , mso4 requirement , nrs on the discharge day , conversion rate to another operation , or complication rate . two minor adverse events developed in the less - p group : one case of transient gross hematuria and one case of an emergency room visit for pain that was not relieved by oral medication after discharge . the success rate was 94% ( 17 of 18 cases ) in the cl - p group ( median 23 months ) and 100% ( 9 of 9 cases ) in the less - p group ( median 14 months ) . there was no significant difference in the success rate between the two groups ( p=1.00 ) . in the past 20 years , accumulating evidence has suggested that laparoscopic pyeloplasty is the standard of care in adults [ 3,13 - 15 ] . several studies have shown a success rate of greater than 90% for the laparoscopic procedure , similar to that achieved with traditional open dismembered pyeloplasty [ 13 - 15 ] . laparoscopic pyeloplasty is associated with a short hospital stay , decreased parenteral narcotic use , good cosmesis , and less postoperative pain [ 13 - 15 ] . these include port site bleeding , incisional hernias , and postoperative pain caused by incisional wounds . less was suggested on the basis of the assumption that reducing the number of port sites would reduce some of the problems with conventional laparoscopic surgery . since then , less has been used for various urological diseases and has been shown to be feasible and safe and to have a success rate comparable to that of conventional laparoscopic surgery . previous reports comparing less with conventional laparoscopic nephrectomy have shown comparable results and potential cosmetic advantages . recently , tracy et al reported the following comparative results between less - p and cl - p : operative time ( 202 vs. 257 minutes ) , estimated blood loss ( 35 vs. 85 ml ) , minor and major complications ( 14.3% vs. 14.3% , 21.4% vs. 10% , respectively ) , and success rates ( 96% vs. 100% ) . these results demonstrated that less - p was safe and feasible and was able to achieve a similar success rate to that of cl - p with potent cosmesis . however , less is still a challenging procedure , especially for reconstructive surgery , because incomplete triangulation and more complex motion of the articulating instruments limits effective suturing . for that reason , an additional 2 mm or 5 mm trocar is generally used during less - p . tracy et al used a temporary 5 mm port in all cases to aid in suturing and an additional 3 mm subxyphoid port for right - sided procedures for liver traction when necessary . stein et al also used a 2 mm grasping instrument to aid in suturing . in our series , we also used a 2 mm needlescopic trocar in all less cases . because the needlescopic trocar is the same size as a veress needle , for which an incision is not needed for insertion , it does not make a puncture site scar after the operation . we assumed that by using a 2 mm needlescopic trocar , the cosmetic objective of umbilical less , a hidden scar , could be achieved . in reality it does not provide enough holding strength , does not have rotation function ( which means that the operator must rotate his or her wrist ) , and requires more effort and concentration to manipulate the instrument because of the small jaw . however , despite these limitations , the instrument provided powerful support during our less - p . this approach supports an environment similar to that of cl - p : it provided effective traction of the bowel or liver and assisted with suturing by making triangulation . the 2 mm trocar made it easy to place a ureteral stent antegradely because it could keep the guide wire and ureter in a line . using a 3 mm instrument instead of a 2 mm one may be a better choice for overcoming the disadvantages of the 2 mm instrument . however , the addition of a 2 mm trocar and grasper is still our routine practice for suturing in less pyeloplasty because use of the 2 mm trocar and grasper is feasible and the cosmetic effect is better . our study showed comparable results between less - p with an additional 2 mm trocar and conventional laparoscopic surgery . there were no significant differences in length of stay , estimated blood loss , mso4 requirement , or nrs on the discharge day . our data also showed that the mean nrs on postoperative day 1 in the less - p group was lower than that in the cl - p group ( 3.7 vs. 5.6 , p=0.024 ) . we can not precisely explain this result , but we assume that the fewer number of incisions helped to decrease the early postoperative pain . however , the pain gradually decreased throughout the hospital stay period , and when the patients were discharged , there was no significant difference . these data suggest that less - p may result in less pain than cl - p during the early postoperative period . in this retrospective study , this use of pca may have affected the difference in postoperative pain between the two groups . if pca had not been used , there might have been a greater difference in pain severity between the groups . in this study , less - p could be effectively performed compared with cl - p with minimal complications and a low conversion rate . the mean operation time in the less group was even shorter than that in the cl - p group ( 252.2 min vs. 309.7 minutes , p=0.044 ) . however , this may be from the time difference in the operation period . accumulated experience with intracorporeal suturing may have a decisive effect on the operation time in the less group . despite the time difference , this result demonstrates that less - p could be performed effectively by experienced laparoscopic surgeons . less - p has a potential cosmetic advantage , but we were unable to assess the cosmetic advantage . despite inserting a jackson - pratt drain at the umbilicus , we effectively hid the surgical scar within the umbilicus during the less - p procedure . furthermore it is obvious that a hidden scar has a cosmetic advantage over an exposed cl - p scar ( fig . 4 ) , but further studies with a validated cosmetic scale are necessary to address this issue . there are several important limitations of our study that should be acknowledged , including the retrospective design with its inherent problems , the small number of patients , and the short - term follow - up period . nevertheless , we consider this study to be valuable because we could show less pain in the less - p group during the early postoperative period in a matched control design study . issues such as return to everyday activities and cosmesis remain important outcomes that should be addressed by future studies to provide further insight . also , the role of less pyeloplasty needs to be further confirmed by a prospective randomized study with a large number of cases and a long - term follow - up . less - p is a technically feasible and safe procedure with the addition of 2 mm instruments to overcome associated difficulties . in our study series , the surgical outcomes of less - p were comparable to those of cl - p . in addition , less - p could decrease early postoperative pain . however , functional outcome data with long - term follow - up are needed to establish whether less pyeloplasty should be the standard option for upjo treatment .

purposedespite a recent surge in the performance of laparoendoscopic single - site surgery ( less ) , concerns remain about performing less pyeloplasty ( less - p ) because of the technical difficulty in suturing . we report our techniques and initial experiences with less - p using additional needlescopic instruments and compare the results with conventional laparoscopic pyeloplasty ( cl - p).materials and methodsnine patients undergoing less - p were matched 2:1 with regard to age and side of surgery to a previous cohort of 18 patients who underwent cl - p . in both groups , the operating procedures were performed equally except for the number of access points . in the less - p group , we made a single 2 cm incision at the umbilicus and used a homemade port . we also used additional 2 mm needlescopic instruments at the subcostal area to facilitate suturing and the ureteral stenting.resultsthe preoperative characteristics were comparable in both groups . postoperatively , no significant differences were noted between the less - p and cl - p cases in regard to length of stay , estimated blood loss , analgesics required , and complications . but , less - p was associated with a shorter operative time ( 252.2 vs. 309.7 minutes , p=0.044 ) and less pain on postoperative day one ( numeric rating scale 3.7 vs. 5.6 , p=0.024 ) . the success rate was 94% with cl - p ( median , 23 months ) and 100% with less - p ( median , 14 months).conclusionsour initial experiences suggest that less - p is a feasible and safe procedure . the use of additional 2 mm instruments can help to overcome the difficulties associated with less surgery .

INTRODUCTION MATERIALS AND METHODS 1. Patients 2. Surgical technique 3. Statistical analysis RESULTS DISCUSSION CONCLUSIONS

PMC3015921

the development of ph does not always necessitate repair , but when surgical intervention is undertaken , recurrence is common . standard surgical approaches include local suture repair , local prosthetic mesh placement , and stoma transposition with or without midline laparotomy . using these approaches favorable outcomes following laparoscopic ( lap ) ph repair with mesh have been reported in case reports and small case series . we report our experience with lap ph repair in a colorectal patient population and propose that lap ph repair is a suitable and effective approach in the management of this disease . a retrospective chart review was performed on all patients with ph at the site of an ileostomy or colostomy who underwent lap or open surgical repair at our institution between november 1999 and november 2006 . data were taken from the time of first open repair in the open group and first lap repair in the lap group . patients were sorted into groups on an intent - to - treat basis , and the one converted lap patient was kept in the lap group . data collected included patient demographics , indication for initial stoma creation , operative time , length of stay ( los ) , postoperative complications , recurrence , and follow - up . a retrospective chart review was performed on all patients with ph at the site of an ileostomy or colostomy who underwent lap or open surgical repair at our institution between november 1999 and november 2006 . data were taken from the time of first open repair in the open group and first lap repair in the lap group . patients were sorted into groups on an intent - to - treat basis , and the one converted lap patient was kept in the lap group . data collected included patient demographics , indication for initial stoma creation , operative time , length of stay ( los ) , postoperative complications , recurrence , and follow - up . all patients were placed in the supine position with an ioban drape ( 3 m , st . peritoneal access was achieved with an 11-mm optical trocar ( optiview , ethicon , cincinnati , oh ) placed diagonally to the hernia . one 5-mm port was placed opposite the 11-mm port , superior to the ostomy , and one 5-mm port was placed inferiorly to the 11-mm port ( figure 1 ) . the herniated bowel and omentum were carefully reduced from the hernia defect ( figure 2a ) . a polytetrafluoroethylene ( ptfe ) mesh ( dual mesh , gore & associates , newark , de ) large enough to create at least a 4-cm overlap in all directions was used for all repairs . the mesh was then rolled and inserted into the abdomen through the 11-mm port . an endoclose device ( us surgical , norwalk , ct ) was used to grasp the prolene sutures for tying over the fascia in the subcutaneous space . for the 7 most recent patients , a nonslit flap configuration was used with the bowel ( stoma ) exiting laterally . a protack device ( us surgical , norwalk , ct ) was used to fix the edges of the mesh circumferentially except laterally , where the bowel came around the mesh . the bowel was pinned against the lateral anterior abdominal wall by the mesh to create a modified sugarbaker hernia repair ( figure 2b ) . alternatively , in the first 3 patients , a keyhole was cut , and a 3-cm aperture for the bowel was created in the mesh prior to implantation . in another patient , also operated on early within the reviewed time period , a diagonal , lateral slit was made to accommodate passage of the intestine . in the 4 patients with cut or slit mesh , a protack device was then used to circumferentially secure the mesh around the bowel and perimeter . tacks were also placed lateral to the mesh to close the slit , thus creating a 3-cm to 4-cm aperture around the bowel . at the conclusion of the repair , the fascia at the optiview port site was closed with figure - of - eight vicryl suture . location of trocar placement for repair of left - sided ph ( a ) and for repair of right - sided ph ( b ) . colostomy ( black arrow ) with associated parastomal fascial defect ( white arrow ) following reduction of herniated bowel ( a ) ; intact gore - tex dual mesh against anterior abdominal wall with underlying bowel limb ( black , dashed line ) obliquely exiting from mesh ( white arrow ) ( b ) . a midline incision through the prior incision site at the level of the stoma or a circumstomal , curvilinear incision outside the limits of the stomal faceplate was created . following subcutaneous dissection and identification of the hernia defect , the hernia sac was entered and freed circumferentially from the stoma . the fascial defect was closed with interrupted figure - of - eight or simple stitches of either # 1 prolene or 0 ethibond . in one case , the soft tissue defect was closed with interrupted vicryl suture , and the skin was closed with running subcuticular vicryl suture . all patients were placed in the supine position with an ioban drape ( 3 m , st . peritoneal access was achieved with an 11-mm optical trocar ( optiview , ethicon , cincinnati , oh ) placed diagonally to the hernia . one 5-mm port was placed opposite the 11-mm port , superior to the ostomy , and one 5-mm port was placed inferiorly to the 11-mm port ( figure 1 ) . the herniated bowel and omentum were carefully reduced from the hernia defect ( figure 2a ) . a polytetrafluoroethylene ( ptfe ) mesh ( dual mesh , gore & associates , newark , de ) large enough to create at least a 4-cm overlap in all directions was used for all repairs . the mesh was then rolled and inserted into the abdomen through the 11-mm port . an endoclose device ( us surgical , norwalk , ct ) was used to grasp the prolene sutures for tying over the fascia in the subcutaneous space . for the 7 most recent patients , a nonslit flap configuration was used with the bowel ( stoma ) exiting laterally . a protack device ( us surgical , norwalk , ct ) was used to fix the edges of the mesh circumferentially except laterally , where the bowel came around the mesh . the bowel was pinned against the lateral anterior abdominal wall by the mesh to create a modified sugarbaker hernia repair ( figure 2b ) . alternatively , in the first 3 patients , a keyhole was cut , and a 3-cm aperture for the bowel was created in the mesh prior to implantation . in another patient , also operated on early within the reviewed time period , a diagonal , lateral slit was made to accommodate passage of the intestine . in the 4 patients with cut or slit mesh , a protack device was then used to circumferentially secure the mesh around the bowel and perimeter . tacks were also placed lateral to the mesh to close the slit , thus creating a 3-cm to 4-cm aperture around the bowel . at the conclusion of the repair , the fascia at the optiview port site was closed with figure - of - eight vicryl suture . location of trocar placement for repair of left - sided ph ( a ) and for repair of right - sided ph ( b ) . colostomy ( black arrow ) with associated parastomal fascial defect ( white arrow ) following reduction of herniated bowel ( a ) ; intact gore - tex dual mesh against anterior abdominal wall with underlying bowel limb ( black , dashed line ) obliquely exiting from mesh ( white arrow ) ( b ) . a midline incision through the prior incision site at the level of the stoma or a circumstomal , curvilinear incision outside the limits of the stomal faceplate was created . following subcutaneous dissection and identification of the hernia defect , the hernia sac was entered and freed circumferentially from the stoma . the fascial defect was closed with interrupted figure - of - eight or simple stitches of either # 1 prolene or 0 ethibond . in one case , the soft tissue defect was closed with interrupted vicryl suture , and the skin was closed with running subcuticular vicryl suture . a total of 25 patients with ileostomies or colostomies underwent ph repair by the open approach or laparoscopic mesh placement between november 1999 and november 2006 . the lap repairs were all performed by 2 surgeons ( rh and ts ) who had completed minimally invasive surgery fellowships . all of the open repairs were performed by 2 board - certified colon and rectal surgeons ( wk and lp ) . twelve patients ( mean age 60 , range 25 to 80 years ) underwent lap approaches , with one patient requiring conversion to laparotomy with primary suture repair of the ph secondary to small - bowel enterotomy . thirteen patients ( mean age 54 ; range , 35 to 71 years ) underwent open ph repair . characterization of 25 patients having undergone laparoscopic or open parastomal repair se = standard error ; los = length of stay . diverticulitis , gist , paget 's disease , radiation proctitis , strangulated abdominal wall abscess , anismus . indications for initial stoma creation were ulcerative colitis ( 3 lap , 6 open ) , crohn 's disease ( 2 open ) , rectal cancer ( 4 lap , 3 open ) , diverticulitis ( 1 lap ) , anismus ( 1 lap ) , radiation proctitis ( 1 lap ) , gastrointestinal stromal tumor ( gist ) ( 1 open ) , paget 's disease ( 1 lap ) , strangulated abdominal wall abscess ( 1 open ) , and rectal cancer in the setting of crohn 's disease ( 1 lap ) . five patients had previously undergone open ph repairs : 4 in the lap group and 1 patient in the open group . twenty - three of the twenty - five ph repairs ( 92% ) were performed electively . indications for elective repair included chronic discomfort or progressive pain ( 7 lap , 7 open ) , bulging contributing to difficult maintenance of stomal appliances ( 4 lap , 3 open ) , and chronic obstruction ( 1 lap , 1 open ) . operative time for repair was not statistically significant between the 2 groups [ 17210.0 min ( lap ) versus 13719.1 min ( open ) ] , p=0.14 . the difference between los for the lap group ( 3.10.4 days ) and open group ( 5.10.8 days ) approached significance , p=0.05 . immediate postoperative complications occurred in 4 lap patients ( 33.3% ) and 2 open patients ( 15.4% ) , p=0.38 . in the lap group , complications were 2 wound infections , parastomal cellulitis , and a seroma beneath the mesh . no complications secondary to mesh erosion into bowel or mesh infection were seen in the lap group . the 2 complications in the open group were wound infections , one of which was a perineal wound infection separate from the ph repair site following a completion proctectomy that was simultaneously performed with the ph repair . the second complication was a parastomal abscess with subsequent fistula development , subsequently repaired by laparotomy , transection of the fistula tract , and resiting of the ileostomy . of these recurrences , 4 ( 33% ) were in the lap group and 7 ( 54% ) were in the open group . recurrences were evenly distributed among ileostomies and colostomies within the 2 groups ( 2 para ileostomal and 2 para - colostomal recurrences in the lap group ; 3 para - colostomal and 4 ileostomal recurrences in the open group ) . in the lap patients repaired by a modified sugarbaker technique , one of these patients developed a respiratory infection following her repair and experienced severe cough , which may have contributed to an increase in intraabdominal pressure , thus promoting her recurrence . this patient underwent a subsequent laparoscopic repair , and it was noted that one transfascial suture appeared to have torn through the mesh . among the 3 patients who had undergone mesh repair utilizing a keyhole - cut mesh , 2 recurred , resulting in a 67% recurrence rate among the total of 3 keyhole repairs . among our 13 patients who had local suture repairs , 7 recurred ( 54% ) , although one recurrence was associated with a motor vehicle collision at 10 months postoperative time in which the patient was a restrained driver . recurrences in the lap group occurred after 10.53.48 months versus 20.36.99 months in the open group . mean length of follow - up was 13.94.50 months for the lap group and 14.04.30 months for the open group . ph , the protrusion of abdominal contents through the fascial defect surrounding a stoma , represents a common complication of stoma construction that remains challenging to manage when they are diagnosed , they can often be managed without surgery using a hernia support belt , weight loss , avoidance of lifting heavy objects , observation , and patient education . larger , symptomatic hernias that cause pain , obstruction , and difficulty with maintenance of the appliance , as well as create cosmetic and other quality of life issues , warrant surgical repair . classically , 3 methods have been utilized to repair ph : local primary fascial repair , local mesh repair , and stoma transposition . recurrence rates of 46% to 76% following simple fascial repair have been described , with associated complication rates up to 71% , including death . in our study , we found a recurrence rate with open fascial repair similar to that reported in the literature at 53.8% , but had a much lower complication rate than that occurring in most studies at 15.4% . the incorporation of synthetic mesh into local ph repairs results in a lower recurrence rate but can be associated with serious complications , such as dense adhesion formation to the mesh , mesh infection , abscess development , and perforation or mesh erosion into the bowel . since the 1970s , the preference for type of mesh used in ph repair has changed from polyethylene to polypropylene to ptfe to expanded polytetrafluoroethylene ( eptfe ) , the last being a softer , smoother , low - porosity membrane that promotes minimal inflammation and adhesion formation while allowing tissue ingrowth . one consideration in mesh repair is whether to create an opening in the mesh through which the bowel may exit or pin the bowel against the abdominal wall with the mesh . several studies utilizing mesh through different open approaches and with alternative modifications have reported varying success rates . tekkis et al described a modified thorlakson technique that incorporated an incomplete circumferential mesh to reinforce fascial repair . this technique was performed on 5 patients in whom , in a short - term follow - up of less than 2 years , ph had not recurred , though 40% experienced complications of hematoma or prolapse . saclarides et al reviewed their experience with in situ y - split mesh repair and noted an 11% recurrence and complications of hematoma and delayed resumption of oral intake . stelzner et al reported a similar recurrence rate of 15% after repair of paracolostomy hernia with prosthetic nonslit mesh , after a mean follow - up of 3.5 years . longman and thompson showed promising results with a modification of in situ split mesh repair that included tacking back the triangular flaps from the opening created in the mesh . over a median follow - up period of 30 months , no recurrences had developed and only one complication of superficial wound breakdown had occurred . alternatives to local repair , such as transposition , or resiting of the stoma with repair of the fascial defect , are also not without challenge . failure rates following stoma resiting are reported to range from 15% to 57% , rates that are improved compared with those associated with fascial repair , but many of which are associated with higher complication rates , up to 88% . regardless of type of ph repair , no one method has emerged as ideal with which to set the standard of surgical treatment . in recent years , expanding knowledge of and experience with laparoscopic techniques have heralded innovative approaches to this dilemma . results of the first laparoscopic approaches to ph repair with mesh reinforcement were case reports published in 1998 and 1999 , by porcheron et al and bickel et al , respectively . a small case series of 4 patients undergoing laparoscopic ph repair with mesh reinforcement was published by kozlowski et al in 2001.operative time was long , an average of 4.3 hours , but no recurrences had developed at 2 months to 33 months . leblanc et al went on to incorporate the use of overlapping prosthesis in a comparison of single mesh onlay with double - patch techniques of 2 different types versus 2 identical pieces of eptfe mesh . of 7 onlay repairs , none recurred ; of 5 2-patch repairs , one parastomal hernia recurred . twenty - five percent of patients experienced complications of intraoperative enterotomy , ileus , seroma , obstruction , and death . in 2007 , hansson et al performed a multi - center trial using funnel - shaped eight cases were converted due to dense adhesions or contamination secondary to full - thickness bowel injury . two patients ( one conversion secondary to bowel injury ) required mesh removal secondary to mesh infection or abscess . in our study , we sought to review our experience with ph repair . early in our laparoscopic experience more recent laparoscopic repairs ( subsequent 7 patients ) were based on a modified sugarbaker technique through which 3 essential elements were incorporated : intraperitoneal placement of nonslit mesh , lateralization of the bowel limb between the mesh and abdominal wall , and fixation of transfascial sutures no more than 5 cm apart . the first was a full thickness small bowel injury necessitating conversion to an open procedure . the second was a serosal thermal injury to the colon that was repaired by oversewing the injury laparoscopically , thus avoiding conversion . the 3 lap patients who developed wound infections or parastomal cellulitis recovered with antibiotics and wound care . no patient in our lap group had mesh infection or mesh erosion into the bowel . it can also be argued that bowel lateralization following mesh placement can potentially lead to bowel obstruction . however , in our study , no postoperative bowel obstructions were seen , suggesting that securing the intestine against the abdominal wall does not cause obstruction and that a slit may be unnecessary . overall , our laparoscopic recurrence rate was 33% . early in our experience , we used a keyhole mesh technique , resulting in a 67% recurrence rate . we then changed our approach to utilize a modified sugarbaker technique and have since experienced a recurrence rate of 29% . we believe that this improvement in recurrence rate is due both to increasing experience and a better technique . our experience with the laparoscopic modified sugarbaker technique in the repair of ph continues to increase . this approach provides an alternative strategy to open repair with which to treat the common and difficult problem of ph .

background / objectives : despite multiple options for operative repair of parastomal hernia , results are frequently disappointing . we review our experience with parastomal hernia repair.methods:a retrospective chart review was performed on all patients with parastomal hernia who underwent lap or open repair at our institution between 1999 and 2006 . information collected included demographics , indication for stoma creation , operative time , length of stay , postoperative complications , and recurrence.results:twenty-five patients who underwent laparoscopic or open parastomal hernia repair were identified . laparoscopic repair was attempted on 12 patients and successfully completed on 11 . thirteen patients underwent open repair . operative time was 17210.0 minutes for laparoscopic and 13719.1 minutes for open cases ( p=0.14 ) . lengths of stay were 3.10.4 days ( laparoscopic ) and 5.10.8 days ( open ) , p=0.05 . immediate postoperative complications occurred in 4 laparoscopic patients ( 33.3% ) and 2 open patients ( 15.4% ) , p=0.38 . parastomal hernia recurred in 4 laparoscopic patients ( 33.3% ) and 7 open patients ( 53.8% ) after 13.94.5 months and 21.44.3 months , respectively , p=0.43.conclusion : laparoscopic modified sugarbaker technique in the repair of parastomal hernia affords an alternative to open repair for treating parastomal hernia .

INTRODUCTION MATERIALS AND METHODS Data Collection OPERATIVE TECHNIQUE Laparoscopic Repair With Slit or Nonslit Mesh Local Fascial Repair RESULTS DISCUSSION CONCLUSION

PMC4252925

atomic force microscope ( afm ) is considered an important and applicable method for ra assessment . rough surface of restorations with loss of contour will lead to high friction and results in high surface wear rate . however , no study was found that investigated the capacity of acidic soft drinks to produce microleakage or evaluating the surface roughness of the eroded tooth - colored restorations . hence , the aims of this study were to evaluate the effect of an acidic soft drink ( lemon juice ) on the surface roughness and microleakage of cavities restored with four types of widely used tooth - colored dental restorative materials . it is hypothesized that there are no changes in the surface roughness and marginal integrity of the eroded tooth - colored dental restorative materials . one hundred and sixty freshly extracted human molars , free from cracks , caries , and fillings were cleaned withultrasonic and hand scalers , and then kept in thymol solution ( 1% ) at room temperature . standardized box - shaped cavity preparations were made on the buccal surfaces of tooth samples with 5.0 mm in mesiodistal width , 3.0 mm in occlusogingival height , and 2.0 mm in depth , measured with periodontal probe . samples were randomly assigned to four groups : group a was restored with conventional glass ionomer restorative material ( ketac molar easymix , 3m - espe , germany ) ; group b with resin - modified glassionomer restorative material ( fuji ii lc , gc corporation , japan ) ; group c with nanoionomer restorative material ( ketac n100 , 3m - espe , usa ) ; and group d with microhybridcr ( filtek z250 , 3m - espe , usa ) . all restorative materials were placed according to manufacturers instructions , and the details of those materials are shown in table 1 . in group a ; ketac molar easymix liquid was applied to the cavity for 10 s , rinsed and dried with cotton pellet , prior to application of ketac molar easymix . in group b ; gc dentin conditioner ( gc corporation , japan;10% polyacrylic acid ) was applied to the cavity for 10 s , rinsed , and dried . fuji ii lc capsule was triturated , and then injected into the cavity preparation , light - cured for 20 s. fuji coat lc ( gc corporation , japan ) was applied to the restoration and light - cured for 10 s. in group c ; gc dentin conditioner was applied , followed by ketac n100 primer ( 3 m espe , usa ) which was light - cured for 10 s. ketac n100 pastes were mixed , and then applied to the preparation and light - cured for 20 s. for group d ; the cavity was acid - etched with scotchbond etchant ( 3 m espe , usa ) and bonded with adper single bond adhesive ( 3 m espe , usa ) before placement of filtek z250 , which was then light - cured for 40 s. all light - curing procedures were performed using light - cure unit ( elipar free light 2 led , 3m - espe , usa ) with a light intensity of 1123 mw / cm . the output from the curing device was validated using a visible curing light meter ( cure rite / caulk ) . all restorations were finished and polished after 24 h using sof - lex ( 3m - espe , usa ) discs . each group was further subdivided into study and control groups ( n = 20 ) . lemon juice purchased from local grocery stores with ph 2.79 measured using an analog ph meter ( hanna microprocessor ph meter , usa ) was used in the study . all areas of the teeth were covered with two coats of acid resistant nail polish except the restoration and 1 mm rim of the tooth structure around . the control samples were kept in deionized distilled water ( ddw ) and were tested for surface roughness at baseline using afm . all study group samples were immersed in the lemon juice at room temperature for 24 h , then washed with ddw and dried with cotton pellets , then immersed in 2% methylene blue dye solution at room temperature for 24 h , removed , and rinsed under running water . each sample was sectioned longitudinally at the middle in a buccolingual direction using a diamond disc ( exakt hard material cutter , germany ) under water spray to give two equal halves ( a and b ) . segment a was once more sectioned to provide a 1.0 mm thickness segment for the surface roughness test ( ra ) , while segment b was used for the microleakage measurement . all samples for ra test were embedded in cylindrical wax block ( collegewax metrowax , england ) , and scanned under afm ( ambios technology inc , usa ) in its contact mode except forketac molars which were measured using profilometer ( s3000 mitutoyo , japan ) . the degree of marginal leakage was evaluated by the penetration of the dye stain from the occlusal and gingival cavosurface margins to the base of the cavity preparation . each specimen was viewed under a stereomicroscope ( leica imaging system , leica , uk ) at 20 magnification by one examiner and photographs taken . the scoring criteria were : 0- no evidence of dye penetration at tooth / restoration interface . data were analyzed with statistical package for social sciences ( spss ) version 18.0 ( spss inc , 2010 ) using mann - whitney and kruskal - wallis statistical tests of the surface roughness ( ra ) and microleakage , with a significance level set at p < 0.05 . each sample was sectioned longitudinally at the middle in a buccolingual direction using a diamond disc ( exakt hard material cutter , germany ) under water spray to give two equal halves ( a and b ) . segment a was once more sectioned to provide a 1.0 mm thickness segment for the surface roughness test ( ra ) , while segment b was used for the microleakage measurement . all samples for ra test were embedded in cylindrical wax block ( collegewax metrowax , england ) , and scanned under afm ( ambios technology inc , usa ) in its contact mode except forketac molars which were measured using profilometer ( s3000 mitutoyo , japan ) . the degree of marginal leakage was evaluated by the penetration of the dye stain from the occlusal and gingival cavosurface margins to the base of the cavity preparation . each specimen was viewed under a stereomicroscope ( leica imaging system , leica , uk ) at 20 magnification by one examiner and photographs taken . the scoring criteria were : 0- no evidence of dye penetration at tooth / restoration interface . data were analyzed with statistical package for social sciences ( spss ) version 18.0 ( spss inc , 2010 ) using mann - whitney and kruskal - wallis statistical tests of the surface roughness ( ra ) and microleakage , with a significance level set at p < 0.05 . there was a significant difference in the surface roughness of ketac molar , fuji ii lc , and ketac n100 ( p < 0.001 ) , while there was no difference in filtek z250 ( p = 0.053 ) , when the study groups were compared to the control groups [ table 2 and figure 1 ] . comparison among study groups showed significant difference between the groups ( p < 0.001 ) . whitney test revealed that ketac molar easymix exhibited the highest ra among the groups , followed byfuji ii lc , ketac n100 , withfiltek z250 being the least ( p < 0.001 ) . comparison of median ( iqr ) of surface roughness ( nm ) between study and control groups of eroded tooth - colored dental restorative materials afm photographs . ( a ) ketac molar before immersion ( b ) fuji ii lc before immersion . ( f ) filtek z250 before immersion.(g ) filtek z250 after immersion in the acidic drink . afm = atomic force microscope the degree of intraexaminer reproducibility of measurements , that is , intra class correlation coefficient ( icc ) was close to 1.00 , indicating high reliability . there were no significant differences in microleakage of ketac molar easymix and fuji ii lc after exposure to the acidic drink ( on both occlusal and gingival margins p > 0.05 ) . in ketac n100 , no significant difference was demonstrated at the occlusal margin ( p > 0.05 ) , but there was a significant difference at the gingival margin ( p = 0.029 ) . in filtek z250 samples ; there was a significant difference in microleakage occlusally ( p = 0.035 ) , but no difference gingivally ( p > 0.05 ) [ table 3 ] . figure 2 shows the microleakage of fuji ii lc in control and study groups . comparison of median ( iqr ) of microleakage scores between study and control groups of eroded tooth - colored dental restorative materials stereomicroscope photographs showing microleakage measurement . ( b ) fuji ii lc after immersion in theacidic drink comparison among study groups showed that ketac molar easymix samples exhibited the highest microleakage scores , followed by filtek z250 and ketac n100 , while the least was demonstrated in fuji ii lc ( p < 0.001 ) . no statistical significant difference was observed between occlusal and gingival margins in ketac molar easymix ( p = 0.524 ) and ketac n100 ( p = 0.906 ) , while the differences were observed in both fuji ii lc ( p = 0.047 ) and filtek z250 ( p = 0.008 ) . there was a significant difference in the surface roughness of ketac molar , fuji ii lc , and ketac n100 ( p < 0.001 ) , while there was no difference in filtek z250 ( p = 0.053 ) , when the study groups were compared to the control groups [ table 2 and figure 1 ] . comparison among study groups showed significant difference between the groups ( p < 0.001 ) . whitney test revealed that ketac molar easymix exhibited the highest ra among the groups , followed byfuji ii lc , ketac n100 , withfiltek z250 being the least ( p < 0.001 ) . comparison of median ( iqr ) of surface roughness ( nm ) between study and control groups of eroded tooth - colored dental restorative materials afm photographs . ( a ) ketac molar before immersion ( b ) fuji ii lc before immersion . ( f ) filtek z250 before immersion.(g ) filtek z250 after immersion in the acidic drink . afm = atomic force microscope the degree of intraexaminer reproducibility of measurements , that is , intra class correlation coefficient ( icc ) was close to 1.00 , indicating high reliability . there were no significant differences in microleakage of ketac molar easymix and fuji ii lc after exposure to the acidic drink ( on both occlusal and gingival margins p > 0.05 ) . in ketac n100 , no significant difference was demonstrated at the occlusal margin ( p > 0.05 ) , but there was a significant difference at the gingival margin ( p = 0.029 ) . in filtek z250 samples ; there was a significant difference in microleakage occlusally ( p = 0.035 ) , but no difference gingivally ( p > 0.05 ) [ table 3 ] . figure 2 shows the microleakage of fuji ii lc in control and study groups . comparison of median ( iqr ) of microleakage scores between study and control groups of eroded tooth - colored dental restorative materials stereomicroscope photographs showing microleakage measurement . ( b ) fuji ii lc after immersion in theacidic drink comparison among study groups showed that ketac molar easymix samples exhibited the highest microleakage scores , followed by filtek z250 and ketac n100 , while the least was demonstrated in fuji ii lc ( p < 0.001 ) . no statistical significant difference was observed between occlusal and gingival margins in ketac molar easymix ( p = 0.524 ) and ketac n100 ( p = 0.906 ) , while the differences were observed in both fuji ii lc ( p = 0.047 ) and filtek z250 ( p = 0.008 ) . after exposure to the acidic drinks , there was a significant change in the ra of ketac molar easymix which exceeded the afm limits , therefore , these samples were measured with profilometer . these changes are due to the dissolution of cement matrix and leaching of the principal forming components ( f , sr , al , ca , and na ) leaving the fillers protruded from the surface . the larger filler particles of this material ( 2.8 m in average ) could be considered as an additional factor for higher ra and it comes in agreement with a previous study . the ra values of fuji ii lc were significantly lower than ketac molar easymix , which may be due to its smaller filler size ( 0.02 - 0.04 m ) . the incorporation of resin has added resin polymerization setting reaction , making the material mature faster . the ra values of ketac n100 were the lowest among all gics tested in this study , due to its nanosized filler of 0.005 - 0.025 m . furthermore , the filler loading is approximately 69% ( by weight ) and all of the nanofillers are further surface - modified with methacrylate silane coupling agents that might be the influencing factor for better interaction between nanofillers and matrix . the ra values of filtek z250 did not differ significantly after exposure to the acidic drink . the resin matrix of filtek z250 is a combination of difunctional monomers bisphenol - glycidyl methacrylate ( bis - gma ) , ethoxylated bisphenol a glycol dimethacrylate ( bis - ema ) , urethane dimethacrylate ( udma ) , triethylene glycol dimethacrylate ( tegdma ) . tegdma decreases the surface softening by acids and increases the degree of polymerization of resin - based materials improving their physical properties , thereby minimizing their abrasion rate . precoating the silica particles with silane coupling agents had resulted in a very strong coherence between these particles and organic matrix which helps against hydrolytic degradation . however , this result is in contrast with the findings of the surface roughness of composites simulated with toothbrush abrasion . for microleakage , there were no significant changes in ketac molar easymix and fuji ii lc at either margin , as well as at the occlusal margin of ketac n100 after exposure to acidic drinks . however , ketac molar easymix showed high microleakage scores both in the control and study groups . the possible explanation for fuji ii lc and ketac n100 could be the two - fold mechanism of bonding for gics ; chemically via ionic interaction of the carboxyl groups of the polyalkenoic acid with calcium ions of hydroxyapatite that remained attached to the collagen fibrils and second is the micromechanical bonding via application of polyalkenoic acid that removes the smear layer and exposes collagen fibrils up to about 0.5 - 1m depth . however , there was a significant difference demonstrated at the gingival margins of ketac n100 samples , which could be attributed to the presence of resin which causeshigher polymerization shrinkage and also due to their superficial mechanical interlocking . microleakage of filtek z250 was higher at occlusal margin after exposure to the acidic drink . there was a possibility of erosion of enamel and subsequent loss of the adhesive material resulting in higher microleakage levels . polymerization shrinkage can also create a gap at tooth / restoration interface resulting in bond failure and subsequent microleakage . the difference of microleakage at the gingival margins of filtek z250 was not significant . here , the major tooth substrate is dentin , which has the ability to buffer the acidic effects and prevent demineralization , thereby preserving the marginal integrity . ketac molar easymix exhibited the highest microleakage scores when comparison was made among study groups . the slow setting mechanism of conventional gic ( acid - base reaction ) may result in the delay of material adhesion to tooth structure . the lowest microleakage was observed in fuji ii lc samples , which could be due to resin incorporation and the polymerization reaction which is considered as dominant reaction . similar results were found in a study , but another study found contrasting results . in fuji ii lc , microleakage was higher gingivally , where dentin represented the major tooth substrate to which the material was bonded . since dentin contains a substantial proportion of water and organic material , the adhesion of a hydrophilic material like fuji ii lc ( due to hydroxyethyl methacrylate ( hema ) incorporation ) could have resulted in water sorption , swelling , weakening of the restorations , and resulting in gaps formation and changed markedly on exposure to moisture . additionally , dentin is more porous , which could have enhanced the permeability to dye penetration compared to enamel . pretreatment of the prepared cavities with ketac n100 primer , where copolymer of acrylic / itaconic acid dissolved in hema and water resulted in partial demineralization of both enamel and dentin . the tooth tissue is easier to be wetted by the restorative material which might improve the adhesion , as supported by a previous study . higher microleakage demonstrated gingivally in filtek z250 could be due to the fact that the gingival margins were located further in dentin . furthermore , the collagen content of dentin is higher than enamel ( 18% ) which is normally inaccessible , owing to the surrounding hydroxyapatite crystals . this study agrees with a study where microleakage was found to be more cervically than occlusally in cr restorations . use of ph cycling or toothbrush abrasion cycling should be used in future studies to directly extrapolate the findings of the study from a clinical standpoint . use of ph cycling or toothbrush abrasion cycling should be used in future studies to directly extrapolate the findings of the study from a clinical standpoint . by the limitation of this study , all gic restorative materials were eroded after exposure to the acidic drink , where ketac molar showed the highest surface roughness , followed by fuji ii lc , and minimum in ketac n100 . however , there was no significant difference in surface roughness of filtek z250 , which shows that it is stable in an acidic environment . fuji ii lc showed the leastmicroleakage followed by ketac n100 and filtek z250 , while the worst material was the conventional gic ( ketac molar easymix ) . in general , all materials showed more microleakage at the gingival margins than occlusal margins .

objectives : to investigate the effect of acidic solution on surface roughness and microleakage of tooth - colored restorative materials.materials and methods : a 160 box - shaped cavities were prepared on the buccal surfaces of 160 human molars , and assigned to four groups : group a restored with ketac molar easymix , group b with fuji ii lc , group c with ketac n100 , and group d with filtek z250 , and subdivided into study and control groups ( n = 20 ) . study groups were immersed in lemon juice ( ph = 2.79 ) for 24 h , whilst controlgroups in deionized distilled water . all samples were immersed in 2% methylene blue dye , sectioned into two equal halves for surface roughness , and microleakage tests . data were analyzed using mann whitney and kruskal wallis tests at p < 0.05.results:there was a significant difference in surface roughness of ketac molar , fuji ii lc , and ketac n100 . no significant difference was found in microleakage of ketac molar and fuji ii lc ; however , there were significant differences in the gingival margin of ketac n100 , and the occlusal margin of filtek z250.conclusions:all glass ionomer cements were eroded after exposure to the acidic drink . filtek z250 and ketac molar easymix showed more microleakage . all materials showed more microleakage at the gingival margins .

INTRODUCTION MATERIALS AND METHODS Assessment of surface roughness and marginal integrity RESULTS Surface roughness Microleakage DISCUSSION Suggestion for future research CONCLUSIONS

PMC4980926

all the experiments were performed after getting the approval from ethical committee of institute ( dean/13 - 14/caec/189 dated 09 - 05 - 2013 ) . experiments were performed on healthy male albino rats ( charles - foster strain ) , weighing between 200 and 300 g. the animals were kept in the 12:12 h light / dark cycle and were provided with ad libitum animal food ( hindustan lever ltd . ) and water . animals were anesthetized with urethane ( merck , germany ) , with an initial dose of 1.5 g / kg body weight intraperitoneally and was maintained by injecting the urethane as required . the trachea was dissected and exposed after a mid - line incision over the neck . a transverse cut was made between the tracheal rings , and a polyethylene tube of appropriate diameter was inserted and secured firmly . tracheal secretions were aspirated from time to time by gentle suction through a fine polyethylene tube . an incision was made on the femoral triangle along the course of the femoral artery . the femoral artery was dissected and skeletonized by clearing the tissues and fascial attachments by blunt dissection . the femoral vein and nerve the cannula was attached with a syringe , containing freshly prepared heparinized saline ( 20 iu / ml ) . a small nick was made in the artery near the proximal end then the cannula was inserted and secured firmly with thread . the cannula was in turn connected through a three - way stopcock to a statham strain gauge pressure transducer ( biodevices , ambala ) . after the cannulation of the proximal part of the femoral artery , the cannulation was done in the distal part of the same artery to inject the venom / drugs / saline in a local segment of the vessel . the pressure transducer was filled with the heparinized saline and the cannula was connected to it . the galvanometer deflections were recorded on a chart paper with the help of a pen . the mean arterial pressure ( map ) the skin over the xiphisternum was secured with the thread and was attached to a force - displacement transducer . heart rate ( hr ) was calculated manually from r - r intervals of the ecg . after the surgical procedures , animals were allowed to stabilize for 30 min followed by the initial recording of blood pressure ( bp ) , ecg , and rr . a volume of 0.10 ml of normal saline was injected in the peripheral end of the femoral artery and the cardiorespiratory parameters were recorded at the interval of 5 min up to 15 min as initial recording . this was followed by the injection of venom ( 1.0 mg / kg ) in the peripheral end of the same femoral artery , and the cardiorespiratory parameters were recorded at the interval of 5 min up to 60 min . stock solution ( 2 mg / ml ) of bt venom was prepared in distilled water and refrigerated . bt venom ( 1 mg / kg ) was used to stimulate the perivascular nociceptors as it produces optimal responses . , hyderabad , india . des - arg ( b1 receptor antagonist ) and hoe-140 ( b2 receptor antagonist ) were obtained from sigma chemical company , st . all the chemicals / venom / saline was injected in the peripheral end of the femoral artery . venom - induced responses were considered as the venom only response . in the subsequent two groups , venom was injected after pretreatment with des - arg ( b1 receptor antagonist ) and hoe-140 ( b2 - receptor antagonist ) . bp , ecg , and rr were recorded at the interval of 5 min up to 60 min . the volume of all the injectables was kept at 0.10 ml and the room temperature was maintained at 25c 0.5c throughout the experiment . in the second group of experiments , i.a . injection of des - arg ( 10 ng / kg ) was given , and the cardiorespiratory parameters were recorded at the interval of 5 min up to 15 min . further , the venom was injected in the same peripheral end of the femoral artery , and the cardiorespiratory parameters were recorded at the interval of 5 min up to 60 min . in the third group of experiments , the animals were pretreated with hoe-140 ( 10 ng / kg ) which was followed by injection of venom and the cardiorespiratory changes were recorded as above . the values at a given time were pooled and were presented as a mean standard error of mean ( sem ) ; the map , hr , and rr responses before venom were taken as initial responses . the comparisons of various groups were made by using the two - way analysis of variance ( anova ) followed by student 's t - test wherever required . all the experiments were performed after getting the approval from ethical committee of institute ( dean/13 - 14/caec/189 dated 09 - 05 - 2013 ) . experiments were performed on healthy male albino rats ( charles - foster strain ) , weighing between 200 and 300 g. the animals were kept in the 12:12 h light / dark cycle and were provided with ad libitum animal food ( hindustan lever ltd . ) and water . animals were anesthetized with urethane ( merck , germany ) , with an initial dose of 1.5 g / kg body weight intraperitoneally and was maintained by injecting the urethane as required . the trachea was dissected and exposed after a mid - line incision over the neck . a transverse cut was made between the tracheal rings , and a polyethylene tube of appropriate diameter was inserted and secured firmly . tracheal secretions were aspirated from time to time by gentle suction through a fine polyethylene tube . an incision was made on the femoral triangle along the course of the femoral artery . the femoral artery was dissected and skeletonized by clearing the tissues and fascial attachments by blunt dissection . the femoral vein and nerve the cannula was attached with a syringe , containing freshly prepared heparinized saline ( 20 iu / ml ) . a small nick was made in the artery near the proximal end then the cannula was inserted and secured firmly with thread . the cannula was in turn connected through a three - way stopcock to a statham strain gauge pressure transducer ( biodevices , ambala ) . after the cannulation of the proximal part of the femoral artery , the cannulation was done in the distal part of the same artery to inject the venom / drugs / saline in a local segment of the vessel . the pressure transducer was filled with the heparinized saline and the cannula was connected to it . the galvanometer deflections were recorded on a chart paper with the help of a pen . the mean arterial pressure ( map ) the skin over the xiphisternum was secured with the thread and was attached to a force - displacement transducer . the respiratory movements were recorded on a chart recorder via a bridge amplifier . respiratory rate ( rr ) was calculated from these recordings . the electrocardiographic ( ecg ) potentials were recorded on a chart recorder . heart rate ( hr ) was calculated manually from r - r intervals of the ecg . after the surgical procedures , animals were allowed to stabilize for 30 min followed by the initial recording of blood pressure ( bp ) , ecg , and rr . a volume of 0.10 ml of normal saline was injected in the peripheral end of the femoral artery and the cardiorespiratory parameters were recorded at the interval of 5 min up to 15 min as initial recording . this was followed by the injection of venom ( 1.0 mg / kg ) in the peripheral end of the same femoral artery , and the cardiorespiratory parameters were recorded at the interval of 5 min up to 60 min . stock solution ( 2 mg / ml ) of bt venom was prepared in distilled water and refrigerated . bt venom ( 1 mg / kg ) was used to stimulate the perivascular nociceptors as it produces optimal responses . hyderabad , india . des - arg ( b1 receptor antagonist ) and hoe-140 ( b2 receptor antagonist ) were obtained from sigma chemical company , st . in this study , 18 animals were used in three different groups ( n = 6 ) . all the chemicals / venom / saline was injected in the peripheral end of the femoral artery . venom - induced responses were considered as the venom only response . in the subsequent two groups , venom was injected after pretreatment with des - arg ( b1 receptor antagonist ) and hoe-140 ( b2 - receptor antagonist ) . bp , ecg , and rr were recorded at the interval of 5 min up to 60 min . the volume of all the injectables was kept at 0.10 ml and the room temperature was maintained at 25c 0.5c throughout the experiment . in the second group of experiments , i.a . injection of des - arg ( 10 ng / kg ) was given , and the cardiorespiratory parameters were recorded at the interval of 5 min up to 15 min . further , the venom was injected in the same peripheral end of the femoral artery , and the cardiorespiratory parameters were recorded at the interval of 5 min up to 60 min . in the third group of experiments , the animals were pretreated with hoe-140 ( 10 ng / kg ) which was followed by injection of venom and the cardiorespiratory changes were recorded as above . the values at a given time were pooled and were presented as a mean standard error of mean ( sem ) ; the map , hr , and rr responses before venom were taken as initial responses . the comparisons of various groups were made by using the two - way analysis of variance ( anova ) followed by student 's t - test wherever required . in our earlier reports , we have shown that 1 mg / kg of venom produced optimal responses on cardiorespiratory parameters . therefore , this concentration was chosen as a tool for the elicitation of vasosensory reflex responses in this study . individual and mean sem values of rr , map , and hr are given after the injection of venom ( 1 mg / kg ) only and after the injection of venom in des - arg and hoe-140 pretreated groups [ figures 1 and 2 ] and the original tracings are also shown in the figures . des - arg ( 10 ng / kg ) pretreatment attenuated cardiovascular changes ( mean arterial pressure and heart rate ) induced by intra - arterial injection of venom . original tracings of respiration ( resp ) , ecg and blood pressure are shown in the top left panel . the dotted line at 0 indicates the time of injection of venom ( 1 mg / kg ) . the horizontal line indicates 5 s for resp and ecg ; 50 s for blood pressure . the mean standard error of mean values from six experiments are shown in line graphs . indicates p < 0.05 ( two - way analysis of variance ) as compared to venom only group from des - arg pretreated group ( des - arg + venom ) hoe-140 ( 10 ng / kg ) pretreatment does not alter the cardiorespiratory responses significantly after intra - arterial injection of venom . original tracings of respiration ( resp ) , ecg and blood pressure are shown in the top left panel . the dotted line at 0 indicates the time of injection of venom ( 1 mg / kg ) . the horizontal line indicates 5 s for resp and ecg ; 50 s for blood pressure . the mean standard error of mean values from six experiments is shown in line graphs . indicates p < 0.05 ( two - way analysis of variance ) as compared to venom only group from hoe-140-pretreated group ( hoe-140 + venom ) an increase in the rate and depth of respiration was seen immediately after the administration of venom in venom only group ( rr from 74 5.1 to 104 10.8/min ) , and it was about 40% of the initial . rr was then decreased below the initial level within 5 min ( 45 10.9/min ) . subsequently , a gradual sustained increase in rr was noticed up to 60 min [ figure 1 ] . bt venom - induced respiratory changes in des - arg pretreated animals were similar to the venom only responses during the entire period of observation [ figure 1 ] . administration of venom in venom only group produced an immediate fall in map ( from 90 4.9 to 87 6.7 mm hg ) followed by a rise and reached the peak ( 131 6.8 mm hg ) within 5 min . subsequently , it decreased but still remained above the initial level [ figure 1 ] . in des - arg pretreated animals , the immediate depressor response was accentuated as compared to venom only group ( from 80 12 to 68.6 10 mm hg ) , but the pressor response was attenuated markedly up to 60 min [ figure 1 ] . the responses were significantly different from the corresponding values in venom only group ( p < 0.05 , two - way anova ) . there was no change in hr immediately after the administration of venom in venom only group . within 5 min , hr began to decrease and the decrease was maximal at 30 min ( 151 18.9 beats / min ) , which remained nearly same up to 60 min [ figure 1 ] . in des - arg pretreated animals , the venom - induced decrease in hr began earlier and reached maximal level at 10 min ( from 320 4 to 175 32.6 beats / min ) . the responses were significantly different from the venom only group [ figure 1 ; p < 0.05 , two - way anova ] . in hoe-140 pretreated group , the venom - induced changes in rr were not statistically different from the venom only group but showed an increasing trend in the rate of respiration in late phase after 30 min [ figure 2 ] . the immediate depressor response was accentuated as compared to venom only group ( from 83 10 to 65 13 mm hg ) and the pressor response was similar to venom only group up to 30 min . however , after 30 min , the pressure response was lower than the venom only group response [ figure 2 ] . the responses were significantly different from the corresponding values in venom only group from 30 to 60 min ( p < 0.05 , two - way anova ) . in hoe-140 pretreated animals , the decrease in hr began immediately after venom and the decrease was about 55% at 5 min ( from 302 31 to 135 24.5 beats / min ) , which remained at the same level up to 60 min [ figure 2 ] . the responses up to 15 min were significantly different from the venom only group an increase in the rate and depth of respiration was seen immediately after the administration of venom in venom only group ( rr from 74 5.1 to 104 10.8/min ) , and it was about 40% of the initial . rr was then decreased below the initial level within 5 min ( 45 10.9/min ) . subsequently , a gradual sustained increase in rr was noticed up to 60 min [ figure 1 ] . bt venom - induced respiratory changes in des - arg pretreated animals were similar to the venom only responses during the entire period of observation [ figure 1 ] . administration of venom in venom only group produced an immediate fall in map ( from 90 4.9 to 87 6.7 mm hg ) followed by a rise and reached the peak ( 131 6.8 mm hg ) within 5 min . subsequently , it decreased but still remained above the initial level [ figure 1 ] . in des - arg pretreated animals , the immediate depressor response was accentuated as compared to venom only group ( from 80 12 to 68.6 10 mm hg ) , but the pressor response was attenuated markedly up to 60 min [ figure 1 ] . the responses were significantly different from the corresponding values in venom only group ( p < 0.05 , two - way anova ) . there was no change in hr immediately after the administration of venom in venom only group . within 5 min , hr began to decrease and the decrease was maximal at 30 min ( 151 18.9 beats / min ) , which remained nearly same up to 60 min [ figure 1 ] . in des - arg pretreated animals , the venom - induced decrease in hr began earlier and reached maximal level at 10 min ( from 320 4 to 175 32.6 beats / min ) . the responses were significantly different from the venom only group [ figure 1 ; p < 0.05 , two - way anova ] . in hoe-140 pretreated group , the venom - induced changes in rr were not statistically different from the venom only group but showed an increasing trend in the rate of respiration in late phase after 30 min [ figure 2 ] . the immediate depressor response was accentuated as compared to venom only group ( from 83 10 to 65 13 mm hg ) and the pressor response was similar to venom only group up to 30 min . however , after 30 min , the pressure response was lower than the venom only group response [ figure 2 ] . the responses were significantly different from the corresponding values in venom only group from 30 to 60 min ( p < 0.05 , two - way anova ) . in hoe-140 pretreated animals , the decrease in hr began immediately after venom and the decrease was about 55% at 5 min ( from 302 31 to 135 24.5 beats / min ) , which remained at the same level up to 60 min [ figure 2 ] . the responses up to 15 min were significantly different from the venom only group ( p < 0.05 , student 's t - test for unpaired observations ) . in our previous studies , venom - induced vasosensory reflexes altering the cardiorespiratory parameters were demonstrated . the afferents were found to be present in the ipsilateral somatic nerves and in nerve plexuses around the blood vessels . the involvement of trpv1 and serotonin receptors ( 5ht3 ) in mediating the vasosensory reflex responses is also shown earlier . further , it has been shown that inflammatory mediators are involved in nociceptive processing at the perivascular nerve endings as the cardiorespiratory parameters of the reflex responses are blocked by the pg synthesis inhibitor . these mediators in turn modulate the trpv1 and serotonin receptors ( 5-ht3 ) to bring about the actions . bt venom contains serotonin , histamine , bk potentiating factor , peptide toxins , etc . bk is an inflammatory mediator and produces the actions via bk-1 ( b1 ) and bk-2 ( b2 ) receptors . the b1 receptors are normally dormant and are expressed after exposure to toxic chemicals / tissue injury products such as cytokines , substance p , neuropeptides , capsaicin , or after repeated exposure to bk . the b2 receptors , on the other hand , are involved in the production of inflammatory edema . local application of exogenous kinins produces pain by stimulating b2 receptors in human skin or in the rat tissues . however , our results suggest that b2 receptors mediate late phase of pressor responses and immediate phase of hr responses produced by venom . the edema sets in after a time period thus the delayed phase of the pressor responses may be mediated via this mechanism . our results also reveal that b2 receptors prevent a sudden decrease in hr produced by venom . the evidence suggests that b1 receptors control pg production locally and pgs are known hyperalgesic substances . the analgesic profile of b1 receptor antagonist resembles to that of nonsteroidal anti - inflammatory drugs , which are agents known to inhibit the fatty acid , cyclooxygenase necessary for pgs synthesis . thus , a chain of mediators ( cytokines , kinins , and eicosanoids ) may be involved in these reactions . the b1 receptors which are located on nonneural cells ( fibroblast , mast cells , endothelial cells , etc . ) produce pgs as a secondary mediator and in turn sensitize the nerve endings in the periphery . the b1 receptor antagonist pretreatment attenuated the venom - induced bp and hr responses [ figure 1 ] . our observation elsewhere with indomethacin is consistent for the attenuation by b1 receptor antagonist . on the respiratory responses , there was no statistical difference between b1/b2 receptors antagonist - treated groups as compared to venom only groups . however , the b2 receptor antagonist increased the rr in the late phase . this may be due to the fact that respiration is influenced by bp changes . thus , the difference in the respiratory responses in b1 and b2 receptor antagonist groups may be due to baroreflex driven increase in rr . further , it is known that the b1 receptors are expressed after latency as mentioned for the nociceptive actions and respiratory responses occurred earlier than their expressions . bt venom - induced vasosensory reflexes modulating cardiovascular parameters were mediated mainly via b1-kinin receptors and partially via b2-kinin receptors in anesthetized rats .

objective : intra - arterial injection of mesobuthus tamulus ( bt ) venom produces reflex vasosensory responses modulating cardiorespiratory parameters in albino rats . the present study was conducted to understand the role of kinin receptors in modulating vasosensory reflexes evoked by bt venom.materials and methods : in urethane - anesthetized rats , tracheostomy was performed to keep the airway patent . the femoral artery was cannulated proximally , as well as distally , to record the blood pressure ( bp ) and to inject the chemicals , respectively . electrocardiographic and respiratory excursions were recorded to compute the heart rate ( hr ) and respiratory rate ( rr ) . a group of animals was pretreated with saline / kinin receptor antagonists intra - arterially ( b1/b2 receptor antagonists ) before the injection of venom.results:after intra - arterial injection of bt venom ( 1 mg / kg ) , there was an immediate increase in rr , which reached to 40% within 30 s , followed by a decrease of 40% . further , there was sustained increase in rr ( 50% ) up to 60 min . the bp started to increase at 40 s , peaking at 5 min ( 50% ) , and remained above the initial level up to 60 min . the bradycardiac response started after 5 min which peaked ( 50% of initial ) at 25 min and remained at that level up to 60 min . in b1 receptor antagonist ( des - arg ) pretreated animals , venom - induced cardiovascular responses were attenuated ( by 2025% in mean arterial pressure and hr ) significantly but not in b2 receptor antagonist ( hoe-140 ) pretreated animals . either of the antagonists failed to alter the rr responses.conclusions:bt venom - induced vasosensory reflex responses modulating cardiovascular parameters are mediated via b1-kinin receptors in anesthetized rats .

Materials and Methods Animals and Anesthesia Dissection and Recordings Drugs and Solutions Experimental Protocol Analysis of Data and Statistics Results Effect of des-Arg on Venom-induced Cardiorespiratory Changes Effect of Hoe-140 on Venom-induced Cardiorespiratory Changes Discussion Conclusions Financial Support and Sponsorship Conflicts of Interest

PMC4500003

human posture is determined and maintained by muscle coordination , proprioception , equilibrioception , and the position and function of the joints1 . the ankle strategy that is applied to the ankle joints is an important2 component of maintaining postural adjustment and includes the proper use of hip joint muscles attached to the ankle joints and appropriate stepping strategies3 . in terms of mechanics , the maintenance of a sufficient range of motion , strength , and proprioception is crucial in providing shock absorption and balance control of movements in the lower extremities while walking , which are the primary functions of the ankle joints5 . functional balance is the ability to maintain a position and adjust posture while moving from one postural position to a different position or a functional motion , such as movement from a certain position to another position6 . the ankle joints are adjusted by the ground reaction force generated by the contractions of the gastrocnemius , peroneus longus , and tibialis anterior muscles7 . small changes to adjustments in balance are accomplished by the action of the muscles associated with the ankle joints ; the body movement around the ankle , acting like an inverted pendulum , corrects the position of the center of gravity2 . isometric , isotonic , and isokinetic exercises are used in resistance training for increasing muscle strength . isotonic exercises are auxotonic contractions in which , despite changes in muscle length , tension in the muscles remains constant and improves muscle strength8 . isokinetic exercises allow muscles to exert maximum strength within the range of all joint movements at a constant speed ; various exercise methods can be used to improve muscle strength9 . isometric exercises are popular and have been performed successfully by physiotherapists for rehabilitation . in particular , they can be adjusted for patients whose range of joint movements is limited , and they are useful when performed in a safe training environment10 . muscle fatigue , which is a hindrance factor during exercise , is defined as a condition that brings about a reduction in maximum voluntary force or power generated by a muscle or muscle group ; the muscle fatigue mechanism is a complex phenomenon in which the central and peripheral nervous systems generally work together11 . in order to evaluate the mechanisms of muscular strength production muscle movement begins with an electrical activity , and this signal is recorded by electromyography ( emg)8 . determining the level of muscle activity is a non - invasive method of studying the electrical and mechanical events of a single muscle excitation - contraction combination ; recording the electrical activity of muscles has been widely used in studies on muscle coordination , muscle contraction characteristics , motor unit recruitment , and the muscle firing rate12 . in previous studies of patients with knee osteoarthritis that compared isometric and isokinetic exercises , it was found that isokinetic exercises were much more effective than isometric exercises for improving muscle strength13 . yang and hwang14 reported that a six - week treatment of neurological physical therapy with afferent knee joint isokinetic exercises in patients with acute stroke and hemiplegia improved balance control and muscle strength in the lower extremities . in addition , an analysis of the muscle activities of volleyball players who received isometric training for six weeks found that there were significant differences in the improvement of muscle strength8 . after isokinetic exercises were performed for four weeks on the ankle joints of adults in their 20s , the subjects were asked to complete a one - leg stance on the same leg , and it was reported that the ankle muscle training helped to improve balance control . the plantar flexor of the ankle joint is considered to be the key regulator in standing15 , and it plays an important role16 in walking efficiency17 . in the current study , isotonic , isokinetic , and isometric exercises were performed on ankle joints to assess their effects on the muscle activity and balance control of the tibialis anterior , peroneus longus , and gastrocnemius muscles . the 30 participants in this study were students at a university in cheonan , republic of korea . the procedures of the experiment were explained to the students , who did not have any problems with the experiment and voluntarily agreed to participate . exclusion criteria included musculoskeletal or neurological diseases ; muscle strength training during the past six months18 ; joint or bone disorders or ankle sprains ; and tendon , fascia , or ligament inflammation19 . the exercises were divided into isotonic , isokinetic , and isometric exercises , and 10 students were randomly assigned to each of the three types of exercise groups . a body composition analyzer ( inbody720 , biospace o. ltd . , seoul , republic of korea ) was used to determine the general characteristics of the subjects . a functional rehab system ( primus rs , bte tech . , hanover , md , usa ) was employed to carry out the isotonic , isokinetic , and isometric exercises . a free emg system ( free emg , bts inc . , milan , italy ) measure the muscle activity of the subjects , and a computerized balance platform ( bt4 , hur labs oy . , tampere , finland ) was used to determine balance control . after being informed about the study and understanding the purpose and content of the experiment , each of the 30 participants signed a consent form . after measuring their physical characteristics , ten students were randomly assigned to each of the three exercise groups : isotonic , isokinetic , and isometric . before conducting the experiment , the dominant limb was identified by having the subjects kick a ball that had been placed in front of them . the limb that was used to kick the ball was identified as the dominant side20 in order to apply the exercise to the non - dominant side ; surface electrodes were then attached to the tibialis anterior , peroneus longus , and gastrocnemius muscles . the subjects were positioned in the physical rehabilitation equipment with their knees fully extended21 , and both sides of the hip joints and the knee joints were fixed into a neutral position using a strap . the exercises were performed after fitting the rotational axis of the equipment along the inner elongation of the anklebone with the foot secured in the isokinetic equipment22 . the isometric exercises were performed three times to induce muscle fatigue in the subjects for maximal voluntary isometric contraction ( mvic)24 . of the three mvics , the one with the highest peak torque was selected as the mvic15 . then the subjects were given verbal reinforcement to encourage them to exert maximum force during the isometric contractions24 . to familiarize the subjects with the experiment before performing the isotonic , isometric , or isokinetic exercises , submaximal contractions were performed five times as a pre - exercise that involved isokinetic exercises26 . there was a five - minute rest period after the pre - exercise to prevent the fatigue effect27 . the isotonic exercise group performed self - paced exercises28 at 50% resistance of mvic29 . ten repetitions were performed per set , followed by a rest ; three sets were performed , with a one - minute rest period between the sets to prevent fatigue30 . in the isokinetic exercise group , changes in the increase of muscle strength occurred according to isokinetic strength exercise implementation , which depends on the dynamic form of exercise , speed , and muscle contraction . in particular , low - velocity exercises generally increase muscle strength , while high - velocity exercises are mainly used for the recovery of muscle endurance . among the most frequently implemented angular velocities of 60 , 180 , and 300 degrees / sec in isokinetic exercises , the angular velocity of 60 degrees / sec was selected ; this velocity is mainly used for improving muscle strength in adults or athletes31 . the ankle was put in a neutral position32 , and the exercise intensity was set within the range of pain tolerance . three sets of ten repetitions of plantar flexion and dorsiflexion were performed , followed by a rest33 . there was a one - minute rest period between sets to prevent the fatigue effect32 . the isometric strength training group used mvic resistance to perform plantar flexion ; dorsiflexion was performed at 70% of mvic resistance21 . plantar flexion and dorsiflexion of the ankle were sustained for 15 seconds33 per repetition for a total of three repetitions per set , and each set was followed by a five - second rest period30 ; three total sets were performed . there was a one - minute rest period between each set to prevent fatigue32 . in order to enable maximum performance , strong verbal reinforcement was provided during all tests , along with auditory and visual feedback15 . to generate the emg data , electrodes were attached to the surface at the state of standard maximum contraction to avoid technical errors that might affect the tests . to minimize errors caused by skin impedance and to keep the impedance below 5 k , the skin was prepared first by exfoliation with sandpaper to remove dead skin cells and debris and then disinfection with sterile alcohol23 . all active electrodes ( carbon electrode ; 3 m , ) were attached parallel to the direction of the muscle fiber at 2-cm intervals34 . a 1-khz sampling rate , 500-hz low - pass filter , and 20-hz high - pass filter were used to measure emg . the emg signals of each muscle measured were treated with the value of root mean square . data for the emg measurements were obtained by eliminating the beginning and end of the emg interval . to test the subjects balance ability , a one - leg stance with eyes open was performed using a computerized balance platform . while standing barefoot with both arms comfortably at their sides35 , the subjects were asked to bend one leg along the anterior - posterior axis of the balance platform at 30 and stand on the opposite leg , to maintain as stable a stance as possible36 with their eyes open for 30 seconds37 , and to gaze at a point on a monitor 65 cm away35 . an oral countdown from 4 seconds to 1 second was given to start , and the word data were obtained by taking the mean value of the measurements , which were repeated twice . the kolmogorov - smirnov test was used to demonstrate a normal distribution , and the levene f - test was used to verify the homogeneity of the subjects . multivariate analysis of variance ( manova ) was used to compare the difference in muscle activity and balance control in the tibialis anterior , medial gastrocnemius , and peroneus longus , depending on the group ( isotonic , isokinetic , or isometric exercise group ) and time ( pre- or post - exercise ) . when there was a significant difference , this study was approved by the institutional review board of namseoul university ( cheonan , korea , nsu-140528 - 1 ) . fifteen of the subjects were male , and 15 were female ; 28 of the subjects were right - side dominant , and two were left - side dominant . information regarding the subjects , such as age , height , weight , and body mass index , is provided in table 1table 1.subject characteristicsgroupnage ( years)height ( cm)weight ( kg)bmi ( kg / m)msdmsdmsdmsdisotonic1020.31.7170.29.970.014.623.92.6isokinetic1020.21.8165.59.965.820.223.64.8isometric1020.11.4166.911.568.518.224.13.9msd : means standard deviation ; bmi : body mass index . msd : means standard deviation ; bmi : body mass index the changes in muscle activity before and after the tests are shown in table 2table 2.comparison of pre- and post - test muscle electromyography ( mv)variablegrouppre - testpost - testmsdmsdnon - ta * ( support by non-)isotonic0.350.300.330.14isokinetic1.471.521.180.83isometric0.520.530.420.18non - gcm * ( support by non-)isotonic1.211.371.862.17isokinetic2.132.312.812.40isometric0.760.540.732.17non - pl * ( support by non-)isotonic0.831.010.940.86isokinetic1.591.261.781.76isometric0.760.540.730.52dom - gcm * ( support by dom-)isotonic 0.570.591.372.14isokinetic2.351.622.422.25isometric0.610.370.760.50dom - pl ( support by dom-)isotonic 0.620.581.010.83isokinetic1.400.901.871.23isometric0.820.751.040.69*p<0.05 ; isotonic ; isokinetic ; isometric ; non- : non - dominant side ; dom- : dominant side ; ta : tibialis anterior ; gcm : gastrocnemius ; pl : peroneus longus . the statistical analysis indicated that no interaction between group and time could be observed in any of the subjects . the main effects according to group could be observed in the non - dominant tibialis anterior , gastrocnemius , and peroneus longus muscles when the non - dominant limb was weight bearing , and in the peroneus longus and gastrocnemius muscles when the dominant limb was weight bearing . a multiple comparison analysis showed that there were significant differences among the isokinetic , isotonic , and isometric exercise groups in the non - dominant tibialis anterior and between the isokinetic and isometric exercise groups in the non - dominant gastrocnemius and peroneus longus . in addition , there were significant differences among the isokinetic , isotonic , and isometric exercise groups in the dominant gastrocnemius and peroneus longus . * p<0.05 ; isotonic ; isokinetic ; isometric ; non- : non - dominant side ; dom- : dominant side ; ta : tibialis anterior ; gcm : gastrocnemius ; pl : peroneus longus the changes in balance control according to group both before and after the tests are shown in table 3table 3.comparison of pre- and post - test balancevariablegrouppre - testpost - testmsdmsdarea ( mm ) ( support by non-)isotonic435.0139.1615.1244.1isokinetic389.3215.6515.6277.7isometric292.6138.1491.5280.5area ( mm ) ( support by dom-)isotonic399.1151.5495.0229.2isokinetic353.5119.8453.4161.6isometric349.2149.4470.2295.8velocity ( mm / s ) ( support by non-)isotonic11.54.012.23.0isokinetic12.33.713.43.6isometric11.42.816.313.8velocity ( mm / s ) ( support by dom-)isotonic11.73.211.12.6isokinetic12.32.814.46.2isometric12.03.514.78.5*p < 0.05 ; < 0.05 : compared with post - test ; non- : non - dominant side ; dom- : dominant side . no main effect according to group was observed , and the main effect of time on surface area was present only when the non - dominant and dominant sides were weight bearing . multiple comparison analysis of the significant differences in surface area showed that when the non - dominant side was weight bearing , there were significant differences in all three exercise groups ; when the dominant side was weight bearing , there were only significant differences in the isokinetic exercise group . * p < 0.05 ; < 0.05 : compared with post - test ; non- : non - dominant side ; dom- : dominant side the ankle strategy requires a normal operating range and muscle strength , and it is used to maintain balance . if the range of movement of the ankle joints becomes constrained , the postural adjustment provided by the ankle joints becomes constrained as well5 . postural adjustments require integration of body segment movement navigation and the information sent through the central nervous system through the proprioception , tactile , visual , and afferent systems ; muscle fatigue influences one or more regulatory systems , such as postural adjustment , and it is typically associated with changes in movement and sensory systems39 . in this study , the subjects were young , healthy adults who performed isotonic , isokinetic , and isometric exercises of the ankle joints ; this study analyzed the effects these exercise methods have on the muscle activity and balance control of the tibialis anterior , gastrocnemius , and peroneus longus muscles . in the case of muscular activity , there were significant differences in the non - dominant tibialis anterior , gastrocnemius , and peroneus muscles when the non - dominant side was weight bearing ; when the dominant - side was weight bearing , there were significant differences in the dominant gastrocnemius and peroneus longus muscles . in the case of balance , there were significant differences in surface area , depending on time when both the non - dominant side and the dominant side were weight bearing ; however , no significant difference in velocity was observed . significant differences were noted in the emg of the non - dominant tibialis anterior when the non - dominant limb was weight bearing . these results are consistent with previous research on the bilateral effects of muscle fatigue of one leg and muscle emg , where it was found that the tibialis anterior muscle of the limb on which the exercise was performed experienced a decline in emg . it was reported that this decline was related to resistance to stretching of the fatigued muscles , as fatigued muscle activity is usually associated with increased passive tension39 . 38 analyzed the muscular activity of the plantar flexor and dorsiflexor muscles in elderly and young adult patients by having them perform a one - leg stance with their eyes closed , and they found that the emg of the tibialis anterior was higher than that of the gastrocnemius in both groups . when a one - leg stance with eyes open was performed , emg was higher in the tibialis anterior of the elderly group and higher in the gastrocnemius of the young adult group . the findings were similar in the current study , in which adults in their 20s were the subjects ; in addition , the emg of the non - dominant gastrocnemius was higher than the non - dominant emg of the tibialis anterior when the non - dominant limb was weight bearing . these results indicate that due to the muscle fatigue induced by the three exercises , the emg activity of the tibialis anterior declined in relation to the resistance to muscle activity by the muscle that had been fatigued by the one - leg stance with eyes open . in a study regarding the effects of fatigue during isometric contractions of both the dominant and non - dominant quadriceps muscles , it was found that initial recovery from the combined effect of the fatigue mechanisms that contribute to the maximum decline in power due to sustained isometric contraction was in the dominant leg . this result occurred even though the combined effect of the peripheral and central fatigue mechanisms that induce the development of muscle fatigue during continuous isometric contraction of one side was not related to the dominant leg40 . in addition , in a study on postural adjustment disturbance of the opposite leg in a one - leg stance brought on by fatigue induced by stimulation and voluntary contraction of the same limb ( the exercised lower extremities ) , it was reported that the cross - over fatigue effect after stimulation and voluntary contraction could interfere with postural adjustment . the training of one weight - bearing side could also be transferred to the untrained side due to the cross - education effect41 . thus , in this study , the unexercised , dominant side showed significant differences in the gastrocnemius and peroneus longus when the dominant leg was weight - bearing due to this effect , while the unexercised dominant gastrocnemius and peroneus longus developed tiredness before muscle fatigue . moreover , the lack of significant differences in the dominant tibialis anterior muscle when the dominant side was weight - bearing might be due to the morphological characteristics of the muscles : the plantar flexor , which is composed of cooperative muscles , has a high compensation potential , but the muscle fatigue recovery rate is slow , while the dorsiflexor has relatively low compensation potential but a fast fatigue recovery rate . in this study , the results of balance ability both before and after exercise in the respective exercise groups showed that there were significant differences in the dominant and non - dominant weight - bearing sides in terms of area . in a previous study , in which young adults performed a 30-second one - leg stance on their dominant leg after inducing isokinetic fatigue in the ankle and hip joints , there was no significant difference in the area of the ankle joint , even though the area tended to increase after the fatigue rather than before the fatigue42 . in a study on changes in balance orientation of healthy elderly women after knee and ankle fatigue , it was found that acute muscle fatigue reduced balance adjustment43 and localized dorsiflexor fatigue from the midsagittal plane was a leading factor in precipitating postural adjustment disturbance ; this was found to produce a higher average disturbance threshold during posture correction than the plantar flexor in a non - fatigued state44 . the analysis of the effects of ankle muscle fatigue on one - leg stance posture control and recovery in adult male and female subjects showed that there were no gender differences ; the fatigue effect appeared in the right and left factors both before and after fatigue in both the male and female groups45 . in a study in which young adults performed a one - leg stance with their eyes open after isokinetic fatigue had been induced in the ankle , knee , and hip joints , it was found that from the coronal plane , knee and hip joint fatigue brought about disturbances to posture adjustment ; however , midsagittally , the fatigue of the three joints produced posture adjustment disturbances46 . similarly , as in previous studies , the lower extremity fatigue induced by the three exercise methods implemented in this study affected the posture adjusting mechanisms . even the non - dominant side , which did not perform the exercises , was affected before the fatigue ; thus , the transferred fatigue caused postural sway to increase significantly . this outcome shows the direct effect of plantar flexor and dorsiflexor fatigue of the ankle joints . in this study , there were no significant differences in velocity before or after the exercises depending upon the group . according to a study on the effects of ankle and hip joint muscle fatigue on postural sway in a one - leg stance , muscle fatigue of the plantar flexor and dorsiflexor of the ankle joints did not increase the sway velocity compared with the muscle fatigue in the flexion and extensor muscles of the hip joints ; muscles that are proximal ( hip and knee ) exert a greater influence on posture adjustment than muscles that are distal ( ankle ) . the use of the hip joint strategy increases according to the difficulty of the tasks however , the ankle strategy is primarily used when standing . thus , the one - leg stance is adjusted by a combination of hip and ankle joints . when the ankle joints are fatigued , decreased ankle joint control ( due to a damaged ankle proprioceptive sense ) can be compensated for by increased dependency on the hip strategy42 . a study analyzing the effects of vision on postural control and muscle fatigue in triceps surae muscles47 reported that vision can bring about postural deficiency related to fatigue in the plantar flexor . in addition , in a study of postural sway during one - leg stance , lower body angle and postural strategy , and the effects of plantar flexor and dorsiflexor fatigue , it was found that visual senses compensated for compromised postural adjustment . a proprioceptive sensory deficit strengthened the role of vision and subsequently reduced the amount of proprioceptive sensory information necessary to stand on one leg , particularly when the support surface decreased37 . in a study of healthy athletes and athletes with functional ankle instability that investigated the effects of fatigue due to treadmill running and sensorimotor control , it was found that fatigue due to treadmill running had negative effects on static and dynamic posture control assessment in all groups . that finding indicates that damage has occurred to muscle activation patterns and reflex activity ; in the state of fatigue , postural adjustment is affected by complex mechanisms of the sensory , neural - muscular , and central nervous systems ; the central nervous system receives more disturbances than the peripheral mechanisms of proprioception48 . moreover , in a study on posture sway and the effects of incremental difficulties in a standing balance task with visual feedback , as the difficulty of the task increased , the amplitude of the postural sway increased , and the fuzzy entropy of postural sway also increased from the anteroposterior axis in the most unstable condition49 . according to a study on visual compensation in accordance with muscle fatigue and postural adjustment , different sensory systems exist in a complex relationship related to postural control . under the condition of muscle fatigue , vision can compensate for the muscle fatigue generated by the triceps surae muscle by increasing the contributions of the various sensory systems for adjusting posture . concurrently , in the state of muscle fatigue , the effectiveness of the visual presentation is important for proper adjustment of support47 . therefore , the reason there was no significant difference in velocity / speed was because the balance was measured in a one - leg stance with eyes open without constraining the subject s vision . due to the muscle fatigue , compensation from visual information regarding postural adjustment and dependence on the hip strategy were amplified ; the impairment of the sensorimotor system and proprioception of the ankle joints for maintaining balance were not sufficient to make a significant impact on velocity . this study analyzed the effects of muscle fatigue on muscle activity and balance control in the lower extremities using three exercise methods . the ankle joint in which this was implemented was limited ; balance was measured with visual information , and the subjects were confined to healthy students . for further research , studies regarding the relationship and compensation potential of various joints , such as hip , knee , and ankle joints ; studies on postural and balance control both with and without visual information , as vision and proprioception are closely related50 ; and research focusing on specialized groups , such as patients and athletes with pain or disease in the ankle joint , are needed to expand the scope and variety of the research . in conclusion , in this study , isotonic , isokinetic , and isometric exercises were applied to the ankle joints of healthy adults in their 20s to investigate the effects of these exercise methods on the muscle activity and balance control of the tibialis anterior , inner gastrocnemius , and peroneus longus muscles . our results indicated that 1 ) there were no significant differences due to the changes of muscle activity before , during , or after the exercise between the interaction of group and time ; however , there were significant differences according to group in the non - dominant tibialis anterior , gastrocnemius , and peroneus longus muscles when the non - dominant side was weight - bearing , as well as in the dominant gastrocnemius and peroneus longus when the dominant side was supported ; 2 ) there were no significant differences before or after exercise in the interaction of group and time due to the change in balance control ; depending on time , there were significant differences in the surface area depending upon whether the non - dominant side or the dominant side was weight - bearing , but there was a significant difference in speed . accordingly , the muscle fatigue generated by the three exercise methods affected the decline in muscle activity and balance control ; it also influenced the side that did not perform the exercise before fatigue due to this effect . the finding that visual compensation occurs to maintain postural balance will further aid future treatment and research regarding muscle activity and balance control in the lower extremities .

[ purpose ] this study analyzed the effects of isotonic , isokinetic , and isometric exercises of ankle joint muscles on lower extremity muscle activity and balance control . [ subjects and methods ] the subjects were 30 healthy adults ( 15 males ) in their 20s who were randomly assigned to three different exercise method groups of 10 people each . the isokinetic exercise group performed three sets at an angular velocity of 60/sec , including a single rest period after every set of 10 repetitions . the isometric exercise group performed three sets consisting of three 15 repetitions of a 15-second exercise followed by a 5-second rest . [ results ] multivariate analysis of variance revealed that depending on the exercise method , the non - dominant tibialis anterior , gastrocnemius muscle , and peroneus longus showed significant differences in muscle activity for weight - bearing non - dominant sides ; when the dominant side was weight - bearing , the dominant gastrocnemius and peroneus longus showed significant differences in muscle activity ; and the non - dominant and dominant sides showed significant differences in balance control depending on the duration of support in the area . [ conclusion ] muscle fatigue from the three exercise methods produced a decline in muscle activity and balance control ; due to the fatigue before exercise , the side that did not perform the exercises was affected .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3765760

the neuropeptide which is most well known in the group is substance p ( sp ) . sp has pronounced pro - inflammatory effects , including the promotion of extravasation and accumulation of leukocytes at sites of injury . sp is also involved in the so - called neurogenic inflammation , wound healing , and angiogenesis . sp has a high affinity for the neurokinin-1 receptor ( nk-1r ) , having its major functions via this receptor . the nk-1r has 407 amino acids and belongs to the g - protein - coupled group of receptors . the nk-1r can play an important role in the modulation of the accumulation of white blood cells that occurs in inflammatory processes . that includes the situation in , for example , ulcerative colitis and experimentally induced acute pancreatitis and associated lung injury . in the latter situation , the nk-1r is considered to play a key role in the damaging process . it is actually a fact that the upregulation of tachykinins that occurs in inflammation in general is associated with increases in nk-1r expression [ 12 , 13 ] . we have in recent experimental studies been using a rabbit model of a marked overuse affecting the triceps surae muscle and found that the overuse led to muscle inflammation ( myositis ) and muscle tissue derangements . this model can be considered to be a suitable animal myositis model [ 1416 ] . there is a great lack of information concerning the involvement of tachykinins in situations with myositis and muscle injury . interestingly , in our studies using the overuse muscle model , it was observed that myositis and muscle tissue derangements not only occurred for the triceps surae muscle in the experimental side but also for this muscle in the nonexercised , contralateral side . this observation of an affection not only for the experimental side but also for the non - experimental contralateral side is completely new information concerning experimentally induced muscle injury / myositis . the information that so far exists concerning tachykinins and muscle inflammation is related to the patterns of sp immunoreactions observed at the spinal cord level and for the neurons innervating the muscles [ 1719 ] . there is no information at all on the expression of the nk-1r in the situation with myositis / marked muscle changes . our recent observations described above for the overuse rabbit model thus reinforced us to investigate the nk-1r patterns during myositis development . the above described experimental model was used , as it has been observed to be suitable in establishing myositis . the triceps surae muscle on both the experimental and non - experimental , contralateral sides were evaluated . the main aim of the study was to examine for the importance of the tachykinin system in the myositis process . the hypothesis was that nk-1r would be highly expressed in the affected areas with myositis . a further aim was to evaluate if there were upregulations of the nk-1r expressions not only in the experimental side but also in the contralateral non - experimental side . female new zealand white rabbits with a weight of approximately 4 kg ( age ranging from 6 to 9 months ) were used for the experiment . in total , samples from 24 animals were evaluated . eighteen animals corresponded to animals that were subjected to an exercise protocol leading to a marked overuse of the triceps surae muscle . they underwent the experimental exercise procedure on their right leg every second day , for a total period of 1 , 3 , and 6 weeks , respectively , ( see further below ) . to induce muscle overuse , a laboratory model ( a kicking machine ) leading to marked repetitive passive flexion and extension of the right ankle was achieved by means of a pneumatic piston and a band was placed around the hip / pelvis to restrict movements on the left side . during the plantar flexion phase , an active contraction was furthermore induced by electrical stimulation via surface electrodes ( pediatric electrodes 40 426a , hewlett packard , andover , ma , usa ) placed 2 cm apart over the triceps surae on the right side . the stimulation was synchronized with the plantar flexion movement of the piston by a microswitch , which trigged the stimulator unit ( disa stimulator type 14e , disa elektronik a / s , herlev , denmark ) . a single impulse of 0.2 ms duration was delivered 85 ms after the initiation of the plantar flexion at an amplitude of 3550 v. the stimulation intensity , which was tested out before the experiment , was submaximal and the intensity was individually corrected to obtain powerful muscle contractions . this exercise session lasted for 2 hours and was repeated every second day for 1 , 3 , or 6 weeks . the frequency and duration of repetitive movements were chosen in order to make a very marked strain on the muscles . the experimental procedures conform to those previously described [ 14 , 15 , 20 ] and do , with some amendments being made , conform to those described in studies by backman and collaborators . all animals were anaesthetized during the exercise procedure , by means of an intramuscular injection of fentanylfluanison ( 0.2 - 0.3 ml / kg ) and diazepam ( 0.2 ml / kg ; 5 mg / ml ) , followed by additional injections of fentanylfluanison ( 0.1 ml / kg ) every 3045 min during the experimental procedure in order to maintain anesthesia . , the rabbits were sacrificed by an intraperitoneal injection of an overdose of pentobarbital natrium and the triceps surae with attached achilles tendon was dissected out . further dissection was made , whereby samples conforming to the distal parts of the soleus and gastrocnemius muscles ( 5 810 mm ) were further processed . these samples were fixed by immersion overnight at 4c in an ice - cold solution of 4% formaldehyde in 0.1 m phosphate buffer ( ph 7.0 ) . they were thereafter thoroughly washed in tyrode 's solution containing 10% sucrose at 4c overnight . the samples were mounted on thin cardboard in oct embedding medium ( miles laboratories , naperville , il , usa ) , frozen in propane chilled with liquid nitrogen , and stored at 80c . the sections were mounted on slides precoated with chrome - alum gelatine and were then processed for immunohistochemistry or morphology . these were directly mounted and frozen in the way described above and were sectioned and processed for the demonstration of tissue morphology . they were then rinsed in distilled water , dipped in 0.1% acetic acid for a few seconds , followed by washing in running water . after washing with pbs 5 min 3 , incubation for 20 min in a 1% solution of triton x-100 ( kebo lab , stockholm , sweden ) in 0.01 m phosphate buffer saline ( pbs ) , ph 7.2 , containing 0,1% sodium azide as preservative , was performed , whereafter followed rinsing in pbs three times , 5 min each time . the sections were then incubated in 5% normal donkey serum ( code no : 017 - 000 - 121 , jackson immune research lab . inc . ) in pbs and were thereafter incubated with the nk-1r primary antibody , diluted in pbs ( ph 7.4 ) , in a humid environment . after incubation with specific antiserum and three 5-minute washes in pbs , another incubation in normal donkey serum followed , after which the sections were incubated with fitc - conjugated affinipure donkey anti - goat igg ( 705 - 095 - 147 ; jackson immunoresearch lab inc , dilution 1 : 100 ) for 30 min at 37. the sections were thereafter washed in pbs and then mounted in vectashield mounting medium ( h-1000 ) ( vector laboratories , burlingame , ca , usa ) . examination was carried out in a zeiss axioscope 2 plus microscope equipped with an olympus dp70 digital camera . as an alternative procedure , sections were initially pretreated with acid potassium permanganate for 2 min , a procedure found to enhance specific immunofluorescence reactions . after this pretreatment , the procedures described above followed . both procedures ( with or without acid potassium treatment ) there were , however , some differences in staining intensities and levels of background reactions . the initial procedures conformed to the procedures described above concerning the staining for the nk-1r antibody . that included the use of fitc - conjugated affinipure donkey anti - goat igg ( 705 - 095 - 147 ) . the sections were then rinsed in pbs 4 2.5 min and incubated in 5% normal donkey serum in pbs for 15 min . the sections were thereafter incubated with a monoclonal tachykinin ( sp ) antibody ( 8450 - 0505 , biogenesis , poole , uk ) , diluted 1 : 50 in pbs with bsa , for 60 min at 37c . after incubation with this antiserum and 4 2.5-minute washes in pbs , a new incubation in normal donkey serum followed , after which the sections were incubated in tritc - conjugated affinipure donkey anti - rat lgg ( 712 - 025 - 150 ) ( jackson immunoresearch , west grove , pa , usa ) , diluted 1 : 40 in pbs supplemented with 0.1% bsa , for 30 min at 37c . the reactions obtained with the tachykinin antibody are further on referred to as sp immunoreactive . the initial procedures conformed also in this case to the procedures described above concerning the staining for the nk-1r antibody . that included the use of fitc - conjugated affinipure donkey anti - goat igg ( 705 - 095 - 147 ) and rinsing in pbs 4 2.5 min , and as normal serum , 5% normal donkey serum in pbs was used . after the procedures for nk-1r immunolabelling were finished , the sections were rinsed in pbs 4 2.5 min and incubated in 5% normal rabbit serum in pbs with bsa for 15 min . after that , the sections were incubated with the other primary antibody to be stained for ( all of which were mouse monoclonal antibodies ) and which was against white blood cell markers , betaiii - tubulin , s-100beta , or desmin ( see further below ) , and diluted in pbs with bsa , in a humid environment . incubation was performed for 60 min at 37c . after incubation with this antiserum and 4 2.5-minute washes in pbs , a new incubation in normal rabbit serum followed , after which the sections were incubated in rabbit anti - mouse immunoglobulins / tritc ( r0276 ) ( dako , denmark ) . as an alternative procedure , normal donkey serum was used instead of rabbit normal serum , and in these cases , donkey anti - mouse immunoglobulins / tritc ( 715 - 295 - 150 ) ( jackson immunoresearch ) were utilized . the secondary antibody was used at a dilution of 1 : 40 ( r0276 ) or 1 : 500 ( 715 - 295 - 150 ) ; the incubation with secondary antiserum in both cases proceeded for 30 min at 37c . the sections were thereafter washed in pbs for 4 2.5 min and were then mounted in vectashield mounting medium ( h-1000 ) or mounting medium with dapi ( h-1500 ) ( vector laboratories , burlingame , ca , usa ) in order to identify nuclei . an nk-1r antibody produced in goats it is an affinity purified polyclonal antibody raised against a peptide mapping within an internal region of nk-1r of human origin . it was regularly used at a dilution of 1 : 501 : 100 in 0.1% in pbs . the tachykinin antibody used in double stainings was an sp monoclonal antibody produced in rats ( 84500505 , biogenesis , poole , uk ) . it was used at a dilution of 1 : 50 in 0.1% bsa in pbs . the antibody 84500505 recognizes the cooh terminal end of sp and has been previously used for the immunohistochemical detection of sp in experimental animals and man . one of these conformed to an antibody against cd68 , ( code no : m0814 ) , from dakocytomation ( glostrup , denmark ) , which was used at a dilution of 1 : 100 in 0.1% bsa in pbs . the antigen for this antibody is a glycosylated transmembrane glycoprotein , which is mainly located in lysosomes . another one was an anti - rabbit t - cell / neutrophil antibody from abd serotec ( oxford , uk ) ( mca805 g ) , which was utilized at a dilution of 1 : 100 in 0.1% bsa in pbs . this antibody is an affinity purified antibody against a cell surface antigen , which is reported to be expressed by a subset of t cells , thymocytes , neutrophils , and platelets . a third antibody used was the antibody mab1087 from chemicon ( temecula , ca , usa ) , which reacts with human eosinophil peroxidase . this antibody was used at a dilution of 1 : 100 in 0.1% bsa in pbs . furthermore , an antibody against betaiii - tubulin ( t8660 , sima aldrich , usa , 1 : 100 ) was used , being utilized at a dilution of 1 : 500 . the antibody specificially recognizes an epitope located on isotype iii of beta - tubulin . an antibody against s-100 ( beta - subunit ) was also utilized ( s 2532 ) ( sigma , new york , ny , usa ) . it was used at a dilution of 1 : 500 . the antibody recognizes an epitope located on the beta - chain , but not the alpha - chain , of s-100 . it is by the supplier ( dakocytomation , glostrup , denmark ) reported to give specific reactivity with desmin in a wide variety of tissues as seen via western blotting experiments . further descriptions of the antibodies are described elsewhere [ 16 , 25 , 26 ] . control stainings concerning the nk-1r antibody included the use of nk-1r blocking substance ( sc-5220p ) ( 50 g / ml antiserum ) . concerning controls for the sp antibody , blocking with sp peptide ( full length sp peptide ) from sigma ( s6883 ) ( 50 g / ml antiserum ) was used . other control stainings conformed to stainings when the primary antibodies were excluded ( buffer instead of the antibody ) . the characteristics of antibodies used have also been evaluated in previous studies [ 2325 ] . in order to complement the results obtained at the protein level ( via immunohistochemistry ) , stainings to show the situation at the mrna level ( via in situ hybridization ) digoxigenin- ( dig- ) hyperlabelled oligonucleotide triple probe cocktail ( ssdna ) for the detection of the nk-1r , also known as tachykinin receptor 1 ( tacr1 ) ( gene detect , new zealand ) , was thus used . a triple probe cocktail was prepared as the exact rabbit tacr1 sequence is not yet cloned . this contained three antisense probes directed towards both human and rat tacr1 mrna , which is considered to have a very high probability of detecting rabbit tacr1 mrna . the antisense probe sequences were - probe # 1 : ggctgcacgaactggttagactcagaggtgttggtggagatgttgggg ,- probe # 2 : tggagctttctgtcatggtcttggagttgctgcgagaggagccgttgg ,- probe # 3 : tgaccaccttgcgcttggcagagacttgctcgtggtagcggtcagagg . probe # 1 : ggctgcacgaactggttagactcagaggtgttggtggagatgttgggg , probe # 2 : tggagctttctgtcatggtcttggagttgctgcgagaggagccgttgg , probe # 3 : tgaccaccttgcgcttggcagagacttgctcgtggtagcggtcagagg . as a negative control , a triple probe cocktail of the corresponding sense dig - hyperlabelled ssdna probes was used . also -actin antisense / sense probes ( gd5000-op ) ( genedetect , new zealand ) were used for control purposes . based on the features noted concerning tissue morphology ( myositis affection ) and nk-1r immunohistochemical reaction patterns , these corresponded to 1 sample from the control nonexercised group , 1 sample from the 1-week group ( exercised side ) , and 2 samples from the 6-week group ( one from nonexercised side and one from exercised side ) . two of the samples were from the soleus muscle and two were from the gastrocnemius muscle . ish was performed according to an established protocol using an alkaline - phosphatase- ( ap- ) labelled anti - dig antibody for detection , with a few modifications [ 28 , 29 ] . the cryostat knife was washed in 70% etoh in diethyl pyrocarbonate [ depc]h2o and the sections were mounted on super frost plus slides ( nr . 041300 , menzel - glser , braunschweig , germany ) . concerning the further procedures , see [ 28 , 29 ] . in brief , an aliquot ( 50 ng ) of the ssdna probe was added to 15 l of hybridisation solution in a 1.5 ml microfuge tube , denatured for 5 min in 80c and then put on ice . the hybridisation solution was as follows : 500 l formamide , 200 l 20x ssc , 50 l of 20x denhardt 's solution , 50 l herring sperm dna [ 10 mg / ml ] heat denatured , 25 l baker 's yeast rna [ 10 mg / ml ] , 175 l dextran sulphate [ 50% ] , and total volume was : 1.0 ml . the probe - containing hybridisation solution was then applied to each section , the sections being covered with cover slips and sealed with nail polish . incubation followed at 56c overnight . in order to complement the nk-1r reaction studies , eia analysis of sp content for the muscle specimens for this purpose , muscle samples were frozen in liquid nitrogen directly after being weighed , whereafter they were homogenized , by the use of precellys 24 tissue homogenizer ( bertin technologies , saint quentin en yvelines , cedex , france ) , and further processed for eia using commercially available enzyme immunoassay sp kit ( phoenix pharmaceuticals , burlingame , ca , usa ) . the assay was conducted in accordance with the instructions from the manufacturer . an sp eia analysis evaluating all the various groups independent t - tests were done concerning evaluations of differences in sp concentration between controls and 6w groups . the statistical software spss pasw statistics 18 ( spss inc , chicago , usa ) was used . a p - value < 0.05 was considered to be significant . the study protocol was approved by the local ethical committee at ume university ( a 34/07 ) . a licensed breeder had bred all animals for the sole purpose of being used in animal experiments . the specimens of the soleus and gastrocnemius muscles showed marked morphological changes in response to the experimental procedure . the changes were especially pronounced after the 6-week experiment period ( figure 1 ) . however , morphological alterations were also to some extent seen in the 1-week and 3-week groups . the morphological changes included an occurrence of marked inflammatory infiltrates , a pronounced variability in muscle fiber size , and occurrence of marked muscle fiber alterations ( morphologically abnormal muscle fibers ) ( figure 1 ) . numerous white blood cells could be seen to be dispersed in the tissue ( figures 1(b ) and 1(c ) ) . the morphological changes were seen in certain parts of the specimens . in other areas of the specimens , importantly , morphological changes occurred to an almost similar extent in the muscles in the contralateral nonexercised side as in the muscles in the exercised side ( figures 1(b ) and 1(c ) ) . the pattern for the morphological changes was the same for the soleus and the gastrocnemius muscles , but the extent of the changes was somewhat more pronounced for the soleus muscle . the findings concerning the morphology are in accordance with previous findings for the triceps surae muscle in response to the experimental regimen here used . immunoreactions for nk-1r occurred in cells that were dispersed in the inflammatory infiltrates ( figure 2 ) . that was the situation for both the exercised ( figures 2(a)2(c ) ) and nonexercised ( figure 2(d ) ) sides and for both muscles . double stainings showed that parts of the nk-1r immunoreactive cells were also sp immunoreactive , whilst others did not show sp immunoreaction ( figures 2(e ) and 2(f ) ) . the nk-1r immunoreactions were abolished in sections processed with nk-1r antiserum that had been preabsorbed with nk-1r antigen ( figures 2(g ) and 2(h ) ) . the nk-1r immunoreactive cells were found to correspond to cd68 immunoreactive cells , that is , macrophages ( figures 3(a ) and 3(b ) ) , or eosinophils ( figure 3(c ) ) . there were no nk-1r immunoreactions in cells showing reactions for the antibody that was a t - cell / neutrophil marker . white blood cells were not only seen to be dispersed in the tissue , but could also be seen to be coalesced into muscle fibers ( morphologically abnormal muscle fibers ) ( c.f . above ) in the myositis areas ( figure 1(a ) ) . the stainings for nk-1r showed that there were frequently nk-1r immunoreactions in the cells that had invaded the muscle fibers ( figures 4(a)4(d ) ) . that was the situation for exercised as well as nonexercised sides and for soleus as well as gastrocnemius muscles . as was the case for the white blood cells that were dispersed in the tissue , white blood cells within the muscle fibers double - staining nk-1r / sp showed that sp immunoreactions were frequently detected in the nk-1r immunoreactive cells within the muscle fibers ( figures 4(d ) and 4(e ) ) . nerve fascicles of various dimensions were seen in specimens of both muscles . based on the morphologic appearance , a large number of these were entirely composed of myelinated nerve fibers . immunohistochemical analysis revealed that nk-1r immunoreactions were never observed within nerve fascicles being entirely built up by myelinated nerve fibers ( figure 5(a ) ) . on the whole , the nerve fascicles of control samples and those located in normally appearing muscle areas of experimental animals were nonimmunoreactive for nk-1r ( figures 5(a ) and 5(b ) ) . occasional immunoreactive nerve fibers with a varicose appearance were , however , noted close to these nonimmunoreactive nerve fascicles ( figure 5(b ) ) . such nerve fibers could also be observed in association with blood vessels in connective tissue spaces . marked immunoreactions for nk-1r were , on the other hand , noted in nerve fascicles of large / considerable sizes that were located in myositis areas and in the close proximities of these areas for both muscles and for both exercised and nonexercised sides . the reactions were point - like but were frequently coalesced . due to the occurrence of marked reaction intensities for these , broad areas were partly seen to exhibit fluorescence ( figures 5(c)5(e ) ) . these types of immunoreactions were especially seen in the 6-week groups ( figures 5(c)5(e ) ) but could also be seen in myositis areas of the 1- and 3-week groups . such immunoreactions for nerve fascicles were never seen in areas of muscles from experimental animals showing normal morphology and were never seen in the specimens of the control nonexperimental animals ( c.f . above ) . double stainings revealed that these nk-1r immunoreactions frequently , but not always , were associated with comparatively marked s100beta immunoreactions ( figure 6 ) . the nuclei of the nerve fascicles in which the nk-1r immunoreactions described above were noted and which thus were located in myositis areas showed , when dapi was used in embedding medium and when s100beta immunolabelling was performed , another colour reaction than the nuclei located outside the nerve fascicles ( figures 6(c ) and 6(f ) ) . the former nuclei showed a pink colour reaction , whilst the latter exhibited a bluish reaction ; that is , a colour that is characteristic of the dapi reaction . the nuclei of nerve fascicles located in normally appearing muscle tissue mainly showed the characteristic bluish dapi reaction . via parallel double stainings for betaiii - tubulin / nk-1r and s100beta / nk-1r and via comparisons with parallel stainings made for white blood cells further information on nerve - related nk-1r reactions was obtained . strong nk-1r immunoreactions that were not present in large nerve fascicles and that were not inflammatory cell related were thus seen in myositis areas of both sides and in close proximities to these areas ( figures 7(a)7(c ) ) . these strong nk-1r immunoreactions were betaiii - tubulin immunoreactive ( figures 8(a ) and 8(b ) ) . the nk-1r immunoreactions were associated with strong s100beta immunoreactions ( figures 7(f ) and 7(g ) ) . parts of the nk-1r immunoreactive axonal profiles were sp immunoreactive ( figures 7(d ) and 7(e ) ) . these axonal nk-1r immunoreactions ( figures 8(c ) and 8(d ) ) , as well as the point - like nk-1r immunoreactions seen in large nerve fascicles ( figures 5(e ) and 5(f ) ) , were abrogated by preabsorption with nk-1r antigen . nk-1r immunoreactions were noted for the endothelial layer of some of the blood vessels in myositis areas and areas located in the close proximities of these areas ( figure 9 ) . blood vessel - related nk-1r immunoreactions were never seen for the walls of blood vessel walls of controls and those in normally appearing muscle tissue of experimental animals and were in principle not seen for the walls of large arteries of either of the groups . the reactions were abrogated via nk-1r preabsorption ( figures 9(c ) and 9(d ) ) . nk-1r immunoreactions were also seen for fibroblast - like cells located in connective tissue spaces ( figure 9(e ) ) . in order to further clarify the nk-1r reactions in muscle fibers , double staining for desmin and nk-1r was performed . in muscle fibers that were heavily infiltrated by white blood cells ( c.f . above ) and for which nk-1r was frequently detected in these cells , there was a marked decrease or a lack of desmin immunoreactivity . the lack of desmin immunoreaction could be seen in parts of the muscle fiber ( figure 10 ) or in the entire muscle fiber . these muscle fibers contained more internal nuclei than normally seen ( figure 11 ) . in these fibers , there was also nk-1r immunoreaction , with the immunoreaction in this case being scattered in the form of punctuate reactions ( figure 11 ) . both types of muscle fibers described above showing nk-1r immunoreactions and an aberrant desmin immunoreaction were observed in myositis areas or in areas that were closely adjacent to these areas . they were very occasionally seen in other areas of muscle samples of experimental animals and were never seen in muscle samples of control animals . other muscle fibers in the myositis areas and in normally appearing muscle tissue of experimental animals and all muscle fibers in the specimens of control animals showed the characteristic desmin immunoreaction pattern , that is , a striated reaction pattern in longitudinally cut muscle fibers ( figure 11 ) . the pattern and magnitude of the nk-1r immunoreactions for all the various structures were similar for exercised and nonexercised sides . the marked nk-1r immunoreactions seen for white blood cells , nerve fascicles , blood vessel walls , and muscle fibers described above were restricted to myositis areas and areas that were very close to these areas . thus , the magnitude of the nk-1r immunoreactions was related to the degree of inflammatory infiltrates ( myositis ) within the specimens , which to some extent varied between the different samples in the experimental groups . the in situ hybridization studies showed that nk-1r mrna reactions were observable in the same cell structures as were found to be nk-1r immunoreactive . that included fibroblast - like cells ( figure 12 ) , cells of blood vessel walls in myositis areas and/or closely adjacent areas ( figures 13 and 14 ) , white blood cells in inflammatory infiltrates ( figure 15 ) , and muscle fibers showing abnormal morphology , being heavily infiltrated by cells ( figure 16 ) or containing frequent internal nuclei . the nk-1r mrna reactions in blood vessel walls were not only localized to the endothelial layer but could also be seen for the smooth muscle part of the walls ( figure 13 ) . the specificity of reactions seen for all the various structures was verified via stainings with sense control probe ( figures 12(b ) , 13(b ) , 14(b ) , and 16(c ) ) . analysis of sp concentration was made for muscles of control animals and animals for which the most pronounced myositis was observed , that is , the 6-week groups . it was found that the concentration was clearly higher in the 6-week groups for both muscles and for both sides . the mean values were thus 1.83 times higher for exercised side and 1.77 times higher for nonexercised side , as compared to control muscle , concerning the soleus muscle ( p = 0.004 in both cases ) , and 3.45 times ( exercised side ) and 3.01 times ( nonexercised side ) higher than the mean value in the control group concerning the gastrocnemius muscle ( p < 0.05 in both cases ) . via evaluations of the nk-1r reaction pattern , the present study indicates that tachykinins are highly involved functionally in the myositis and muscle affection process in our rabbit model of muscle overuse . an upregulation of the expression of nk-1r was observed in both the exercised as well as the nonexercised sides . nk-1r immunoreactions and nk-1r mrna reactions were seen for cells of the inflammatory infiltrates and for blood vessel walls within the inflamed and affected areas . strong nk-1r immunoreactions in axonal profiles and fine nk-1r immunoreactions in large - sized nerve fascicles were furthermore frequently detected in the areas influenced by the myositis process . additionally , morphologically abnormal muscle fibers in these areas displayed nk-1r immunoreactions and nk-1r mrna reactions . the nk-1r reactions were thus in principle restricted to the myositis areas and the areas located very close to these areas . when interpreting the significance of the nk-1r immunoexpressions , one should be aware of the fact that not only sp but also endokinins and hemokinins show a remarkable potency for nk-1 receptors . nevertheless , in the tachykinin stainings performed in the present study antibodies against sp ( monoclonal antibodies ) were used . an interesting aspect in the current study is that the tachykinin system becomes involved in a process that occurs bilaterally in response to a unilateral experiment . there was thus an upregulation of nk-1r expression bilaterally , as well as an increase in sp levels bilaterally as seen via eia analyses . similar crossover effects were reported in a recent study where retinal laser burn - induced neuropathy lead to an increase in sp - inducible nk-1r first in the retina of the burned eye and then in the contralateral eye . the authors of this study , who had previously noted that unilateral retinal laser burn leads to inflammation not only in this eye but also in the nonburned eye , suggested that sp had transmitted early inflammatory signals from the affected eye to the contralateral eye . in the same context , chang and collaborators demonstrated that unilateral burn injury in a limb can have a long - lasting allodynia that spreads to the contralateral limb . it was concluded that central neuropathic mechanisms , via the occurrence of the hyperexcitability of second - order neurons and microglial activation , were responsible for the cross - transfer effect . it has previously been shown that unilateral experiments and unilateral inflammation in limbs can lead to contralateral effects and in which cases neurological mechanisms are considered to be very important [ 3538 ] . occurrence of contralateral responses following unilateral stimuli in the form of intradermal injections of capsaicin has also been shown for humans and contralateral increases in sensory neural activity are suggested to be of importance for the symmetry concerning the inflammation in rheumatoid arthritis . it is furthermore known that electrical stimulation can lead to orthodromic activation of afferent nerve fibers which can initiate the release of sp and changes in both the stimulated and the nonstimulated contralateral muscles . bilateral effects concerning sp have previously been seen after experiments using heat injury and unilateral formaldehyde injections and in response to craniofacial inflammation as well . it should here be stressed that the nk-1r expressions seen in our model of myositis / muscle injury were not only related to nerve structures but also inflammatory cells , blood vessel walls , and morphologically abnormal muscle fibers . it is also noteworthy that sp in experimental studies has been found to have a systemically acting wound messenger effect in the early wound healing processes after an injury to the conjunctiva . in future studies using the currently used model , the bases for the contralateral effects it should be recalled that affections occurred bilaterally for the tendons in studies focusing on the achilles tendons using the unilateral setup utilized in the present study . it is well known that there is a widespread expression of nk-1r in the central nervous system . it is also shown that unmyelinated axons in nerve bundles in peripheral locations can display nk-1r , and that nk-1rs are found on sensory nerves in various locations [ 47 , 48 ] . however , nerve - related nk-1r immunoreactions were very infrequently seen in the controls and areas showing normal muscle morphology in experimental animals in this study . the only profiles that were encountered occurred as varicose profiles . on the other hand , nerve - related nk-1r immunoreactions were frequently detected in myositis areas and areas that were very close to these areas . nk-1r immunoreactions were thus seen in the form of fine distinct point - like reactions that were coalesced in large nerve fascicles and in the form of strong immunoreactive axonal profiles . the double stainings we performed showed that these nk-1r immunoactions were often associated with markedly s100beta immunoreactive schwann cells . this observation suggests that these nk-1r expressing profiles are enclosed by schwann cells . in accordance with this interpretation , previous studies made at the ultrastructural levels in the rat dental mucosa have shown that nk-1rs are present in vesicular structures and the axoplasm of axons that are enclosed by schwann cells . our double stainings also showed that the large nerve fascicles / axonal profiles harbouring these nk-1r reactions were related to nuclei that showed another type of fluorescence reactions than nuclei located outside the nerve fascicles and than those usually seen for nerve fascicles of normal muscle regions . the former fluorescence reactions are related to a combination of the dapi reaction and s-100beta immunoreaction . it , therefore , seems likely that these nerve fascicles / axons are in a phase of regeneration . it is thus known that the s-100beta protein in principle is detected in the cytoplasm and the membranes of the schwann cells , and not the nuclei , as seen in immunohistochemical stainings , in normal situations . furthermore , it is known that schwann cells can be activated when there is a nerve damage and that the s-100beta protein can be involved in axonal regeneration [ 51 , 52 ] and that schwann cells ensheath regenerating axons . the nk-1r , as well as tachykinins like sp , has been detected in several types of white blood cells . in the present study , nk-1r the sp / nk-1r system may thus have an important role for the accumulation of these cells in the muscle tissue in the myositis process . concerning macrophages , these findings are in accordance with a report saying that nk-1rs are involved in the accumulation of macrophages in the airways in the development of smoking - induced emphysema and in response to cigarette smoking exposure . the nk-1r reactions in the cells in the inflammatory infiltrates showed frequently an intracellular location . this is likely to be due to the nk-1rs being internalized after binding to released sp or that it represents newly synthesized receptors . it is well known that the nk-1r in neurons undergoes rapid internalization after binding to sp has occurred and that the receptor thereafter recycles to the plasma membrane . the intracellular location of nk-1rs has been reported to be transport vesicles and endosomes . also in nonneuronal cells , there is a process of endocytosis and recycling of the nk-1r . it might be that the cells in the inflammatory infiltrates are under the continuous influence of sp in an autocrine / paracrine fashion and that there is a continuous nk-1r internalization but also a continuous synthesis . sp and the nk-1r are actually reported to be internalized in the same vesicles and then sorted into independent endosomes , leading to the eventual degradation of sp and recycling of the nk-1r . nk-1r immunoreactions and nk-1r mrna reactions were seen in the endothelium of blood vessels in myositis areas . it is well known that sp induces an increased vascular permeability , vasodilatation , and hyperthermia via binding to nk-1rs on the endothelial cells of the blood vessel walls . the vasodilator effect by sp is thus considered to be dependent upon the endothelium and to be mediated by the nk-1r . we also sometimes noted reactions for nk-1r mrna for the smooth muscle layer . in accordance with this finding , it has been found that smooth muscle cells of airways in rats show mrna transcripts for the nk-1r . nk-1r was not only detected in white blood cells that were dispersed in the tissue but also in white blood cells that heavily had infiltrated muscle fibers . based on the complete or relative loss of desmin immunoreactivity in these muscle fibers , they can be considered to represent degenerating / necrotic muscle fibers . it is thus known that desmin is undetectable or much decreased in necrotic muscle fibers . on the other hand , we noted that other muscle fibers in myositis areas and adjacent areas showed a very marked desmin immunoreaction . this is in accordance with the known fact that there is an overexpression of desmin during muscle regeneration processes . also these fibers exhibited nk-1r immunoreaction , the reactions in this case showing a punctuate pattern . this indicates that both degenerative and regenerative events occur in the myositis process and that the nk-1r is involved in both types of processes . as discussed above , there were marked nk-1r reactions in myositis areas and areas adjacent to these areas , whilst there were almost no nk-1r reactions at all in areas with normal morphology . the reactions were seen for nerve structures , white blood cells , blood vessel walls , and abnormal muscle fibers . the observations of a very marked nk-1r expression in myositis specimens , in parallel with an increased content of sp in these specimens ( as seen for specimens of the 6-week groups ) , are comparable to findings made in other situations for tissues exhibiting inflammation and tissue damage / reorganization . that includes colonic fibrosis , immunodeficiency virus lesions , airway inflammation , and sarcoidosis . it has since long been considered that the occurrence of interactions between tachykinins and inflammatory cells is of great importance in pathological conditions and that the nk-1r has important roles in these conditions . there is a reason to believe that tachykinin effects hereby are related to interactive effects with other signal substances . the fact that sp and nk-1r sometimes were found to be colocalized suggests that autocrine / paracrine sp effects may occur in the tissue . this can be related to an auto - regulatory mechanism for sp in relation to the nk-1rs . autoreceptor functions for sp affecting nk-1rs have since long been suggested to occur in other situations . it has also previously been suggested that nk-1rs found on sensory neurons are autoreceptors , being related to the modulation of peripheral pathophysiological effector functions . autocrine / paracrine effects by sp has on the whole been considered to take place in several conditions , including in leukemia and various inflammatory situations . one can ask wheter the marked nk-1r expression in the myositis areas is not only related to proinflammatory effects but also to healing effects . it should here be recalled that there occurs an overexpression of the nk-1r not only in response to inflammation and tissue damage , but also in situations with tissue repair and wound healing . an inflammatory component is actually described to be involved in healing effects for the skin . topical treatment of sp to skin wounds in genetically diabetic mice thus leads to an increase in inflammatory density as a part of the healing process . sp is considered to be involved not only in cutaneous healing , but also in corneal wound healing and to be involved in processes of tendon repair [ 7375 ] . concerning tendinopathy , sp can accelerate the processes of hypercellularity and angiogenesis in tendon tissue in this condition . it is , for example , reported that nk-1r - mediated functions have protective roles in acute hyperoxic lung injury . the present study shows that the tachykinin system is markedly involved in the processes of muscle injury / myositis that occur in the overuse model here used . to what extent the effects of tachykinin there is a reason to believe that tachykinins show interference effects with other signal substances in the processes that occur . the tachykinin system is upregulated on both sides , that is , in the myositis process that occurs in the experimental side as well as the one that occurs contralaterally . the upregulation is for both sides not only restricted to nerve structures but also to cells of the inflammatory infiltrates , the blood vessels , and morphologically changed muscle fibers . it has not previously been demonstrated that tachykinins have the marked effects that are shown here for muscle tissue . the currently used model can be utilized in further studies evaluating the features of interactions between tachykinins and other signal substances in myositis and degenerative / regenerative muscle processes and to evaluate if interference with tachykinin effects can be of value for these processes . the possible effectiveness of nk-1r blocking has been evaluated for various inflammatory conditions [ 78 , 79 ] . it remains for the future to evaluate if interference with effects at the nk-1r can have positive effects in situations with myositis and muscle injury .

muscle injury and inflammation ( myositis ) in a rabbit model of an unilateral muscle overuse were examined . it is unknown if the tachykinin system has a functional role in this situation . in this study , therefore , the neurokinin-1 receptor ( nk-1r ) expression patterns were evaluated . white blood cells , nerve fascicles , fine nerve fibers , and blood vessel walls in myositis areas showed nk-1r immunoreaction . nk-1r mrna reactions were observable for white blood cells and blood vessel walls of these areas . nk-1r immunoreaction and nk-1r mrna reactions were also seen for muscle fibers showing degenerative and regenerative features . there were almost no nk-1r immunoreactions in normal muscle tissue . interestingly , marked nk-1r expressions were seen for myositis areas of both the experimental side and the contralateral nonexperimental side . eia analyses showed that the concentration of substance p in the muscle tissue was clearly increased bilaterally at the experimental end stage , as compared to the situation for normal muscle tissue . these observations show that the tachykinin system is very much involved in the processes that occur in muscle injury / myositis . the effects can be related to proinflammatory effects and/or tissue repair . the fact that there are also marked nk-1r expressions contralaterally indicate that the tachykinin system has crossover effects .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusions

PMC3238378

glucokinase ( gk ) ( ec 2.7.1.1 ) is expressed in glucosensitive cells of the pancreas , liver , hypothalamus , anterior pituitary gland , and enteroendocrine k and l cells [ 13 ] . gk has a prominent role as a glucose sensor because of its specific kinetic properties , including an affinity for glucose that is within the physiological plasma concentration range ( half - saturation level for glucose [ s0.5 ] of ~8 mmol / l ) , positive cooperativity , and lack of inhibition by glucose-6-phosphate . in liver and brain , gk is controlled by an endogenous inhibitor , the glucokinase regulatory protein ( gkrp ) [ 4 , 5 ] . during starvation , the enzyme after refeeding , glucose mediates the dissociation of the gk - gkrp complex and gk translocates to the cytoplasm . also , several naturally occurring activating mutations have been described that induce a conformational change in gk , which results in higher affinity for glucose , and reduce the interaction between gk and gkrp [ 6 , 7 ] . similarly , synthetic gk activators ( gkas ) enhance gk activity and induce its translocation to the cytosol . there is evidence that a defect in gkrp regulation of gk , resulting in increased liver gk activity probably as a result of increased cytosolic translocation , has consequences on glucose and triglyceride homeostasis in humans , even though these patients have reduced risk to type 2 diabetes [ 13 , 14 ] . we aimed to study the metabolic consequences of gk - gkrp deregulation by overexpressing a gk activating mutant ( gka456v ) in the liver of insulin - deficient mice ( lacking endogenous gk ) . transfected gk456v is maintained in the cytosol of hepatocytes in vivo leading to improved glycemia in the absence of dyslipidemia . these data provide novel insights into the consequences of gk - gkrp derangement and widen the scope for gka research in the liver . site - directed mutagenesis to generate gk - a456v cdna was performed using the quickchange site - directed mutagenesis kit ( strategene ) , and specific primers containing the desired mutation ( 3ctggtctctgcggcggcctgcaagaag5 and 5gaccagagacgccgccggtgcaagaacg3 ) from dharmacon . the cdnas for gk and gk - a456v were cloned into ecori - bglii of a pcaggs vector which contains the cag ( cytomegalovirus immediate - early enhancer - chicken -actin hybrid ) promoter . the resulting plasmids pgk and pgka456v were amplified in e. coli jm109 cells and extracted using nucleobond pc 2000 ef mega prep kit ( macherey - nagel ) . dna for animal injection was digested with sali and hindiii which cut on both sides of the expression cassette [ 16 , 17 ] in order to extend expression in vivo . expression plasmids were transfected in huh7 cells for kinetic analysis ( supplementary figure 1 available at doi : 10.1155/2011/707928 ) . male icr mice purchased from harlan interfarma iberica s.l were maintained in a constant 12-hour light - dark cycle and fed standard rodent chow and water ad libitum . all animal protocols were approved by the ethics committee at the university of barcelona . to obtain diabetic animals , mice weighing approximately 22 g were injected i.p . with a single dose of streptozotocin ( stz ) ( 200 mg / kg ) dissolved in 100 mm citric / citrate buffer , ph 4.5 . digested plasmid dna was introduced into murine livers using a hydrodynamic - based gene transfer technique via rapid injection of a large volume of dna solution through the tail vein . briefly , 60 g / mouse dna was diluted in 2.0 to 2.5 ml of apyrogenic ringer - lactate solution ( fresenius kabi ) ( 0.1 ml / g body weight ) and injected into the tail vein using a 27-gauge needle and syringe within 5 to 10 seconds . typically , the mice recovered from the injection within 2 to 10 min . at the end of the experiment , animals were sacrificed under xylazine / ketamine anesthesia . blood samples were taken by inferior cava vein puncture and serum was obtained by centrifugation at 700 g at 4c for 15 min . liver was dissected , snap frozen in liquid nitrogen , and subsequently stored at 80c until analysis . for gk immunodetection , livers from 5-hour fasted animals were fixed in a 4% pfa solution and frozen in oct compound . 7 m cryosections were obtained in a cryostat and immunostained with a 1 : 50 dilution of gk antibody ( ap7901c , abgen ) followed by an anti - rabbit antibody alexa fluor 488 ( molecular probes ) . blood glucose levels were measured using a glucocard memory 2 apparatus ( menarini ) from a blood drop collected from the tail tip . the veterinarian clinical biochemistry service from the veterinary hospital of the universitat autnoma de barcelona , bellaterra , spain , measured serum metabolites . cdna synthesis was performed using ready - to - go you - prime first strand beads ( amersham biosciences ) with random hexamers . mrna levels of the transgenic rat gk ( r - gk ) were quantified with specie - specific primers ( applied biosystems ) and normalized using -2-microglobulin as an internal control in an ht7700 real time - pcr system ( applied biosystems ) . mrna levels of selected genes of interest were measured running a low density array ( applied biosystems ) in an ht7900 real time - pcr system ( applied biosystems ) . frozen liver samples were homogenized in ice - cold homogenization buffer ( hepes 50 mm ph 7.4 , kcl 100 mm , edta 1 mm , mgcl2 5 mm , dtt 2.5 mm ) using a polytron pt 3000 and centrifuged at 100,000 g for 1 hour at 4c . supernatants were used for the determination of gk activity following the spectrophotometric method described by davidson and arion . briefly , the assays were carried out in a solution containing hepes 50 mm ph 7.4 , kcl 100 mm , mgcl2 7.5 mm , nad 1 mm , atp 5 mm , bsa 1% and indicated amounts of glucose . the reactions were initiated by adding 5 u / ml of glucose-6-phosphate dehydrogenase ( g6pdh ) from leuconostoc mesenteroides ( roche ) and the rate of increase in absorbance at 340 nm was measured . the gk activity was calculated as the difference between the kinase activities measured at different glucose concentrations and 0.5 mm glucose ( hexokinase activity ) , and was expressed as munits / mg liver . frozen liver samples were homogenized in ice - cold buffer , which contained hepes 50 mm ph 7.4 , kcl 100 mm , edta 1 mm , mgcl2 5 mm , dtt 2.5 mm , and 1% pluronic f-127 ( calbiochem ) , and centrifuged at 20,000 g for 20 min at 4c . g6pdh activity was assayed by measuring the rate of nadph production from nadp 0.5 mm and glucose-6-phosphate 2 mm , at ph 7.6 . frozen tissue was homogenized in radioimmunoprecipitation assay buffer ( ripa ) supplemented with protease and phosphatase inhibitors and centrifuged at 15,000 g for 15 min at 4c . 30 g of tissue extract was resolved by sds - page ( 812% gel ) and electrotransferred onto a hybond - p membrane ( amersham biosciences ) . / fructose-2,6-biphosphatase ( ub - pfk2 ) and l - pyruvate kinase ( l - pk ) ( gifts from ramon bartrons , barcelona university ) , and carbohydrate response element binding protein ( chrebp ) ( novus biologicals ) . acc and acc - p ser-79 ( upstate ) were used at 1/2000 . sheep anti - pepck - c antiserum ( a gift from daryl granner , vandervilt university , usa ) was used at 1 : 20,000 dilution . all membranes were normalized using monoclonal anti--tubulin ( sigma ) at 1 : 10,000 dilution . horseradish peroxidase activity linked to secondary antibody was detected with ecl substrate ( pierce ) in a fujifilm las 300 intelligent dark box iv imaging system . densitometry was performed using multi gauge software . to measure hepatic free glucose , glucose-6-phosphate ( g6p ) , and lactate briefly , perchloric acid 10% was added to frozen pieces of liver ( 5 : 1 v : w ) and the mixture was homogenized in a potter elvehjem apparatus at 1,500 rpm . the homogenate was then centrifuged at 20,000 g for 10 min at 4c . the supernatant was brought to ph 7 with k2co3 4 n and centrifuged again to remove the kclo4 formed during neutralization . g6p was measured following the spectrofluorometric method described by lang & michal , which is based in the reaction catalyzed by g6pdh . briefly , the assay solution contained triethanolamine 0.2 m ph 7.6 , nadp 0.2 m , mgcl2 5 mm , and g6pdh 0.17 u / ml . lactate was determined by a spectrophotometric method based on the reaction catalyzed by lactate dehydrogenase , following gutmann & wahlefeld . briefly , the reaction cuvette contained hydrazine / glycine buffer 0.25 m/0.7 m , ph 9.3 , edta 0.15 mm , nad 2 mm , and 35 u / ml of lactate dehydrogenase . for fructose-2,6-bisphosphatase ( f2,6bp ) determination , frozen livers were homogenized in 0.1 m naoh , heated to 80c for 15 min , and centrifuged at 12,000 g for 5 min . f2,6bp was determined in supernatants by its ability to activate pyrophosphate - dependent 6-phosphofructo-1-kinase from potato tubers , as described by van schaftingen et al . . the hepatic triacylglyceride ( tag ) content was measured in 3 m koh , 65% ethanol extracts using a tag kit ( sigma ) , based on the method of salmon and flatt for liver saponification . a solution of u - c - glucose and glucose was intraperitoneally injected into 5-hour fasted stz - diabetic mice ( 0.2 mg glucose , 10 ci per animal ) . blood , liver , brain , and muscle were obtained 30 minutes after injection and dissolved into solvable ( perkin elmer ) . blood samples were bleached with edta 0.1 m and h2o2 ( 30% ) , and liver and brain samples were bleached with h2o2 ( 30% ) alone . radioactivity was quantified by adding scintillation liquid ( ecoscint h ) and counted in a wallac 1409 - 001 counter . pgka456v , wild - type gk ( pgk ) and an empty vector ( pcontrol ) , were introduced into the liver of streptozotocin ( stz- ) treated mice . hydrodynamic - based delivery of plasmid dna led to specific transfection of hepatocytes around the central vein [ 23 , 24 ] , reproducing the physiologic distribution of gk in the liver acinus . this technique induces a transient release of liver enzymes that returns to control values by the second day after injection . consistently , transaminase levels were low at the end - point of analysis , and similar in all treatment groups ( pcontrol 207.5 29.88 u / l , pgk 121.0 48.43 u / l , pgka456v 117.5 40.35 u / l ; n = 49 ) . at the time of injection mice were hyperglycemic ( > 350 mg / dl ) and their insulin level was undetectable ( < 0.1 forty - eight hours postinjection the expression of transfected rat gk and gka456v was detected by rt - pcr ( data not shown ) . further confirmation of gk overexpression was obtained by western blot from liver extracts ( figure 1(a ) ) and gk activity assay ( figure 1(b ) ) . gk activity was undetectable in pcontrol transfected livers irrespective of glucose concentration , as expected in long - term stz diabetic animals . in contrast , both pgk and pgka456v transfected groups had similar gk activity at 100 mm glucose ( mu / g fresh ) , demonstrating that both treatment groups had similar amounts of gk protein . consistent with the s0.5 for glucose of gka456v , liver extracts from pgka456v - injected animals had twice the gk activity of livers from pgk - injected animals at physiological glucose concentrations ( 5 mm ) ( figure 1(b ) ) . gkrp retains gk in the nucleus at low glucose concentrations and releases gk when insulin , high glucose , or fructose-1p are present [ 26 , 27 ] . different studies have suggested that the a456v mutation impairs gkrp regulation in vitro [ 6 , 28 ] . therefore , we assessed gkrp - dependent nuclear sequestration in primary hepatocytes from stz - treated rats transfected with pgk and pgka456v , in the presence or absence of pgkrp . densitometry analysis of western blots from nuclear and cytosolic fractions showed that cellular gka456v distribution was not affected by the presence of gkrp ( data not shown ) . pgk - treated mice showed gk immunoreactivity throughout the liver , especially in nuclei ( figure 2 ) . these data demonstrates , that impaired gkrp binding to gka456v changes its compartmentalization in vivo . the impact of both gk enzymes on glucose metabolism was first evaluated in fed animals . both gk and gka456v slightly reduced glycemia ( figure 3(a ) ) . to explore the fate of glucose in the liver fed pgk - treated mice showed significantly higher f2,6bp and glycogen contents ( figure 4(a ) ) , together with a rise in lactate , indicating a higher glycolytic flux . gka456v - expressing livers also showed higher hepatic free glucose than both control and gk groups , even though the content of g6p was similar in all three groups . glycogen content was significantly higher than control but similar to pgk - treated group ( figure 4(a ) ) . pgka456v - treated animals showed a marked reduction of glycemia after a 5-hour fast ( figure 3(a ) ) . lactate was only increased after pgk treatment ( pcontrol , 7.296 0.772 mm ; pgka456v , 8.778 0.649 mm ; pgk , 10.62 0.608 mm hepatic free glucose content was low in the pgka456v treatment group ( table 1 ) . g6p levels were reduced to the same extent in control and gka456v groups , whereas gk - expressing animals showed similar g6p and atp concentrations to well - fed animals . glycogen content in gk - expressing animals was significantly higher than control and gka456v - expressing groups . fed - to - fast transition produced a marked reduction of glycogen stores in pgka456v - treated animals as compared to pcontrol and pgk , suggesting an increased rate of glycogenolysis in gka456v - expressing animals ( figure 4(b ) ) . in order to clarify whether glucose distribution in vivo favored the liver , we quantitated c radioactivity derived from uptake and metabolism of uc - glucose in various tissues 30 minutes after injection . c label was increased exclusively in the liver of both pgk and pgka456v treatment groups ( control , 100 7.88% ; gk , 138.73 14.63% ; gka456v 144.1 8.99% ; p = 0.037 gk versus control and p = 0.003 pgka456v versus control ) . to assess whether pgka456v expression would affect glucose clearance in healthy mice , we performed a glucose tolerance test 1 week after transfection ( figure 3(b ) ) . consistently , our data demonstrates a significant increase in serum tag ( pcontrol 33.5 7.72 mg / dl , pgka456v 38.13 7.66 mg / dl , pgk 74.00 14.76 mg / dl , p < 0.05 versus pcontrol and pgka456v ) and nefa ( pcontrol 0.325 0.09 mm , pgka456v 0.4013 0.05 mm , pgk 0.66 0.10 mm p < 0.05 versus pcontrol ) only in the postabsorptive state . besides , gk overexpressing mice showed a tendency to increase hepatic tag content , which is consistent with the literature [ 30 , 31 ] . in contrast , gka456v overexpression did not promote dislipemia , as evidenced by maintained levels of circulating nefa and tag . there was a significant increase in -hydroxybutyrate levels in the pgka456v group ( pcontrol , 0.18 0.03 mm ; pgka456v , 0.27 0.03 mm in order to elucidate the mechanism responsible for the metabolic profile observed , we analyzed protein and mrna levels for key enzymes and transcription factors . both gk and gka456v treatments were accompanied by increases in l - pk protein ( figures 5(a ) and 5(b ) ) . pgk - treatment resulted in higher levels of ubiquitous pfk2 ( pfkfb3 ) protein ( figures 5(a ) and 5(b ) ) , but not mrna ( table 2 ) , consistent with elevated f2,6bp observed in this treatment group ( table 1 ) . also , endogenous , mouse gk mrna expression was induced in the livers of pgk - injected mice , as detected by species - specific qrt - pcr . correlating with the induction of the expression of endogenous gk , cmyc mrna was only increased in pgk - treated animals . other regulatory factors implicated in the regulation of endogenous gk expression , such as srebp and lxr , were not affected by treatment with pgk and pgka456v ( table 2 ) . as an increment in gk expression in the liver is commonly associated with increased lipogenesis [ 32 , 33 ] , enzymes and transcription factors involved in de novo lipogenesis were analyzed . gk- and pgka456v - expressing groups presented increased mrna content for fasn and mod1 that were more pronounced in pgk - treated animals . also , acc1 and chrebp were more induced by the overexpression of gk than by gka456v . gk , through the production of xylulose-5p , mediates the inhibition of gluconeogenesis by glucose [ 34 , 35 ] . f2,6bp has also been associated with the inhibitory effect of glucose metabolism on gluconeogenesis . consistently , we observed a reduction of pepck at the protein level in both pgk- and pgka456v - treated mice . similarly , pgc-1 and hnf4 mrnas were reduced in pgk - injected mice , whereas gka456v - overexpressing livers only showed reduced pgc-1 mrna . in contrast , the level of mrna for the catalytic subunit of glucose-6-phosphatase ( glc6pase ) were strikingly higher in gka456v - overexpressing animals , consistent with more glycogen breakdown observed after a 5-hour fast . similar results were observed in healthy mice overexpressing pgka456v after a short fast ( data not shown ) . to evaluate the consequences of gk activation and deregulation by gkrp in the liver , we hydrodynamically injected pgka456v in the liver of stz - diabetic mice . this strategy allowed us to obtain a mouse model where the effects of exogenous gks are isolated from those of endogenous gk ( undetectable levels of mouse gk mrna and activity in pcontrol - treated animals ) and insulin action ( insulin levels in addition , the use of hydrodynamic gene transfer provides proper zonal expression of gk and gka456v in perivenous hepatocytes [ 23 , 25 ] . therefore , only animals that were positive for exogenous expression of gk as determined by qrt - pcr ( 3x sd of controls ) were selected for further analysis ( data not shown ) . after selection , both gk - overexpressing groups had identical protein levels and maximal activity ( 100 mm glucose ) figures 1(a)1(b ) . these results are not in agreement with the reported instability of liver gk protein in a phhi - gka456v transgenic mouse and the gkrp knock - out model [ 37 , 38 ] , probably because of the lack of endogenous transcriptional control in pgka456v . also , gka456v activity at 5 mm glucose was higher in liver extracts , as expected from the kinetic changes associated with the mutation . the gka456v mutation resides on a hinge of the enzyme responsible for the conformational changes associated with gkrp interaction and allosteric activation . gkrp regulates gk action in hepatocytes by retaining the enzyme in the nucleus at low glucose concentrations and liberating gk when insulin , high glucose levels , or fructose-1p is present [ 26 , 27 ] . we show here that gka456v is predominantly cytosolic in the liver of transfected animals , contrary to the expected nuclear compartmentalization of wild - type gk . in addition , gka456v was not appropriately translocated to the nucleus of diabetic primary hepatocytes in the presence of excess gkrp ( data not shown ) . lack of gkrp binding to gka456v has been shown by heredia et al . in vitro and inferred from the structural characterization of gk bound to synthetic activators [ 12 , 39 ] . recent reports on the p446l mutation in gkrp also suggest that a decrease in gk inhibition results in increased gk activity , suggesting that gk localizes to the cytosol . an analysis of gk and gka456v overexpression in well - fed animals demonstrated small but significant changes in glycemia , together with increased hepatic glucose metabolism , as suggested by higher f2,6bp and glycogen content , in the absence of alterations to lipid serum metabolites . no changes were observed in enzymes or regulatory proteins involved in gluconeogenesis in fed animals ( data not shown ) . [ 31 , 33 , 40 ] , who used a low dose of gk - expressing adenovirus ( expressed largely in periportal hepatocytes , [ 41 , 42 ] ) but differ from those obtained using high doses of adenovirus , indicating that dose and/or zonal expression of gk influences the metabolic impact of gk overexpression . when animals were examined after a short fast ( postabsorptive ) , marked differences were observed among groups . ( 1 ) gk overexpression reduced glycemia ~15% , consistent with enhanced glucose metabolism in the liver , as shown by increased g6p , f2,6bp , glycogen content , and glycolytic and lipogenic enzymes together with higher serum lactate , nefa and tag concentrations . have shown that a fed - to - fast transition in stz - diabetic mice results in increased gluconeogenic potential and fatty acid oxidation in the liver , consistent with results obtained in pcontrol - treated animals in the present study . conversely , pgk treatment resulted in reduced pepck and pgc1 mrna , supporting the view that gk overexpression indirectly inhibits gluconeogenesis and fatty - acid oxidation . consistently , we measured higher nefa and tag serum levels in the postabsorptive state although no changes in -hydroxybutyrate were observed . these results indicate that enhanced glucose metabolism resulting from gk overexpression had a small effect on glycemia , possibly because pgk dose was low . ( 2 ) gka456v overexpression led to a significant reduction of serum glucose of ~60% that was consistent with higher glucose clearance on a glucose tolerance test in fasting , healthy mice . fasting hypoglycemia is also observed in human carriers of the p446l gkrp mutation [ 14 , 44 ] . gka456v overexpression did not impact the concentration of serum lipids whereas -hydroxybutyrate was increased , contrary to the dyslipidemia observed in gk overexpressing animals in this and earlier studies , including human carriers of the p446l gkrp mutation [ 14 , 44 ] . hepatic levels of f2,6bp and l - pk protein were increased but other indicators ( g6p , fasn mrna , chrebp protein , and mrna and glycogen content ) of glucose utilization were lower than in gk - expressing livers . importantly , glycogen balance ( fed - to - fast ) was significantly lower in gka456v - expressing animals , indicating an important net loss of glycogen . g6pase expression was also higher in this group of animals , providing an explanation for increased glycogen loss observed in gka456v - containing livers . the involvement of g6pase in the positive regulation of glycogenolysis has been shown in several animal models overexpressing p36 or p46 components of the g6pase system , resulting in decreased glycogen levels in the hepatocyte [ 4548 ] . moreover , a lack of g6pase system activity observed in glycogen storage disease type 1a , or in a g6pase ko promotes glycogen accumulation . activation of the nonoxidative branch of the pentose phosphate pathway , as a result of high exogenous glucose , transiently upmodulates g6pase mrna levels and stability in an insulin - independent manner [ 34 , 35 ] . gka456v - expressing livers showed higher g6pdh activity and gsh / gssg ratio during the fed - to - fast transition than control or gk groups ( figures 4(c)-4(d ) and table 1 ) . increased g6pase mrna and protein in the presence of gka456v would suggest an induction of glucose cycling . it is , however , clear that this counter regulation shall have consequential effects on glucose homeostasis if persistent , as occurs in the gka456v model . all in all , the present data confirms that pgk overexpression in the liver results in relatively small changes in glycemia but overt dislypidemia . the present model represents the first attempt to overexpress pgk in perivenous hepatocytes , and confirms previous observations using adenovirus . in contrast to the work of scott et al . and morral et al . , gk overexpression in perivenous hepatocytes does not significantly affect glc6pase expression , suggesting that zonation is an important experimental variable not sufficiently addressed to date in the field . in addition , we have demonstrated that gka456v overexpression reduces blood glucose to a great extent , probably due to its capacity to induce glc6pase . we conclude that impaired gkrp regulation and gka456v altered kinetics greatly influence liver metabolism , in line with results obtained in humans with a mutant gkrp . besides , it suggests that activating gk exclusively in the liver could be a feasible strategy for diabetes , widening the scope for gka research . similar conclusions , based on its potent induction of glucose clearance by the liver , have been obtained after liver - specific gk activation in chicken in a recent study by rideau et al . . studies on animal models of type 2 diabetes are under way in our laboratory in order to evaluate the metabolic impact in the context of insulin resistance and obesity .

recent reports point out the importance of the complex gk - gkrp in controlling glucose and lipid homeostasis . several gk mutations affect gkrp binding , resulting in permanent activation of the enzyme . we hypothesize that hepatic overexpression of a mutated form of gk , gka456v , described in a patient with persistent hyperinsulinemic hypoglycemia of infancy ( phhi ) and could provide a model to study the consequences of gk - gkrp deregulation in vivo . gka456v was overexpressed in the liver of streptozotocin diabetic mice . metabolite profiling in serum and liver extracts , together with changes in key components of glucose and lipid homeostasis , were analyzed and compared to gk wild - type transfected livers . cell compartmentalization of the mutant but not the wild - type gk was clearly affected in vivo , demonstrating impaired gkrp regulation . gka456v overexpression markedly reduced blood glucose in the absence of dyslipidemia , in contrast to wild - type gk - overexpressing mice . evidence in glucose utilization did not correlate with increased glycogen nor lactate levels in the liver . pepck mrna was not affected , whereas the mrna for the catalytic subunit of glucose-6-phosphatase was upregulated ~4 folds in the liver of gka456v - treated animals , suggesting that glucose cycling was stimulated . our results provide new insights into the complex gk regulatory network and validate liver - specific gk activation as a strategy for diabetes therapy .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

PMC4243745

porcelains are highly esthetic materials extensively used in dentistry to construct various types of restorations and prostheses such as porcelain fused to metal crowns , veneers , inlays , onlays and all ceramic restorations . they fulfill the esthetic and functional demands of the patients by their superior properties when compared to other restorative materials . the new glass ceramics ( ips d.sign ; ivoclar vivadent ag , schaan , liechtenstein ) have become popular for porcelain - fused - to - metal restorations . ips d.sign is a new type feldspathic - based porcelain containing dispersed fluorapatite and leucite crystals in a feldspathic glassy matrix . the leucite crystals ( <3 m ) present in the ips d.sign porcelain also contribute to the overall strength . recently , the new all ceramic systems ( ips e.max ; ivoclar vivadent ag , ) have been introduced into the market . ips e.max ceram is a veneering porcelain of this system which is a feldspathic - based porcelain having a microstructure unlike ips d.sign . this porcelain only consists of dispersed fluorapatite crystals in a feldspathic glassy matrix ; thus , having a microstructure unlike that of any other commercially available dental porcelains . fluorapatite crystals , 2 - 5 m in length and 300 nm in diameter of needle - like morphology , are known to be contained in natural bone and teeth . these very small crystals in dental microstructures result in very special optical properties such as translucence and opalescence , which also result the same properties as in dental restorations . despite the outstanding esthetic quality of the porcelains , these characteristics impair their physical properties such as surface roughness , surface hardness , strength and influence the clinical success and failure of porcelain restorations . crack propagation and degradation of dental porcelains occur when porcelains are exposed to aqueous solutions or erosive agents . alkaline metal ions are far less stable in the glass phase than in the crystalline phase . therefore , some alkaline ions in porcelains were leached after being exposed to acidic solutions . variation in ph , solution chemistry , wear and mechanical load makes the oral cavity a complex environment . consequences of porcelain degradation include coarseness of exposed surface , promoting plaque accumulation and wear to antagonist materials . the potential erosive effect of these acidic food and beverages on enamel occurs primarily by the dissolution of apatite crystals . however , their effect on the porcelain restorations has not been clearly documented . therefore , the present in vitro study was designed to evaluate changes of the flexural strength and surface of fluorapatite - leucite and fluorapatite porcelains after being exposed to erosive agents ( pineapple juice , green mango juice , coca soft drink , citrate buffer solution and 4% acetic acid ) in cyclic immersion . the null hypothesis was that there was no significant difference in flexural strength of each type of dental porcelain tested after being exposed to erosive agents . two commercial dentin shade a3 porcelain powders were used : ips d.sign and ips e.max ceram ( ivoclar vivadent ag ) ( figure 1 ) . ips d.sign and ips e.max ceram are indicated to be used as veneering porcelain for porcelain fused to metal and all ceramic restorations , respectively . one hundred and twenty bar specimens from each of the 2 porcelains were fabricated using the 26.0x6.0x3.0 mm silicone mold ( provil novo putty ; heraeus kulzer gmbh , d-63450 hanau , germany ) . the porcelain powders were mixed with deionized water , filled in the silicone mold and condensed with a condenser ( ceramosonic ii ; shofu inc , higashiyama - ku , kyoto , japan ) . the specimens were then fired according to the manufacturer 's instructions ( table 1 ) . after firing , the specimens were polished ( model phoenix 4000 ; buehler gmbh , 40599 dsseldorf , germany ) under running water using 600- and 1,200-grit silicon carbide paper ( 3 m espe , st . paul , mn , usa ) to the dimensions of 25.0x5.0x2.0 mm , following the guidelines of the iso 6872 standard . then , the specimens were ultrasonically cleaned in distilled water for 10 min , and subjected to self - glazing according to the manufacturer s instructions ( table 1 ) . porcelains used in the present study firing schedules for the porcelains used in the present study the porcelain bars were divided into 8 groups of 15 specimens each . subsequently , the specimens were alternately immersed in 25 ml of an erosive agent for 30 min and in 25 ml of deionized water for 5 min for 7 cycles at 37c . this amount of erosive agent ( 25 ml ) was a sufficient volume to completely cover the specimen . in order to maintain the original ph level of the erosive agent , the same protocol was used with different types of 5 erosive solutions included in the study ( citrate buffer solution , pineapple juice , green mango juice , cola soft drink and 4% acetic acid ; see figure 2 ) and deionized water ( control ) . the specimens immersion protocol simulated an individual eating acidic food , sour fruits and drinks . seventh group was continuously immersed in 4% acetic acid at 80c for 16 h ( as modified from iso 6872 ) in order to examine the extensive effect which could occur . a 8 group was kept dry at 37c for 245 min in order to compare the effect of moisture condition . after the immersion sequence was completed , the specimens were rinsed with deionized water , blotted dry and subjected to flexural strength testing . erosive agents used in the present study the flexural strength was measured with the universal testing machine ( model lrx - plus ; ametek lloyd instruments , farnborough , hampshire , uk ) . bar - shaped specimens were centered and placed on two steel spheres ( 1.6 mm in diameter ) of a supporter part positioned 12 mm apart from each other . three point bending tests were carried out using a 250 n load cell at crosshead speed 0.25 mm / min . the load at failure was recorded in newton and converted to flexural strength in mpa ( 3wl/2bd ; w = failure load , l = span length , b = specimen s width , and d = specimen s thickness ) . two - way anova was analyzed to measure statistically significant differences among the types of erosive agents and the type of porcelains after being exposed to erosive agents . tukey s honestly significant difference ( hsd ) tests were used for post hoc comparisons ( =0.05 ) . the t - test was used for comparing the flexural strength between the two types of porcelain for each erosive agent ( =0.05 ) . two commercial dentin shade a3 porcelain powders were used : ips d.sign and ips e.max ceram ( ivoclar vivadent ag ) ( figure 1 ) . ips d.sign and ips e.max ceram are indicated to be used as veneering porcelain for porcelain fused to metal and all ceramic restorations , respectively . one hundred and twenty bar specimens from each of the 2 porcelains were fabricated using the 26.0x6.0x3.0 mm silicone mold ( provil novo putty ; heraeus kulzer gmbh , d-63450 hanau , germany ) . the porcelain powders were mixed with deionized water , filled in the silicone mold and condensed with a condenser ( ceramosonic ii ; shofu inc , higashiyama - ku , kyoto , japan ) . the specimens were then fired according to the manufacturer 's instructions ( table 1 ) . after firing , the specimens were polished ( model phoenix 4000 ; buehler gmbh , 40599 dsseldorf , germany ) under running water using 600- and 1,200-grit silicon carbide paper ( 3 m espe , st . paul , mn , usa ) to the dimensions of 25.0x5.0x2.0 mm , following the guidelines of the iso 6872 standard . then , the specimens were ultrasonically cleaned in distilled water for 10 min , and subjected to self - glazing according to the manufacturer s instructions ( table 1 ) . porcelains used in the present study firing schedules for the porcelains used in the present study subsequently , the specimens were alternately immersed in 25 ml of an erosive agent for 30 min and in 25 ml of deionized water for 5 min for 7 cycles at 37c . this amount of erosive agent ( 25 ml ) was a sufficient volume to completely cover the specimen . in order to maintain the original ph level of the erosive agent , the same protocol was used with different types of 5 erosive solutions included in the study ( citrate buffer solution , pineapple juice , green mango juice , cola soft drink and 4% acetic acid ; see figure 2 ) and deionized water ( control ) . the specimens immersion protocol simulated an individual eating acidic food , sour fruits and drinks . seventh group was continuously immersed in 4% acetic acid at 80c for 16 h ( as modified from iso 6872 ) in order to examine the extensive effect which could occur . a 8 group was kept dry at 37c for 245 min in order to compare the effect of moisture condition . after the immersion sequence was completed , the specimens were rinsed with deionized water , blotted dry and subjected to flexural strength testing . the flexural strength was measured with the universal testing machine ( model lrx - plus ; ametek lloyd instruments , farnborough , hampshire , uk ) . bar - shaped specimens were centered and placed on two steel spheres ( 1.6 mm in diameter ) of a supporter part positioned 12 mm apart from each other . three point bending tests were carried out using a 250 n load cell at crosshead speed 0.25 mm / min . the load at failure was recorded in newton and converted to flexural strength in mpa ( 3wl/2bd ; w = failure load , l = span length , b = specimen s width , and d = specimen s thickness ) . two - way anova was analyzed to measure statistically significant differences among the types of erosive agents and the type of porcelains after being exposed to erosive agents . tukey s honestly significant difference ( hsd ) tests were used for post hoc comparisons ( =0.05 ) . the t - test was used for comparing the flexural strength between the two types of porcelain for each erosive agent ( =0.05 ) . the flexural strength values of the two types of porcelain were showed in table 2 . anova results showed that the interaction between the two variables ( type of porcelain and erosive agent ) found statistically significant difference ( p=0.02 ) . between the two dental porcelains , a statistically significant difference was also found ( p=0.01 ) , but none was found among the types of erosive agents ( p=0.46 ) . mean flexural strength standard deviations ( sd ) of ips d.sign and ips e.max ceram same superscript letters in columns indicate no significant difference ( tukey 's hsd test ; =0.05 ) . when comparing the mean flexural strength values between the porcelains for each group , the results of the t - test showed that all ips e.max ceram groups yielded a significantly higher mean flexural strength ( p<0.05 ) than that of ips d.sign groups . the results of this study support acceptance of the null hypothesis , as the flexural strength of both porcelains was not affected by the erosive agents . it is noticed that the highest flexural strength was found in the dry condition group ( stored at 37c ) of both porcelains and decreased in all groups when the porcelains were immersed in erosive agents as well as in water . the possible explanation for these results could be the effect of glazing in determining flexural strength values . porcelains , by nature , exhibit inherent flaws or defects on their surface and internal body . these flaws could impair their physical properties . however , the surface flaws are covered by the glazes , either self - glazing or overglazing . re - firing the porcelain prior to final restoration produces a self - glaze layer . this layer may increase the strength of the porcelain restoration from two possible mechanisms . firstly , when the restoration is heated , the self - glaze layer fills in surface flaws , reducing their depth and blunting the flaw tips . this should increase strength because , for given porcelains , strength increases with decreasing sharpness and flaw depth . secondly , for feldspathic - based porcelains , the self - glaze layer has a lower coefficient of thermal expansion than the leucite - rich interior . the compressive stress state diminishes the local tensile stress produced from applied loading at surface flaws , thereby needing application of increased load to initiate flaw propagation from the external surface . the ips e.max ceram had higher flexural strength than the ips d.sign in all groups . a possible explanation for this result could be the microstructure of these porcelains . the ips d.sign , feldspathic - based porcelain , is unique and distinct from other porcelains since its microstructure consists of fluorapatite crystal phases in addition to having leucite particles in a feldspathic glassy matrix , while the ips e.max ceram consists of only dispersed fluorapatite crystals in a feldspathic glassy matrix . in feldspathic - based porcelains , the leucite particles contract more than the surrounding glass upon cooling . above a critical particle size , the stresses created during cooling can induce microcracks circumferential to the leucite particles . previous studies have documented that the size of leucite particles in feldspathic porcelain increases during heat treatment within the normal porcelain firing range . in contrast to fluorapatite porcelains , the fluorapatite phase particles are needle - like and contribute to high flexural strength as well as high chemical durability . the erosive agents used in this present study , pineapple juice and green mango juice , are favorite sour fruit juices in many asia countries . they consist of citric acid and other organic acid , which give an acidic ph . however , in the present study , these juices did not affect the flexural strength of the tested porcelain after immersion , which do not agree with the findings of previous studies that showed an impact of acidic agents on porcelains . this study was a short - term experiment and could be the reason to explain why there was no significant difference among the acidic agents and this aspect should be explored . so , a long - term evaluation of the effect of erosive agents on porcelains is required . for example , the presence of water , temperature change , the ph level and the role of saliva in the oral cavity may considerably influence strengths of restorations . in addition , the present study evaluated only fluorapatite - leucite and fluorapatite porcelains . within the limitations of this study , the flexural strengths of the porcelains ( fluorapatite - leucite and fluorapatite porcelains ) after exposure to erosive agents in cyclic immersion were not significantly different . for both types of porcelain , dry storage at 37c yielded the highest flexural strength , though without significant difference from the other groups . the flexural strengths of all groups of fluorapatite porcelains were significantly higher than those of the fluorapatite - leucite porcelains .

objectivethe aim of this study was to evaluate the flexural strength of two porcelain materials ( ips d.sign and ips e.max ceram ) exposed to erosive agentsmaterial and methodsone hundred and twenty bar - shaped specimens were made from each of fluorapatite - leucite porcelain ( ips d.sign ) and fluorapatite porcelain ( ips e.max ceram ) and divided into 8 groups of 15 specimens each . six groups were alternately immersed in the following storage agents for 30 min : deionized water ( control ) , citrate buffer solution , pineapple juice , green mango juice , cola soft drink and 4% acetic acid . then , they were immersed for 5 min in deionized water at 37c . seven cycles were completed , totalizing 245 min . a 7th group was continuously immersed in 4% acetic acid at 80c for 16 h. the final , 8th , group was stored dry at 37c for 245 min . three - point bending tests were performed in a universal testing machine . the data were analyzed statistically by 2-way anova , tukey s hsd test and t - test at significance level of 0.05.resultsthe flexural strengths of all groups of each porcelain after exposure to erosive agents in cyclic immersion did not differ significantly ( p>0.05 ) . for both types of porcelain , dry storage at 37c yielded the highest flexural strength , though without significant difference from the other groups ( p>0.05 ) . the flexural strengths of all groups of fluorapatite porcelains were significantly higher ( p<0.05 ) than those of the fluorapatite - leucite porcelains.conclusionsthis study demonstrated that the erosive agents evaluated did not affect the flexural strength of the tested dental porcelains .

INTRODUCTION MATERIAL AND METHODS Specimen Preparation Erosive Agents Exposure Flexural Strength Measurements Statistical Analysis RESULTS DISCUSSION CONCLUSIONS

PMC3525969

docking simulations and virtual screening are being routinely used in drug design , enabling rapid identification of hits and lead compounds [ 1 - 3 ] . the goal of docking simulations is to determine the binding mode ( bound conformation ) and the strength of binding ( binding affinity ) between a ligand , which is typically assumed to be a small molecule , and a macromolecular receptor , such as a protein . given a resolved or modelled structure of a target receptor , virtual screening involves performing docking simulations for a large number of candidate compounds in order to identify putative leads . these candidates can subsequently be characterised and validated by empirical binding and activity assays , and by assessing their toxicity , pharmacokinetics and other properties for further drug development [ 7 - 9 ] . many methods for molecular docking and virtual screening have been developed to date , including autodock , dock,[12 - 14 ] flex , glide , gold , rosettadock , slide and surflex . these methods introduce various approximations to simplify the problem -- for example , assuming a rigid body docking model in which the receptor structure is fixed . rigid body docking allows one to speed - up computations by comparison with flexible docking ( in which the receptor structure is allowed to move ) by precomputing the forces experienced by the ligand on a grid . in general , docking simulations involve two main components : sampling algorithms to find plausible conformations of the complex , and scoring functions to estimate relative binding affinities and rank ligand poses . the sampling of alternative conformation is coupled with the search for the optimal solution --that is , poses with the highest binding affinity ( or score ) , which typically involves solving a global optimisation problem . consequently , various optimisation techniques -- such as monte carlo , simulated annealing or genetic algorithms -- are used in the context of docking simulations . atomic force fields that describe both intra- and intermolecular interactions in the system , simplified solvation potentials and empirical scoring functions are typically used to estimate the strength of interactions between the ligand and receptor and to score alternative poses of the ligand . docking methods are being constantly improved in each of these aspects , with newer implementations also seeking to take advantage of current computing architectures . following the progress in the field , reviews and comparisons of docking methods these surveys and evaluations focus primarily on re - docking experiments , benchmarking performance on sets of compounds with known binding affinities , and assessment of enrichment ( into true binders ) in the context of virtual screening [ 2,3,5,9,24 - 26 ] . a general conclusion from these studies is that no single method outperforms other methods consistently , suggesting that different targets may require different combinations of sampling approaches and scoring functions for optimal performance . this review focuses on several widely used public domain packages for docking and virtual screening . with recent improvements , these packages are becoming better integrated , more accurate and faster , and are poised to increase their impact as platforms of choice , especially within the academic community . these include three well - benchmarked and widely used public packages -- namely , autodock , dock and slide -- as well as the recently developed autodock vina ( also referred to as ad vina ) . below , some of the characteristics and distinct features of each program are summarised briefly . for detailed descriptions and documentation , public domain programs for small molecule docking and virtual screening assessed in this survey , with the most current version , parallel capabilities and the primary website shown mpi- and cuda - enabled versions available while each of the programs surveyed here offers different solutions to both the sampling and scoring problem , they provide a similar overall functionality and range of options reflecting improvements in docking methods . in particular , ' flexible ' models of both ligands and receptors are available in each of these programs , allowing one to sample not only different conformations of the ligand , but also of the receptor -- for example , by means of introducing a subset of residues ( active space ) for which alternative conformations can also be sampled to find optimal complex structure . in addition , these public domain docking programs have evolved to take advantage of the prevalence of distributed computing and the emergence of new computing platforms , as discussed in the next section . autodock offers well - benchmarked force fields ( including the widely used amber molecular force field in the latest version ) , efficient ( grid - based ) implementation of rigid body docking , a flexible ( receptor ) docking protocol with an active space consisting of residues which are allowed to undergo conformational changes , and efficient implementations of the lamarckian genetic algorithm ( ga ) to search for the ligand pose with the highest binding affinity , while sampling and ' mixing ' suboptimal solutions to solve the underlying global optimisation problem . in spite of its name , ad vina uses a very different search strategy , which couples newton - type local minimisation steps with global optimisation , involving concurrent ( on multicore central processing units [ cpus ] ) runs of a number of local minimisations which can be combined to improve sampling . dock uses a well - tested shape - matching approach to sample alternative poses of the ligand , given the receptor structure . these alternative conformations of the complex subsequently can be assessed and scored using the amber molecular force field coupled with several alternative implicit solvent models . some of the new features added in dock 6 include conjugate gradient minimisation and molecular dynamics simulation capabilities , ligand conformational entropy corrections , ligand and receptor desolvation and receptor flexibility . the mean - field theory - based model used in slide implies that multiple conformations of the receptor contribute to the interactions with the ligand , as the search proceeds through alternative conformations of the complex . this strategy allows one to account implicitly for receptor flexibility and induced - fit complementarity between the ligand and receptor . another advantage of slide is that its force field accounts explicitly for hydrogen bonds of different types . the speed of docking is clearly very important for virtual screening , and many recent improvements have aimed at taking full advantage of current distributed computing platforms . in this regard , dock ( as of version 5 ) offers an efficient mpi - based parallel implementation which can be used on computational clusters . an mpi version of autodock has also been developed , although it does not appear to be widely used . the advent of a novel parallel computing architecture -- compute unified device architecture ( cuda ) -- which utilises graphics processing units ( gpus ) , spurred the development of molecular modelling software for this new platform as well , including a recently developed cuda - enabled version of autodock . further improvements and developments in this regard are likely to improve the accuracy of virtual screening by enabling more extensive sampling and testing of the results . it should be noted , however , that even serial codes can be used efficiently for virtual screening by exploiting the ' embarrassing parallelism ' of docking ; this involves simply screening subsets of compounds from in silico libraries on individual cluster nodes . in this context , it has been reported to achieve a near 60-fold increase in speed ( with a further near - linear decrease in cpu time when using multiple cores ) , compared with autodock , with similar ( or better ) accuracy . in our own tests , similar increases were observed using the xeon(r ) x5570 2.93 ghz quad - core server , with the depth of sampling that allows one to achieve correlations between the predicted and experimental binding affinities reported by fang et al . for a benchmark set of 232 oestrogens . that benchmark is used to illustrate and compare the performance of the methods surveyed here . limitations of the current methods , such as those concerning scoring functions for docking , make the analysis of predicted complexes ( eg in terms of consistency of ligand poses across multiple runs ) an integral part of docking simulations and virtual screening . assessment of the results usually starts with a visual inspection of the predicted binding modes for hits , and subsequently can be extended using structural analysis , geometric clustering of predicted poses , re - scoring and cheminformatic approaches . the output from ligand docking programs usually includes a coordinate file ( in pdb format or other standard formats ) and supplementary data , such as estimated binding affinity , clustering of alternative poses , etc . to view coordinate files , commonly used macromolecular visualisation programs , such as jmol , pymol , visual molecular dynamics ( vmd ) and others can be employed , whereas additional information may require ad hoc programs to be used for data analysis . table 2 summarises several ( free for academic use , with the exception of more advanced functionality in dockingserver ) tools that enable the visualisation and assessment of predicted ligand poses in the protein - ligand complex , and provide other specifically designed features for the analysis of docking results . surveyed tools for the analysis of docking simulations and protein - ligand complexes ( presented in alphabetical order ) most tools discussed here are coupled with a specific docking program to facilitate setting up simulations and subsequent analysis of the results for that program . in this review , however , the survey of visualisation and analysis methods starts with ligplot , which is a simple tool that can be used in conjunction with any docking program ( ligplot does not provide any specific interfaces and the results are assumed to be in the pdb format ) . ligplot automatically generates a detailed two - dimensional diagram of protein - ligand interactions , with distances between interacting atoms and classification of interactions according to the type of interaction centres involved . therefore , ligplot can be easily coupled with docking pipelines to enable visualisation of docking poses . adt provides the possibility of viewing clustered alternative poses of a ligand accompanied by estimated docked energy , cluster size and the root mean square difference ( rmsd ) of the poses within a given cluster , as well as rmsd to the input ( reference ) structure . adt can also represent the ligand using the isocontour rendering that facilitates the visual evaluation of the model as to whether pairwise interactions between atoms of the receptor and ligand are reasonable . vslab is another tool that provides an improved interface to autodock , including setting the docking simulations and analysing them . results are represented using both tabular , easy - to - navigate views and graphical rendering by vmd . dockingserver automatically generates images of the docked ligands using vmd and provides the possibility for an interactive view using the jmol applet . in addition , the server generates summary tables that can be used to assess the main forces driving a specific protein - ligand interaction . viewdock combines the chimera rendering of docking models ( including animation ) with the interface for interactive analysis of multiple ligands docked to the receptor . the list of compounds and alternative poses can be sorted by the estimated binding energy , number of hydrogen bonds formed and other characteristics . the polyview - mm server combines multiple annotations for both the receptor structure and the protein - ligand interaction . alternative poses of the ligand can be visualised and analysed using the jmol applet or high - quality pymol rendering , as both static slides and animations . the server also enables analysis of clustering patterns computed by autodock for potential re - scoring of docking poses , provides a quick interface interactively to map residues in contact with the ligand for each pose and can be used to visualise distances for atom - atom pairs in multiple poses . additionally , the structure of the receptor ( and ligand binding sites in particular ) can automatically be annotated by mapping the location of structural domains , conserved residues , known ( derived from structurally resolved complexes in pdb ) and predicted ( using sppider ) protein interaction sites and other structural and functional features . nuclear receptors ( nrs ) are transcription factors that are activated by sex hormones and play major roles in embryogenesis , development and tumorigenesis . many such drugs have been specifically designed to compete with natural hormones for binding to ligand binding domains of nrs , including tamoxifen and raloxifen used in breast cancer . nrs are also activated by numerous environmental factors , adding to the importance of studying their interactions with small molecules . in particular , ers can be activated by many naturally occurring substances ( eg genistein found in soybean ) and industrially made compounds ( eg bisphenol a [ bpa ] ) which exhibit oestrogenic effects . in this context , this paper will now assess whether current docking programs are sufficiently accurate to provide a basis for the prediction of activity and toxicity risks for potential oestrogens , using the national center for toxicological research ( nctr ) er set of 232 structurally diverse natural , synthetic and environmental compounds with a wide range of biological activities that are potentially mediated by ers . this set provides an excellent benchmark for docking and structure - activity relationship ( sar ) models , by virtue of comparing predicted er binding affinities with experimental binding data obtained using a well validated in vitro rat uterine cytosol er alpha competitive binding assay . the nctr er benchmark also provides an opportunity to illustrate some of the features of the docking programs surveyed here . based on experimentally observed er relative binding affinities ( er - rba ) , each chemical was classified as active ( 131 chemicals ) or inactive ( 101 chemicals ) . the er - rba was calculated by dividing the concentration of chemical that causes 50 per cent inhibition of e2 binding ( ic50 ) of the natural er ligand , e2 , by the ic50 of the competitor , and multiplying the ratio obtained by 100 . thus , er - rba > 100 means a binding affinity greater than that of e2 . a compound was designated as inactive if 50 per cent inhibition was not reached within concentrations of competitor ranging from 1 nm to 1 mm ( versus 1 nm e2 ) , or if no activity was observed . this classification provides an opportunity further to assess the capacity of docking programs successfully to discriminate true hits and inactive compounds , with implications for virtual screening . the docking simulations for programs surveyed here were performed using e2- , raloxifen- and tamoxifen - bound crystal structures of the ligand binding domain of er alpha ( pdb codes 1ere , 1err and 3ert ) , using re - docking of native ligands to test if user - defined parameters and the simulation systems ( including the grid box ) had been properly set up for each of the programs . default values were used for most parameters , with the exception of those effectively defining the depth of the search , which were gradually increased to provide better sampling until no further improvement , in terms of overall correlations , was obtained . for example , the results reported here for autodock were obtained using 50 lamarckian ga runs , 10 million energy evaluation steps and a population size of 150 . with this somewhat extensive sampling , the average running time per compound was 2,987 cpu seconds on a xeon(r ) x5570 2.93 ghz quad - core server ( when using 1 cpu ) . an example of the top scoring pose for tamoxifen , obtained using dock and the tamoxifen - bound structure of the receptor , is shown in figure 1 . as can be seen from the figure , the predicted pose is very close to the native structure ( rmsd of about 1.9 for all atoms of the ligand ) . in addition , the predicted binding affinities for e2 , the er antagonist tamoxifen and the weak oestrogen bpa were found to follow a clear trend , consistent with experimental data . the strongest binding was found for tamoxifen , followed by relatively strong binding for e2 and much weaker binding for bpa . for example , rigid body docking with autodock yielded inhibition constants of 7.51 nm , 357 nm and 6.35 m for tamoxifen , e2 and bpa , using tamoxifen- , e2- and raloxifen - bound structures , respectively . binding of tamoxifen to the ligand binding domain of er alpha showing the native pose from a crystal structure of er alpha resolved in complex with tamoxifen ( pdb i d : 3ert ) in blue and the pose obtained by the re - docking of tamoxifen using dock in red . an all - atom rmsd of about 1.9 between the native and docking poses is obtained in this case , which is consistent with the level of success typically observed in re - docking experiments . the results of docking for nctr er compounds obtained using an ' intermediate ' er alpha structure with a binding pocket similar ( but not identical ) to that of e2 ( pdb code 1err ) are summarised in figures 2 and 3 . for active compounds , pearson correlation coefficients ( ccs ) between predicted and experimentally measured binding affinities of about 0.6 were obtained using slide and rigid body autodock ( see figure 2 ) , with somewhat lower ccs for flexible autodock , vina and dock . it should be noted , however , that each method failed to generate results for a number of compounds . therefore , these ccs can not be compared directly , as they were computed on a slightly different subset of compounds in each case . a direct comparison of ( rigid body ) autodock and slide is shown in figure 2 , and indicates high concordance between these two well - performing methods ( cc of 73 ) . correlations between experimental and predicted binding affinities using autodock and rigid - body docking affinity scores ( a ) and between predicted binding affinities using slide and autodock ( b ) . histograms of predicted binding affinities using slide binding affinity scores for active ( white bars ) and inactive ( crossed bars ) oestrogen - like compounds from the nctr er set . as can be seen from figure 3 , the distributions of scores for active and inactive compounds are shifted and , in fact , their means are significantly different , as assessed by a t - test ( p = 0.00001 ) . qualitatively similar results are obtained for other programs , although slide yields the best overall separation . applying a threshold of 8 for a scaled slide inhibition constant results in selecting about 80 per cent true positives ( active compounds ) , as opposed to about 56 per cent expected for a randomly selected subset . at the same time , about 20 per cent of inactive compounds are predicted to be active ( predicted scores larger than 8) . thus , a significant false - positive rate is observed ( even taking into account that some of the compounds classified as inactive may , in fact , be weak binders ) , reflecting the limitations of current docking methods . it should also be noted that the false - positive rate is likely to increase when docking arbitrary rather than potentially oestrogenic ligands , or when using receptor conformations that are different from a targeted bound state , as these factors contribute to rates of false positives ( and true negatives ) observed in virtual screening [ 2,6,24 - 27 ] . in this survey , several public domain docking programs , including autodock , autodock vina , dock and slide , were briefly described and their performance compared using the nctr er set , yielding encouraging ( albeit moderate ) correlations between predicted and experimentally observed binding affinities for active compounds ( with ccs of up to 0.6 ) . at the same time , however , all of the methods tested result in significant false - positive rates when used for discrimination between active and inactive compounds , underscoring the limitations of current docking methods . the importance of induced fit in the binding of oestrogens to ers , inherent limitations of scoring functions used by docking programs along with other factors are likely to contribute to the difficulties in virtual screening for candidate inhibitors of ers and pose limitations for the prediction of oestrogenicity . the field of toxicogenomics examines cellular responses to xenoestrogens , such as bpa , in order to provide a basis for the understanding of their mode(s ) of action and for improved risk assessment . assessing the health risk of environmental chemicals remains challenging , however , as multiple complex pathways may be involved in integrating the responses to chemical exposures . therefore , sar models are widely used to circumvent these limitations , and to predict the activity of chemical compounds based on their physicochemical and structural characteristics . sar methods typically rely on statistical and machine learning techniques to capture complex correlations between relevant descriptors and outcomes to be predicted . in this context , molecular docking could provide estimates of the strength of physical interactions with relevant receptors , to be used as complementary features for the development of more accurate sar models , even if the accuracy of binding affinity prediction is far from perfect . in conclusion , while limitations remain ( especially with regard to scoring functions ) , methodological advances and the development of more integrated , faster and easier - to - use current generation software packages for docking simulations , including the public domain programs surveyed here , are likely to expand further the realm and scope of applications of docking methods . these applications include virtual screening and drug development , functional annotations for apo - proteins , as well as the elucidation of the modes of action , and assessment of the toxicity , of environmental factors . computational resources were made available by cincinnati children 's hospital research foundation and university of cincinnati college of medicine .

progress in functional genomics and structural studies on biological macromolecules are generating a growing number of potential targets for therapeutics , adding to the importance of computational approaches for small molecule docking and virtual screening of candidate compounds . in this review , recent improvements in several public domain packages that are widely used in the context of drug development , including dock , autodock , autodock vina and screening for ligands by induced - fit docking efficiently ( slide ) are surveyed . the authors also survey methods for the analysis and visualisation of docking simulations , as an important step in the overall assessment of the results . in order to illustrate the performance and limitations of current docking programs , the authors used the national center for toxicological research ( nctr ) oestrogen receptor benchmark set of 232 oestrogenic compounds with experimentally measured strength of binding to oestrogen receptor alpha . the methods tested here yielded a correlation coefficient of up to 0.6 between the predicted and observed binding affinities for active compounds in this benchmark .

Introduction Docking programs surveyed Fast implementations of docking for computational clusters Tools for the visualisation and analysis of protein-ligand docking Oestrogen receptor (ER) binding benchmark Summary and conclusions Acknowledgements

PMC3424898

we present a case of incomplete vesical septum in a transverse plain encountered in a tertiary care center . a 52-year - old normotensive , nondiabetic married male having borne children , presented with low grade fever , progressively increasing frequency and urgency of urination , dull ache in left flank , and intermittent gross hematuria for the last 1 year . he also suffered from right flank pain 5 years ago and was treated by indigenous medication for suspected stone disease . he had received various antibiotics for documented urinary tract infections over the last few months . on further questioning , he was found to be suffering from nausea , hiccoughs , progressive weakness , easy fatigability , loss of weight and lack of appetite . on examination apart from mild suprapubic tenderness , the rest of abdominal examination including external genitalia was unremarkable . in addition to confirming anemia , ( hemoglobin 9.3 g% ) , his hematological investigations revealed polymorphonuclear leucocytosis ( 11 , 300/mm , polymorphs 92% ) . his renal parameters were deranged ( serum creatinine 8.9 mg% ) . despite microscopic pyuria and microhematuria , sample of his urine ziel neilson staining of centrifuged urine was found to contain acid fast bacilli suggestive of mycobacterium tuberculosis . ultrasonogram of abdomen and later on a nonenhanced ct scan confirmed multiple areas of calcification in right kidney and there was evidence of severe infundibular stenosis and thinning of renal parenchyma and collapsed right ureter in continuation with a small intra renal pelvis . left renal pelvis and ureter were dilated and had thick contents ( haunsfield value 25 ) . there was an unusual ledge of soft tissue seen running transversely in the anterior half of urinary bladder [ figure 1 ] . radionuclide scan could not be done due to high serum creatinine ( 8.9 mg% ) to document upper urinary tract transport disturbances . reformatted saggital image of ct scan showing vesical septum in the anterior half of urinary bladder a working diagnosis of tuberculosis of urinary tract with infected left hydroureteronephrosis , right thinned out and calcified renal parenchyma with deranged renal function was made . the patient underwent hemodialysis and was administered standard four drugs antitubercular treatment with dose modification as per the renal impairment . after stabilizing the general condition , he was taken up for cystoscopy , left retrograde ureteropyelogram and left dj stenting under spinal anesthesia . on cystopanendoscopy , there was evidence of pan urethritis , dilated prostatic urethra with no visible verumontanum . there was a transverse ledge of tissue connecting the two lateral walls of the bladder approximately 4 cm proximal to the bladder neck dividing the caudal vesical cavity into anterior and posterior parts [ figure 2 ] . these two parts were found to become one cavity crainially as could be seen in the reformatted saggital image of ct scan . the left ureteric orifice was placed anteriorly as anticipated and the entry to this orifice was also guarded by a transverse bar of mucosa , giving it a double barrel look . however , on cannulation with a soft 5 f ureteric catheter this was found to be a single ureter with bifid meatus ( see video clip in the supplementary material ) . left retrograde ureteropyelogram confirmed obstruction at 4 cm proximal to the ureterovesical junction with proximal hydroureteronephrosis . a gush of turbid urine escaped from the left ureter as the ureteric catheter was advanced into the kidney . a double j stent was placed into left pelvicalyceal system and multiple bladder biopsies were taken . postoperatively , the patient had brisk dieresis with fall in serum creatinine to 1.9 mg% in 72 h. his fluid and electrolytes were monitored meticulously and managed accordingly . the cystoscopic picture showing transverse ledge of tissue dividing the caudal vesical cavity into two antero - posterior parts histopathological examination of the bladder tissue obtained by cold cup biopsy confirmed tuberculosis . he has been advised to undergo right nephrectomy and follow up for left ureteric obstruction which might require ureteroneocystostomy . vesical septae and duplication of bladder appear to be rare congenital anomalies of the urinary bladder . various degrees of duplication of hindgut and bladder are thought to result from partial twinning of the tail portion of the embryo . duplication of bladder could be complete or incomplete with varying degrees of septa formation within the bladder . to our knowledge the present case is the only one with a transverse vesical septum reported in literature . these anomalies are usually associated with variations in other genitourinary organs like partial or complete duplication of urethra , congenital urethral valves , unilateral renal dysplasia , and unilateral gonadal agenesis , and many others . there might be associated congenital anomalies of the other organ systems like duplication of gut . in the present case , there was an incomplete transverse septum joining the two lateral walls of the bladder with a partial septum like mucosal fold causing left bifid ureteric orifice . with modern diagnostic tools these anomalies may be picked up in utero . our patient has presented with this congenital anomaly in the sixth decade of life and that too because of coexisting tuberculosis affecting the urinary system . the cystoscopic absence of verumontanum and the ct findings suggestive of small sized seminal vesicles have raised the doubts about his paternity status . he was advised to get a semen analysis done , which he refused . due to paucity of cases in printed literature , there is no uniformity in the treatment plan . most often than not , the septum has been resected to improve the lower urinary tract symptoms . in our case the patient became symptomatic only after 50 years of age and that to can be attributed to the coexisting tuberculosis . the septum which was running from one lateral wall to the other and the unusual bifid ureteric orifices , which appeared to have the same anomalous basis , i.e. , a transverse ledge of mucosa splitting the ureteric meatus into anterior and posterior parts ( as is evident in the video ) can not be explained on the basis of tuberculosis alone . since the patient remained asymptomatic till almost 50 years of life , it can be assumed that this anomaly was not per se , a cause of symptoms and therefore he may not have required any treatment , had he not contracted tuberculosis . after adequate anti tubercular cover , he was to undergo right nephrectomy and follow up management of left lower ureteric obstruction probably an ureteroneo cystostomy . he was not very keen to pursue allopathic treatment and decided to go for alternative medicine . therefore the overall outcome could not be assessed as he was lost to follow up . in view of varied morphological presentation , there is a need to review the classification of vesical septae especially in light of presence of incomplete septum as in the present case . in addition , the presence of bifid ureteric orifice raises interesting embryological issues surrounding the morphogenesis as this appears to be an incomplete septum of the ureteric orifice itself .

a rare case of incomplete transverse vesical septum with urinary tract tuberculosis is being reported . a 52-year - old male presented with lower urinary tract and systemic symptoms . on investigation he had anemia , leucocytosis , and high serum creatinine . on cystopanendoscopy , there was a transverse ledge of tissue connecting the two lateral walls of the bladder approximately 4 cm proximal to the bladder neck dividing the caudal vesical cavity into anterior and posterior parts . also , the left ureteric orifice had double barrel look . to our knowledge this is the first ever reported case of incomplete transverse vesical septum .

INTRODUCTION CASE REPORT DISCUSSION See video online:

PMC2868996

during the last decade , several new chemotherapeutic agents have been evaluated for their beneficial effect in patients with non - small - cell lung cancer ( nsclc ) . so far , gemcitabine plus cisplatin has emerged as one of the standard regimens for the treatment of advanced nsclc . gemcitabine ( 2,2-difluorodeoxycytidine ; dfdc ) is a fluoropyrimidine antimetabolite that is transported into cells by equilibrative nucleoside transporters . within the cell , gemcitabine is phosphorylated into gemcitabine monophosphate ( dfdcmp ) by deoxycytidine kinase ( dck ) and thereafter into di- and triphosphates . gemcitabine triphosphate ( dfdctp ) is incorporated in dna , and subsequently only one deoxynucleotide molecule more can be inserted , which leads to the halt of dna synthesis . this process is amplified through inhibition of the enzyme ribonucleotide reductase ( rnr ) by gemcitabine diphosphate ( dfdcdp ) . inhibition of rnr leads to intracellular depletion of dcdp and dctp , which favours the incorporation of dfdctp into dna [ 6 , 7 ] . only a small part of the gemcitabine dose is responsible for the cytotoxic effects , since more than 90% of the dose is inactivated by the enzyme cytidine deaminase ( cda ) into 2,2-difluorodeoxyuridine ( dfdu ) . cda is ubiquitous in the human body , catalyzing the hydrolytic deamination of ( deoxy-)cytidine to ( deoxy-)uridine . the total enzyme capacity of cda in all organs and tissues determines the biotransformation rate of gemcitabine into dfdu and thereby indirectly the duration of exposure to gemcitabine . interestingly , cloning of human cda has revealed a 79a > c nonsynonymous coding single nucleotide polymorphism ( csnp ) in exon 1 of the cda gene corresponding with two protein variants with more than twofold difference in vitro deamination rates [ 8 , 9 ] . the a genotype corresponds to the wild type lys - carrying enzyme and the c genotype to a gln - carrying variant ( gln27 cda ) . the wild type cda enzyme has been reported to exert a 1.3- to 3.3-fold higher deamination rate of cytarabine than gln27 cda . the aim of the current study was to evaluate the impact of the common 79a > c csnp in the cda gene on the pharmacokinetics of gemcitabine and its metabolite dfdu in gemcitabine - treated nsclc patients . this study was performed as a site study of a phase iii trial of gemcitabine plus epirubicin versus gemcitabine plus cisplatin in advanced nsclc patients . radiotherapy should have been completed at least 4 weeks before inclusion , and patients should have recovered from any toxic side effect . all patients had to have a performance status 2 according to the eastern cooperative oncology group ( ecog ) scale and a life expectancy of at least 12 weeks . an adequate bone marrow reserve and normal renal ( creatinine clearance 60 ml / min ) and liver function were required . the glomerular filtration rate ( gfr ) was calculated according to the formula of cockcroft and gault . patients were excluded if they had active infections , second primary malignancies , uncorrected hypercalcemia , or an lvef 45% measured by multiple gated acquisition ( muga ) scan . gemcitabine ( gemzar , lilly , nieuwegein , the netherlands ) in a dose of 1,125 mg / m in 250 ml 0.9% nacl solution was administered intravenously as a 30 min infusion . patients co - treated with epirubicin received the epirubicin dose after completion of the gemcitabine infusion . blood sampling blood sampling was carried out on the first day of the first chemotherapy cycle . for pharmacokinetic sampling , a cannula was placed intravenously in the arm of the patient contralateral to the side of drug administration . blood samples of 9 ml were collected in heparinized tubes containing 0.25 mg tetrahydrouridine ( thu ) in 50 l water , just before chemotherapy and at t = 25 , 40 , 50 , 60 , 75 , 90 , 105 , 120 , 150 , 180 , 270 , and 360 min after the start of the gemcitabine infusion . blood samples were immediately placed on ice and centrifuged at 2,500 g for 10 min within 1 h after collection . genotyping material harvesting of oral mucosa cells was performed before chemotherapy by thorough mouth rinsing for 30 s with 5 ml 0.9 % nacl solution . the cell suspension was collected in plastic cups and subsequently placed on ice . within 1 h after collection , the cell suspension was centrifuged for 10 min at 190 g at 4c . the supernatant was discarded and cells were washed in 10 ml ice cold phosphate buffered saline , ph = 7.40 ( pbs ) and centrifuged for 10 min at 190 g at 4c . the remaining suspension was transferred to a microcentrifuge cup and centrifuged briefly ( 15 s ) at 10,000 g. the supernatant was discarded , and the pellet was kept frozen at 80c until further analysis . gemcitabine hydrochloride and dfdu the analysis of gemcitabine and dfdu in plasma was carried out by high performance liquid chromatography as described by freeman et al . . the cda exon 1 polymorphism ( c / a ) at codon position 27 was genotyped by direct sequencing , in both directions , of pcr - amplified genomic dna . first , the cda region flanking the polymorphic site was amplified using pcr with forward primer 5- agtagcttccccttccagtagc and reverse primer 5-cctcttcctgtacatcttcctctg . the 25 l reactions contained 2.5 units taq polymerase ( amersham biosciences , uppsala , sweden ) ; 0.5 mm dntp mix ( roche diagnostics , mannheim , germany ) ; 1x pcr buffer ( roche diagnostics ) , 0.05 mm mgcl2 ; 0.2 m of each primer ; and approximately 50 ng genomic dna . the amplification was performed on a ptc-225 thermal cycler ( mj research , waltham , ma ) , using a stepdown protocol . the first five cycles were carried out at 94c , 65c , and 72c , each for 30 s. the next five cyles were carried out at 94c , 63c , and 72c , each for 30 s. the last 25 cyles were at 94c , 60c , and 72c , each for 30 s. following cycling the pcr products were purified with the qiagen qiaquick pcr purification kit ( westburg , leusden , the netherlands ) . subsequently , 100 ng of the purified pcr product was cycle sequenced with a dyeterminator kit ( us81090 , amersham biosciences , roosendaal , the netherlands ) in a thermal cycler ( mj research ) , using 0.05 mm sequencing primer . for the reverse reaction , the same primer was used as in the pcr , but for the forward reaction an internal primer was used ( 5-ggtaccaacatggcccagaag ) . after the cycle reaction the sequencing products were cleaned on a sephadex plate ( amersham biosciences ) by centrifugation for 5 min at 910 g. the eluted sample was vacuum dried for 45 min at 65c . finally , 20 l of loading solution ( amersham biosciences ) was added to dissolve the sequencing products . the samples were analyzed on a megabace 1000 capillary sequencer ( amersham biosciences ) by injecting the samples for 45 s at 3 kv and running them for 5 h at 4 kv . the data were processed with sequence analyzer 3.0 ( amersham biosciences ) and seqman ii ( dnastar , madison , wi ) . pharmacokinetic data were analyzed with the mw\pharm software package ( version 3.5 ; mediware , groningen , the netherlands ) using the kinfit module . for gemcitabine and its metabolite dfdu , the auc ( using the trapezoid rule ) , clearance , distribution volume , and elimination half - lives were calculated by non - compartmental analysis . differences in laboratory and demographic data between groups were tested using the mann - whitney test . cda genotype - related differences in pharmacokinetic data were investigated by analyzing the wild type aa genotype patient group against the combined heterozygote ac and homozygote cc genotype patient group using the mann - whitney test . we assumed an effect of 30% of the alternative genotype on the gemcitabine auc as clinically relevant . from the literature , the allele frequency of the wild type genotype was estimated at 0.7 , and therefore an almost equal distribution of patients in aa wild type ( 49% ) and combined homozygote cc plus heterozygote ac ( 51% ) patient groups was expected . we calculated that group sample sizes of 10 and 10 were needed to achieve 92% power to detect a difference of 30% between the null hypothesis that both group means are equal and the alternative hypothesis that the mean auc of the mutant group is 30% larger , with group standard deviations of 20% and with a significance level of 0.05 using a two - side mann - whitney test assuming that the actual distribution is logistic . additionally , differences between uncombined cda genotype groups regarding gemcitabine auc , clearance , and half - life were also analyzed using the kruskall - wallis test . statistical analyses were performed using the spss 14.0 statistical package ( spss 2005 , chicago , il ) . power calculations were performed using ncss 2004 statistical & power analysis software ( ncss , kaysville , ut ) . this study was performed as a site study of a phase iii trial of gemcitabine plus epirubicin versus gemcitabine plus cisplatin in advanced nsclc patients . radiotherapy should have been completed at least 4 weeks before inclusion , and patients should have recovered from any toxic side effect . all patients had to have a performance status 2 according to the eastern cooperative oncology group ( ecog ) scale and a life expectancy of at least 12 weeks . an adequate bone marrow reserve and normal renal ( creatinine clearance 60 ml / min ) and liver function were required . the glomerular filtration rate ( gfr ) was calculated according to the formula of cockcroft and gault . patients were excluded if they had active infections , second primary malignancies , uncorrected hypercalcemia , or an lvef 45% measured by multiple gated acquisition ( muga ) scan . gemcitabine ( gemzar , lilly , nieuwegein , the netherlands ) in a dose of 1,125 mg / m in 250 ml 0.9% nacl solution was administered intravenously as a 30 min infusion . patients co - treated with epirubicin received the epirubicin dose after completion of the gemcitabine infusion . blood sampling blood sampling was carried out on the first day of the first chemotherapy cycle . for pharmacokinetic sampling , a cannula was placed intravenously in the arm of the patient contralateral to the side of drug administration . blood samples of 9 ml were collected in heparinized tubes containing 0.25 mg tetrahydrouridine ( thu ) in 50 l water , just before chemotherapy and at t = 25 , 40 , 50 , 60 , 75 , 90 , 105 , 120 , 150 , 180 , 270 , and 360 min after the start of the gemcitabine infusion . blood samples were immediately placed on ice and centrifuged at 2,500 g for 10 min within 1 h after collection . genotyping material harvesting of oral mucosa cells was performed before chemotherapy by thorough mouth rinsing for 30 s with 5 ml 0.9 % nacl solution . the cell suspension was collected in plastic cups and subsequently placed on ice . within 1 h after collection , the cell suspension was centrifuged for 10 min at 190 g at 4c . the supernatant was discarded and cells were washed in 10 ml ice cold phosphate buffered saline , ph = 7.40 ( pbs ) and centrifuged for 10 min at 190 g at 4c . the remaining suspension was transferred to a microcentrifuge cup and centrifuged briefly ( 15 s ) at 10,000 g. the supernatant was discarded , and the pellet was kept frozen at 80c until further analysis . the analysis of gemcitabine and dfdu in plasma was carried out by high performance liquid chromatography as described by freeman et al . . the cda exon 1 polymorphism ( c / a ) at codon position 27 was genotyped by direct sequencing , in both directions , of pcr - amplified genomic dna . first , the cda region flanking the polymorphic site was amplified using pcr with forward primer 5- agtagcttccccttccagtagc and reverse primer 5-cctcttcctgtacatcttcctctg . the 25 l reactions contained 2.5 units taq polymerase ( amersham biosciences , uppsala , sweden ) ; 0.5 mm dntp mix ( roche diagnostics , mannheim , germany ) ; 1x pcr buffer ( roche diagnostics ) , 0.05 mm mgcl2 ; 0.2 m of each primer ; and approximately 50 ng genomic dna . the amplification was performed on a ptc-225 thermal cycler ( mj research , waltham , ma ) , using a stepdown protocol . the first five cycles were carried out at 94c , 65c , and 72c , each for 30 s. the next five cyles were carried out at 94c , 63c , and 72c , each for 30 s. the last 25 cyles were at 94c , 60c , and 72c , each for 30 s. following cycling the pcr products were purified with the qiagen qiaquick pcr purification kit ( westburg , leusden , the netherlands ) . subsequently , 100 ng of the purified pcr product was cycle sequenced with a dyeterminator kit ( us81090 , amersham biosciences , roosendaal , the netherlands ) in a thermal cycler ( mj research ) , using 0.05 mm sequencing primer . for the reverse reaction , the same primer was used as in the pcr , but for the forward reaction an internal primer was used ( 5-ggtaccaacatggcccagaag ) . after the cycle reaction the sequencing products were cleaned on a sephadex plate ( amersham biosciences ) by centrifugation for 5 min at 910 g. the eluted sample was vacuum dried for 45 min at 65c . finally , 20 l of loading solution ( amersham biosciences ) was added to dissolve the sequencing products . the samples were analyzed on a megabace 1000 capillary sequencer ( amersham biosciences ) by injecting the samples for 45 s at 3 kv and running them for 5 h at 4 kv . the data were processed with sequence analyzer 3.0 ( amersham biosciences ) and seqman ii ( dnastar , madison , wi ) . pharmacokinetic data were analyzed with the mw\pharm software package ( version 3.5 ; mediware , groningen , the netherlands ) using the kinfit module . for gemcitabine and its metabolite dfdu , the auc ( using the trapezoid rule ) , clearance , distribution volume , and elimination half - lives were calculated by non - compartmental analysis . differences in laboratory and demographic data between groups were tested using the mann - whitney test . cda genotype - related differences in pharmacokinetic data were investigated by analyzing the wild type aa genotype patient group against the combined heterozygote ac and homozygote cc genotype patient group using the mann - whitney test . we assumed an effect of 30% of the alternative genotype on the gemcitabine auc as clinically relevant . from the literature , the allele frequency of the wild type genotype was estimated at 0.7 , and therefore an almost equal distribution of patients in aa wild type ( 49% ) and combined homozygote cc plus heterozygote ac ( 51% ) patient groups was expected . we calculated that group sample sizes of 10 and 10 were needed to achieve 92% power to detect a difference of 30% between the null hypothesis that both group means are equal and the alternative hypothesis that the mean auc of the mutant group is 30% larger , with group standard deviations of 20% and with a significance level of 0.05 using a two - side mann - whitney test assuming that the actual distribution is logistic . additionally , differences between uncombined cda genotype groups regarding gemcitabine auc , clearance , and half - life were also analyzed using the kruskall - wallis test . statistical analyses were performed using the spss 14.0 statistical package ( spss 2005 , chicago , il ) . power calculations were performed using ncss 2004 statistical & power analysis software ( ncss , kaysville , ut ) . table 1patient characteristicsall patients ( n = 20)cytidine deaminase genotype subgroupsa / a ( n = 8)a / c ( n = 8)c / c ( n = 4)gender ( m / f)17/38/06/23/1age ( years)65 ( 4473)67 ( 5573)65 ( 5773)59 ( 4473)weight ( kg)77 ( 5897)81(6097)75 ( 5887)85 ( 6388)length ( cm)175 ( 161192)177 ( 168193)173 ( 161185)182 ( 169192)bsa ( m)1.93 ( 1.612.27)2.00 ( 1.692.27)1.90 ( 1.612.03)2.09 ( 1.722.13)creatinine ( mol / l)78 ( 49119)80 ( 60119)75 ( 4999)81 ( 6692)crcl ( ml / min ) ( normal : > 60)97 ( 51134)97 ( 51130)88 ( 64106)104 ( 59134)ast ( u / l ) ( normal : < 45)22 ( 1535)22 ( 1235)22 ( 1528)21 ( 1727)alt ( u / l ) ( normal : < 45)27 ( 747)23 ( 732)29 ( 1547)26 ( 1236)bilirubin ( mol / l ) ( normal : < 20)10 ( 617)10 ( 616)10 ( 617)10 ( 911)co - treatmentepirubicin10532cisplatin10352values are medians with the ranges in parentheses patient characteristics values are medians with the ranges in parentheses the groups were comparable with respect to demographic parameters , renal function , and liver enzymes . all patients , except one , had ast levels below the upper limit of normal . two patients had a slightly reduced creatinine clearance ( crcl ) , between 50 and 60 ml / min . mean pharmacokinetic curves of gemcitabine and dfdu in plasma are presented in fig . 1 according to the different genotypes . gemcitabine was rapidly cleared from plasma and undetectable after 3 h in all patients . 1pharmacokinetics of gemcitabine and its metabolite dfdu after a 30 min infusion of 1,125 mg / m gemcitabine grouped according to genotype of the cytidine deaminase 79a > c polymorphismtable 2gemcitabine pharmacokineticsall patients ( n = 20)cytidine deaminase genotype subgroupsp - value ( mw)p - value ( kw)a / a ( n = 8)a / c ( n = 8)c / c ( n = 4)a / c+c / c ( n = 12)gemcitabinedose ( mg)2,098 ( 1,9352,800)2,199 ( 1,9702,400)2,024 ( 2,0002,200)2,359 ( 1,9352,800)2,093 ( 1,9352,800)auc03 h ( mgh / l)8.6 ( 6.022.2)8.7 ( 6.122.2)8.6 ( 7.117.3)9.7 ( 6.012.9)8.6 ( 6.017.3)0.9390.988vdistribution ( l)72 ( 37120)83 ( 37120)75 ( 59105)52 ( 3978)70 ( 39105)0.3160.137clmetabolic ( l / h)246 ( 106466)251 ( 106328)232 ( 118300)248 ( 164466)232 ( 118466)0.5370.736t1/2 elimination ( h)0.22 ( 0.080.48)0.26 ( 0.080.48)0.23 ( 0.170.31)0.16 ( 0.080.21)0.21 ( 0.080.31)0.3160.094dfduauc03 h ( mgh / l)60.5 ( 44.698.6)64.7 ( 46.898.6)55.8 ( 44.671.8)60.6 ( 53.981.1)56.1 ( 44.681.1)0.5890.648 t 1/2 elimination ( h)2.43 ( 1.625.32)2.49 ( 2.095.32)2.14 ( 1.624.25)2.21 ( 1.673.80)2.13 ( 1.624.25)0.1900.419values are medians with the ranges in parenthesesp - values resulting from the mann - whitney ( mw ) test on the difference between aa versus combined ac+cc genotypep - values resulting from the kruskal - wallis ( kw ) test on differences among aa , ac , and cc genotypes pharmacokinetics of gemcitabine and its metabolite dfdu after a 30 min infusion of 1,125 mg / m gemcitabine grouped according to genotype of the cytidine deaminase 79a > c polymorphism gemcitabine pharmacokinetics values are medians with the ranges in parentheses p - values resulting from the mann - whitney ( mw ) test on the difference between aa versus combined ac+cc genotype p - values resulting from the kruskal - wallis ( kw ) test on differences among aa , ac , and cc genotypes the influence of the presence of epirubine on gemcitabine pharmacokinetics was checked by grouping the patients according to treatment schedule ( gemcitabine plus cisplatin or gemcitabine plus epirubicine ) . no difference in gemcitabine pharmacokinetics was measured between the two groups using the mann - whitney u - test . the gemcitabine pharmacokinetic data of all 20 patients were analyzed with respect to the impact of the 79a > c polymorphism in the cda gene . the frequencies of the a / a , a / c , and c / c genotypes were 0.40 , 0.40 , and 0.20 respectively . the allele frequency of the 79a > c polymorphism was 0.40 in our study . the mean pharmacokinetic curves of gemcitabine and dfdu according to cda genotype are presented in fig . 1 . no differences in pharmacokinetic parameters were observed between the wild type aa genotype group and the combined heterozygote ac and homozygote cc genotype group , using the mann - whitney test . additionally , in the kruskal - wallis analysis , we found no significant differences in gemcitabine auc , clearance , or serum half - life among the different cda genotypes ( see table 2 ) . table 1patient characteristicsall patients ( n = 20)cytidine deaminase genotype subgroupsa / a ( n = 8)a / c ( n = 8)c / c ( n = 4)gender ( m / f)17/38/06/23/1age ( years)65 ( 4473)67 ( 5573)65 ( 5773)59 ( 4473)weight ( kg)77 ( 5897)81(6097)75 ( 5887)85 ( 6388)length ( cm)175 ( 161192)177 ( 168193)173 ( 161185)182 ( 169192)bsa ( m)1.93 ( 1.612.27)2.00 ( 1.692.27)1.90 ( 1.612.03)2.09 ( 1.722.13)creatinine ( mol / l)78 ( 49119)80 ( 60119)75 ( 4999)81 ( 6692)crcl ( ml / min ) ( normal : > 60)97 ( 51134)97 ( 51130)88 ( 64106)104 ( 59134)ast ( u / l ) ( normal : < 45)22 ( 1535)22 ( 1235)22 ( 1528)21 ( 1727)alt ( u / l ) ( normal : < 45)27 ( 747)23 ( 732)29 ( 1547)26 ( 1236)bilirubin ( mol / l ) ( normal : < 20)10 ( 617)10 ( 616)10 ( 617)10 ( 911)co - treatmentepirubicin10532cisplatin10352values are medians with the ranges in parentheses patient characteristics values are medians with the ranges in parentheses the groups were comparable with respect to demographic parameters , renal function , and liver enzymes . all patients , except one , had ast levels below the upper limit of normal . two patients had a slightly reduced creatinine clearance ( crcl ) , between 50 and 60 ml / min . mean pharmacokinetic curves of gemcitabine and dfdu in plasma are presented in fig . 1 according to the different genotypes . gemcitabine was rapidly cleared from plasma and undetectable after 3 h in all patients . 1pharmacokinetics of gemcitabine and its metabolite dfdu after a 30 min infusion of 1,125 mg / m gemcitabine grouped according to genotype of the cytidine deaminase 79a > c polymorphismtable 2gemcitabine pharmacokineticsall patients ( n = 20)cytidine deaminase genotype subgroupsp - value ( mw)p - value ( kw)a / a ( n = 8)a / c ( n = 8)c / c ( n = 4)a / c+c / c ( n = 12)gemcitabinedose ( mg)2,098 ( 1,9352,800)2,199 ( 1,9702,400)2,024 ( 2,0002,200)2,359 ( 1,9352,800)2,093 ( 1,9352,800)auc03 h ( mgh / l)8.6 ( 6.022.2)8.7 ( 6.122.2)8.6 ( 7.117.3)9.7 ( 6.012.9)8.6 ( 6.017.3)0.9390.988vdistribution ( l)72 ( 37120)83 ( 37120)75 ( 59105)52 ( 3978)70 ( 39105)0.3160.137clmetabolic ( l / h)246 ( 106466)251 ( 106328)232 ( 118300)248 ( 164466)232 ( 118466)0.5370.736t1/2 elimination ( h)0.22 ( 0.080.48)0.26 ( 0.080.48)0.23 ( 0.170.31)0.16 ( 0.080.21)0.21 ( 0.080.31)0.3160.094dfduauc03 h ( mgh / l)60.5 ( 44.698.6)64.7 ( 46.898.6)55.8 ( 44.671.8)60.6 ( 53.981.1)56.1 ( 44.681.1)0.5890.648 t 1/2 elimination ( h)2.43 ( 1.625.32)2.49 ( 2.095.32)2.14 ( 1.624.25)2.21 ( 1.673.80)2.13 ( 1.624.25)0.1900.419values are medians with the ranges in parenthesesp - values resulting from the mann - whitney ( mw ) test on the difference between aa versus combined ac+cc genotypep - values resulting from the kruskal - wallis ( kw ) test on differences among aa , ac , and cc genotypes pharmacokinetics of gemcitabine and its metabolite dfdu after a 30 min infusion of 1,125 mg / m gemcitabine grouped according to genotype of the cytidine deaminase 79a > c polymorphism gemcitabine pharmacokinetics values are medians with the ranges in parentheses p - values resulting from the mann - whitney ( mw ) test on the difference between aa versus combined ac+cc genotype p - values resulting from the kruskal - wallis ( kw ) test on differences among aa , ac , and cc genotypes the influence of the presence of epirubine on gemcitabine pharmacokinetics was checked by grouping the patients according to treatment schedule ( gemcitabine plus cisplatin or gemcitabine plus epirubicine ) . no difference in gemcitabine pharmacokinetics the gemcitabine pharmacokinetic data of all 20 patients were analyzed with respect to the impact of the 79a > c polymorphism in the cda gene . the frequencies of the a / a , a / c , and c / c genotypes were 0.40 , 0.40 , and 0.20 respectively . the allele frequency of the 79a > c polymorphism was 0.40 in our study . the mean pharmacokinetic curves of gemcitabine and dfdu according to cda genotype are presented in fig . 1 . no differences in pharmacokinetic parameters were observed between the wild type aa genotype group and the combined heterozygote ac and homozygote cc genotype group , using the mann - whitney test . additionally , in the kruskal - wallis analysis , we found no significant differences in gemcitabine auc , clearance , or serum half - life among the different cda genotypes ( see table 2 ) . in this study , we investigated the impact of the common 79a > c nonsynonymous single nucleotide polymorphism in the cytidine deaminase gene on gemcitabine and dfdu pharmacokinetics in nsclc patients . in all patients gemcitabine was rapidly cleared from plasma , as was expected from previous reports . the most important factor that determines the clearance rate of gemcitabine is the activity of cda . the total enzyme capacity of cda in all organs and tissues determines the biotransformation rate of gemcitabine into dfdu and thus the duration of exposure to gemcitabine . despite increasing interest in dna polymorphisms related to gemcitabine metabolism , so far only a few reports have been published on the relation between cda polymorphisms and gemcitabine pharmacokinetics . the 79a > c csnp is the most common cda polymorphism with previously reported allele frequencies of 0.20 in japanese , 0.11 in african americans , 0.04 in africans , 0.30 in caucasian americans , and 0.36 in european caucasians [ 8 , 1416 ] . the polymorphism at nucleotide position 79 corresponds to codon27 , which is located within an n - terminal core domain and might be involved in binding pocket loops that cooperate with zinc ligands [ 15 , 17 ] . the a genotype corresponds to the wild type lys - carrying enzyme and the c genotype to a gln - carrying variant ( gln27 cda ) . kirch et al . found that the deamination rate of cytarabine by bacterial wild type cda enzyme was a 1.33.3 fold higher than by gln27 cda . recently reported in a functional genomics study in mammalian cells that the activity of human recombinant gln27 cda was about 66% of the wild type cda activity for gemcitabine . thus , assuming highest cda activity in a / a , intermediate activity in a / c , and lowest in c / c diplotypes , we studied the consequences of this polymorphism on gemcitabine auc , clearance , and serum half - life . all patients in our study were caucasian europeans , and in this group we found an allele frequency of 0.40 , which is in line with the allele frequencies of 0.30 and 0.36 earlier reported for caucasians [ 8 , 16 ] . obviously , our study was powered to detect differences of at least 30% in population means and smaller differences in pharmacokinetic parameters may have gone unnoticed . in the post - hoc statistical analysis , we found a much larger variation in gemcitabine auc values , ranging from 3050% , than the initially expected 20% . as a result we achieved only 25% power to detect a difference . we reasoned however that , due to the fact that the data in both groups largely overlap , it seems very unlikely that dose adjustment according to cda genotype will become clinically feasible , if a difference might exist . interestingly , this matter has recently been elucidated , as , in parallel with our study , sugiyama et al . investigated the 79a > c polymorphism in a population of 251 japanese patients . in line with our data , they could not detect any effect of the 79a > c polymorphism on the gemcitabine pharmacokinetics after a 30 min 8001,000 mg / m infusion . it is important to realize that the two studies differ with respect to administered gemcitabine dose and patient ethnicity . the mean dose in our study was 38% higher as a result of both a higher standard dose ( 1,125 mg / m ) and generally higher body surface area ( bsa ) values in caucasians . obviously , these factors do not change or modulate the observed lack of impact on pharmacokinetic outcomes of the 79a > c polymorphism . one explanation for our findings may be a far more complex regulation of cda gene transcription , leading to substantial interindividual differences in quantities of cda enzyme , unrelated to the 79a > c polymorphism . demonstrated reduced enzyme activity with the 79a > c polymorphism using in vitro deamination of ara - c and dfdc , respectively , it is possible that additional polymorphisms within cda also influence enzyme activity or that variants in other enzymes in gemcitabine metabolism play a greater than expected role . 5 flanking region contains numerous potential transcription factor binding sites that can cause variations in transcription . unfortunately , we did not perform direct measurement of cda activity in our patients and thus could not check the genotype - phenotype relation at the enzyme level . moreover , we hypothesized that possibly the total capacity of cda in the body may far exceed the amount needed to cope with plasma levels resulting from 8001,125 mg / m gemcitabine doses . in that case , differences in pharmacokinetics may only become manifest at much higher plasma concentrations when michaelis - menten enzyme saturation is becoming relevant . finally , the pharmacokinetic results might have been dispersed as a result of co - administration of other chemotherapeutic agents . indeed , all patients in our study received combined chemotherapy , but cisplatin was administered on day 2 and therefore did not interact with our results . we analyzed that co - administration of epirubicin did not influence the pharmacokinetics of gemcitabine , which is in line with previously reported findings regarding the pharmacokinetics of both drugs when combined in treatment schedules [ 1921 ] . > c polymorphism on gemcitabine pharmacokinetics , the polymorphism still may be relevant at the cellular level . recently demonstrated that the post - induction treatment - related mortality in children treated for acute myeloid leukemia with high dose cytarabine was significantly higher in children bearing the cc compared to the ac or aa genotype . furthermore , tibaldi et al . showed that the wild type cda lys27lys ( aa ) genotype was associated with better clinical outcome , longer time to progression , and overall survival . interestingly , they also found an increased cda enzymatic activity in red blood cells associated with the cda gln / gln ( cc ) genotype . although these findings contradict previous results from in - vitro functional genomics experiments by kirch et al . and gilbert et al . , this clinical study illustrates that the association between genotype and treatment efficacy may be quite complex . recently two new polymorphisms in the cda gene were reported . these concern the 208g > a and 435t > c polymorphism with allele frequencies of 0.04 and 0.30 in japanese patients , respectively . the 208g > a polymorphism produces an alanine - to - threonine substitution ( ala70thr ) within the conserved catalytic domain . introduction of this gene in yeast null mutants resulted in a 20% reduction of the 50% inhibitory concentration value for cytarabine . sugiyama et al . also studied both variants and found a decreased gemcitabine clearance in patients with the 208g > a polymorphism . however , the 208g > a polymorphism has not been detected so far in caucasians and was therefore not included in our study . the 435t > c polymorphism ( thr145thr ) does not encode for an amino acid change . since an impact of this silent 425t > c polymorphism on cda enzyme activity was expected to be less probable , we did not include this polymorphism in our investigations . we conclude that the common 79a > c cda polymorphism does not have a major consistent , significant effect on gemcitabine plasma clearance . the full pharmacogenetic picture of cda in relation to gemcitabine clearance and gemcitabine pharmacodynamics may nevertheless be quite complex and can only be elucidated in large clinical trials .

objectiveto study the impact of the 79a > c polymorphism in the cytidine deaminase ( cda ) gene on the pharmacokinetics of gemcitabine and its metabolite 2,2-difluorodeoxyuridine ( dfdu ) in non - small - cell lung cancer ( nsclc ) patients.patients and methodspatients ( n = 20 ) received gemcitabine 1,125 mg / m2 as a 30 min i.v . infusion as part of treatment for nsclc . plasma samples were collected during 06 h after gemcitabine administration . gemcitabine and dfdu were quantified by high performance liquid chromatography with ultraviolet detection . the cda 79a > c genotype was determined with pcr and dna sequencing.resultsgemcitabine was rapidly cleared from plasma and undetectable after 3 h. the allele frequency of the 79a > c polymorphism was 0.40 . diplotypes were distributed as a / a n = 8 , a / c n = 8 , and c / c n = 4 . no significant differences were found between the different cda genotypes and gemcitabine or dfdu auc , clearance , or half-life.conclusionthe 79a > c polymorphism in the cda gene does not have a major consistent and signficant impact on gemcitabine pharmacokinetics .

Introduction Patients and methods Patient selection Treatment and sample collection Pharmacokinetic analysis Cytidine deaminase genotyping Pharmacokinetic modelling Statistical analysis Results Patients Pharmacokinetics Pharmacogenetic analysis Discussion Conclusions

PMC4299867

the issue of enhancement has been tested in the sports arena around the debate about what technology might give an unfair performance enhancement . the most high profile example of which is that of south african sprinter oscar pistorius . when he was a competitive sprinter pistorius was known as the fastest man on no legs since he is a double below knee amputee ( zehr , 2011 ) . pistorius was born with fibular hemimelia ( or longitudinal fibular deficiency ) meaning he did not have fibulas in his legs . based on this , the chances of pistorius being able to stand and walk were considered very low . accordingly , his parents took the decision when he was about 11 months old to allow surgical amputation of his lower legs . the functional implication of this bilateral double leg amputation was to allow oscar the use of appropriate prosthetics that could carry his weight during walking and running . for running he used prosthetic legs called blades , so named because the physical shape of the carbon fiber prosthetic looks like a blade . the prosthetics were produced by ossur and the model was the ossur flex - foot cheetah ( curran & hirons , 2012 ) . eventually he became the most accomplished and dominant double - amputee sprinter the world has ever seen . the flex - foot part of the prosthetic has caused controversy , though , because that it has been implicated with facilitating his running stride . this is also coupled with the fact that the prosthetic blades are lighter than an intact human lower leg would be . the sum combination of the prosthetic limbs and superb sprinting biomechanics created considerable controversy when oscar competed in an international association of athletics federations ( iaaf ) event held in rome in 2007 . some observers suggested that oscar pistorius could actually run too fast and that his special carbon fiber prosthetic legs actually gave him an advantage over able - bodied runners ( marcellini et al . , 2012 ) . in 2007 the iaaf introduced rule # 144.2 , which prohibits any technical device incorporating springs that provides the user with an advantage over another athlete not using such a device ( http://www.iaaf.org/about-iaaf/documents/rules-regulations ) . this prohibition included the blades that oscar pistorius used and ended his quest for competing in the 2008 olympics . however , the court of arbitration for sport based in lausanne , switzerland , reversed this iaaf decision in may of 2008 . this decision was based on the argument that the iaaf failed to prove that the carbon fiber prosthetic blades provided an unfair advantage ( camporesi , 2008 ) . scientific analysis seems to show that the carbon fiber blades significantly enhance performance by improving running efficiency ( weyand , bundle , kram , et al . , 2009 ; weyand , bundle , mcgowan , et al . , 2009 ) . since the blades are actually much lighter than the lower legs they replaced , they can be moved about 15% faster than in the highest performance of sprinters with intact legs ( including olympic gold medalist usain bolt of jamaica ) . the blades also required 20% less force than was needed to achieve similar running speeds . using the blades meant that the prosthetic legs only needed about one half of the muscle force needed for sprinting at the same speeds as intact limbs . based on legal challenges , the iaaf recanted its ban on use of the blades and the most recent turn in this tale came at the london games in 2012 . in the summer of 2011 , pistorius ran a personal best of 45.07 s in the 400 m , thus satisfying the a standard 45.25 s. this result got him on the south african team for the 2011 iaaf world championships in daegu , korea , where he ran a 45.39 in one of the heats . he did not make it to the final , though , where that same mark would have officially qualified him for the olympics . in july of 2012 pistorius was selected to compete for south africa in the 400 m and 400 m relay at the london summer games . in so doing , although the south african relay team finished next to last in the final , the achievement of pistorius in making it to and running competitively in the olympic games was recognized with oscar pistorius serving as closing ceremonies flag bearer for his country ( johnson , 2012 ) . these amazing achievements again raise the question of whether or not the blades actually did provide a performance enhancement . could someone with something that should be an obvious physical barrier to sprinting no lower legs actually exceed able - bodied runners by using special equipment ? brendan burkett , professor biomechanics at the university of the sunshine coast in australia has raised many very interesting dilemmas when trying to work out the issue of technology in worldwide sports competitions like the olympic and paralympic games ( burkett , 2010 ) . if technology can amplify human performance and play an important role in outcomes , what about the problem of equal access to all competitors ? burkett pointed to the rather stunning fact that the champion of the marathon at the 1960 rome olympics , abebe bikila of ethiopia , actually ran the entire race barefoot . what would his time have been like with access to such technological advances as running shoes ? , the example of oscar pistorius and the use of prosthetic limbs to enhance performance , does raise the rather surreal idea that a present barrier to performance could in fact become the basis for a performance enhancement . we can also consider extending this argument to an extreme example of a talented runner with normal legs who wants to pursue surgical procedures to remove his lower legs so s / he can be fitted with a performance - enhancing prosthetic . i think the major point to take away from this part of the discussion is that amplifying or enhancing human ability carries with it some ethical and societal implications . this is obviously the case for even more conventional applications in the form of mechanical prostheses like that found in pistorius situation . this kind of scenario needs thinking about as we continue to move toward more technologically complex and integrated prosthetics such as brain machine interface or implanted stimulators . it seems fairly clear that even conventional more mechanically driven technology that was originally designed for restoration of function could be applied to actually enhance performance beyond the range of natural performance . where is the line between what are acceptable human abilities and what are not ? and what happens to that line if we use technology to change the human inside as well as out ? the concept that direct action in the nervous system could be had by electrically stimulating the brain has been with us for a very long time but was clearly established by fritsch and hitzig in 1864 ( see for review mussa - ivaldi & miller , 2003 ; schwalb & hamani , 2008 ) . until recently applications of brain stimulation have been largely in the realm of neuroscience research and some clinical diagnostic applications . now , though , take for example the current interest in repetitive transcranial magnetic stimulation ( rtms ) . a quick search on pubmed ( january 24 , 2014 ) revealed that rtms is now seeing applications in : chronic pain syndromes , depression , parkinson 's disease , personality disorder , posttraumatic stress disorder , stroke , bipolar disorder , and , enhancing motor learning . many of the foregoing are clinical examples that would be in the restoration range as outlined in figure 1 . yet it is not a big step toward shifting applications to , for example , enhancing attention and simply improving performance in those who already operate in the natural range shown in figure 1 . indeed , clark and parasuraman ( 2014 ) in an editorial on enhancing brain function explicitly state that tms and related brain stimulation methodologies can be used to improve attention , perception , memory and other forms of cognition in healthy individuals nelson , mckinley , golob , warm , and parasuraman ( 2014 ) also showed recently that transcranial direct current stimulation could be used to enhance vigilance in neurologically unimpaired participants . at this stage is it acceptable in our society for someone to seek a performance advantage by replacing healthy parts of their bodies or enhance brain function with technological constructs ? camporesi ( 2008 ) raised a related question : what are the ethics of enhancement using assistive device technology ( as could be possible with many current neuroprosthetics ) or genetic intervention ( as we soon will be able to do ) ? even a decade ago these questions had relevance , but given the pace of advances , they are now critical . they are all the more timely , given that the parts of human function that we are able to modify and restore or enhance continues to expand rapidly ( stieglitz , 2007 ) . the diagram in figure 2 ( inspired by that of stieglitz ) , illustrates some head to toe examples of research or commercially available technological devices that can be implanted or worn by humans now . neural enhancement technologies for rehabilitation that could also be used to exceed the range of typical human functional ability . inspired by stieglitz ( 2007 ) . neural enhancement technologies for rehabilitation that could also be used to exceed the range of typical human functional ability . some , such as the google glass , were initially launched as enhancement or entertainment tools , which are now being viewed with an eye to medical applications ( glauser , 2013 ) . regardless , many if not all of these could be used as functional enhancements that take us beyond the accepted range indicated at the far right of the performance continuum in figure 1 . of course it becomes much more difficult to evaluate what is the range of normal or natural human ability when technology enters the fray . cybernetics is another term that has been used and more directly suggests a level of control systems involved in combining artificial intelligence and machine - biological interfaces . from cybernetics all of these jumps , of course , take us further and further away from the human range of ability . they do , though , provide a frame of reference for what society has come to accept . doctor who the longest running science fiction show in tv history , according to the guinness book of world records in the form of cybermen . the cybermen and recently cyberwomen are discussed in entertaining detail along with everything else in the doctor who universe by paul parsons in the science of doctor who ( parsons , 2010 ) . the cybermen and cyberwomen of doctor who represent the extremes of biological and machine connection . they have a significant biological base , even including an artificial nervous system , surrounded by an iron robotic exoskeleton . the rise of the cybermen is described as a parallel humanoid species that began implanting technology and artificial parts into their bodies until they one day crossed from humanoid species to cyborgs . these are admittedly examples culled from popular culture and clearly we are not that far down that road with current approaches in neurorehabilitation . just as clearly , the rate of those steps is also increasing rapidly . in 2013 , the london science museum unveiled robotic exoskeleton ( rex ) , a completely manufactured cyborg consisting of organs and organ systems from laboratories and companies around the world , including artificial eye and kidney ( university of california , los angeles , ca ) , ear ( macquarie university , sydney , australia ) , trachea ( royal free hospital , london , england ) , heart ( syncardia , tucson , az ) , spleen ( yale university , new haven , ct ) , pancreas ( de montfort university , leicester , england ) , hand and arm ( touch bionics , livingston , scotland ; johns hopkins university , baltimore , md ) , blood ( sheffield university , sheffield , england ) , and foot and ankle ( mit , cambridge , ma ) . rex stands with the aid of a bilateral leg robotic exoskeleton ( payne , 2013 ) . in truth , i remain stunned by how rapidly research applications are progressing . in inventing iron man ( zehr , 2011 ) i made the argument that for the exoskeleton of marvel 's character to truly function as we see it in movies and in graphic novels ( mangels , 2008 ) , it would have to be a neuroprosthetic controlled by neural commands from the spinal cord and brain . that is , the ultimate brain - machine interface connecting a human to a powered robotic exoskeleton . in framing a technological superhero in this way , i made a number of , what i believed at the time to be speculative and prescient predictions . one was that to truly function seamlessly , a human exoskeleton like that of iron man needed to have sensory feedback . this would come from the suit like a kind of synthetic sensory skin and would feed back into the sensory cortices via the brain machine implant . the second was that connecting a neurological implant in the brain to control an exoskeletal system means a possibility for bidirectional information flow . namely , that if the controllers for the exoskeleton were hacked ( as happens routinely in comic books and science fiction movies ! ) someone would be able to control the human user through the same interface . these were predictions i thought would take some years to achieve , but i was unable to bask in the glory of these predictions for very long . in 2011 before inventing iron man was released by johns hopkins university press , odoherty et al . ( 2011 ) published an article in nature demonstrating enhanced learning of brain machine interface control in the mouse when sensory stimulation was included in the design . this was followed by the same group demonstrating real - time sharing of behaviourally relevant sensory information between the brains of two rats located in separate geographical regions ( natal , brazil ; durham , nc ; pais - vieira , lebedev , kunicki , wang , & nicolelis , 2013 ) . while this design did not actually use the hack of remote controlling one rat from a distance , this was proof principle of exactly this concept . of course a major challenge for neuroprosthetics is also the power supply issue for any controllers , devices , or effectors that might be implanted or worn . just early in 2014 , the laboratory of john rogers ( dagdeviren et al . , 2014 ) provided a proof of principle demonstration of a complete , flexible , and integrated system that is capable of harvesting and storing energy from the natural contractile and relaxation motions of the heart , lung , and diaphragm at levels that meet requirements for practical applications i must admit that in all reality we are left with more questions than answers . the critical piece , though , is that we continue to ask these questions and strive for answers while we continue to advance the field of neurorehabilitation . to return to the quote from the novel amped ( wilson , 2012 ) that led off this essay , wilson 's science fiction novel takes place in a dystopian future where neural implants ( most notably something called the neural - autofocus that is used to sharpen concentration and intelligence ) become widely available . in the beginning , these devices were introduced for use in those with cognitive disabilities , mental challenges , or health risks ( e.g. , to control epilepsy ) , but they eventually see widespread application throughout the population . subsequently a two - tiered class of humans emerges those who are amped and those who are not . shortly many of the technologies may no longer be fictional and many of the ethical issues will require solutions . as we move forward in the fields of neuroprosthetics and neural enhancement that truly are still in their infancy , i suggest it is critical that we allocate some of our attention and some mindfulness on what the future may hold . through our use of technology we are potentially at the threshold of scientific advances that could fundamentally change who we are as a species . will our species of homo sapiens ( literally wise men ) use our technology to transform our species into technical man ? currently our technologies are still relatively nascent and typically applied in the case of neurorestoration . as the field matures , however , to what ends will these technologies be applied , and using what means ? i suggest that presently we are on the path toward the superman of nietzsche by overcoming the limits of our species through application and internalization of our swiftly increasing technological ability . the issue of enhancement has been tested in the sports arena around the debate about what technology might give an unfair performance enhancement . the most high profile example of which is that of south african sprinter oscar pistorius . when he was a competitive sprinter pistorius was known as the fastest man on no legs since he is a double below knee amputee ( zehr , 2011 ) . pistorius was born with fibular hemimelia ( or longitudinal fibular deficiency ) meaning he did not have fibulas in his legs . based on this , the chances of pistorius being able to stand and walk were considered very low . accordingly , his parents took the decision when he was about 11 months old to allow surgical amputation of his lower legs . the functional implication of this bilateral double leg amputation was to allow oscar the use of appropriate prosthetics that could carry his weight during walking and running . for running he used prosthetic legs called blades , so named because the physical shape of the carbon fiber prosthetic looks like a blade . the prosthetics were produced by ossur and the model was the ossur flex - foot cheetah ( curran & hirons , 2012 ) . eventually he became the most accomplished and dominant double - amputee sprinter the world has ever seen . the flex - foot part of the prosthetic has caused controversy , though , because that it has been implicated with facilitating his running stride . this is also coupled with the fact that the prosthetic blades are lighter than an intact human lower leg would be . the sum combination of the prosthetic limbs and superb sprinting biomechanics created considerable controversy when oscar competed in an international association of athletics federations ( iaaf ) event held in rome in 2007 . some observers suggested that oscar pistorius could actually run too fast and that his special carbon fiber prosthetic legs actually gave him an advantage over able - bodied runners ( marcellini et al . , 2012 ) . in 2007 the iaaf introduced rule # 144.2 , which prohibits any technical device incorporating springs that provides the user with an advantage over another athlete not using such a device ( http://www.iaaf.org/about-iaaf/documents/rules-regulations ) . this prohibition included the blades that oscar pistorius used and ended his quest for competing in the 2008 olympics . however , the court of arbitration for sport based in lausanne , switzerland , reversed this iaaf decision in may of 2008 . this decision was based on the argument that the iaaf failed to prove that the carbon fiber prosthetic blades provided an unfair advantage ( camporesi , 2008 ) . scientific analysis seems to show that the carbon fiber blades significantly enhance performance by improving running efficiency ( weyand , bundle , kram , et al . , 2009 ; weyand , bundle , mcgowan , et al . , 2009 ) . since the blades are actually much lighter than the lower legs they replaced , they can be moved about 15% faster than in the highest performance of sprinters with intact legs ( including olympic gold medalist usain bolt of jamaica ) . the blades also required 20% less force than was needed to achieve similar running speeds . using the blades meant that the prosthetic legs only needed about one half of the muscle force needed for sprinting at the same speeds as intact limbs . based on legal challenges , the iaaf recanted its ban on use of the blades and the most recent turn in this tale came at the london games in 2012 . in the summer of 2011 , pistorius ran a personal best of 45.07 s in the 400 m , thus satisfying the a standard 45.25 s. this result got him on the south african team for the 2011 iaaf world championships in daegu , korea , where he ran a 45.39 in one of the heats . he did not make it to the final , though , where that same mark would have officially qualified him for the olympics . in july of 2012 pistorius was selected to compete for south africa in the 400 m and 400 m relay at the london summer games . in so doing , although the south african relay team finished next to last in the final , the achievement of pistorius in making it to and running competitively in the olympic games was recognized with oscar pistorius serving as closing ceremonies flag bearer for his country ( johnson , 2012 ) . these amazing achievements again raise the question of whether or not the blades actually did provide a performance enhancement . could someone with something that should be an obvious physical barrier to sprinting no lower legs actually exceed able - bodied runners by using special equipment ? brendan burkett , professor biomechanics at the university of the sunshine coast in australia has raised many very interesting dilemmas when trying to work out the issue of technology in worldwide sports competitions like the olympic and paralympic games ( burkett , 2010 ) . if technology can amplify human performance and play an important role in outcomes , what about the problem of equal access to all competitors ? burkett pointed to the rather stunning fact that the champion of the marathon at the 1960 rome olympics , abebe bikila of ethiopia , actually ran the entire race barefoot . what would his time have been like with access to such technological advances as running shoes ? , the example of oscar pistorius and the use of prosthetic limbs to enhance performance , does raise the rather surreal idea that a present barrier to performance could in fact become the basis for a performance enhancement . we can also consider extending this argument to an extreme example of a talented runner with normal legs who wants to pursue surgical procedures to remove his lower legs so s / he can be fitted with a performance - enhancing prosthetic . i think the major point to take away from this part of the discussion is that amplifying or enhancing human ability carries with it some ethical and societal implications . this is obviously the case for even more conventional applications in the form of mechanical prostheses like that found in pistorius situation . this kind of scenario needs thinking about as we continue to move toward more technologically complex and integrated prosthetics such as brain machine interface or implanted stimulators . it seems fairly clear that even conventional more mechanically driven technology that was originally designed for restoration of function could be applied to actually enhance performance beyond the range of natural performance . where is the line between what are acceptable human abilities and what are not ? and what happens to that line if we use technology to change the human inside as well as out ? the concept that direct action in the nervous system could be had by electrically stimulating the brain has been with us for a very long time but was clearly established by fritsch and hitzig in 1864 ( see for review mussa - ivaldi & miller , 2003 ; schwalb & hamani , 2008 ) . until recently applications of brain stimulation have been largely in the realm of neuroscience research and some clinical diagnostic applications . now , though , take for example the current interest in repetitive transcranial magnetic stimulation ( rtms ) . a quick search on pubmed ( january 24 , 2014 ) revealed that rtms is now seeing applications in : chronic pain syndromes , depression , parkinson 's disease , personality disorder , posttraumatic stress disorder , stroke , bipolar disorder , and , enhancing motor learning . many of the foregoing are clinical examples that would be in the restoration range as outlined in figure 1 . yet it is not a big step toward shifting applications to , for example , enhancing attention and simply improving performance in those who already operate in the natural range shown in figure 1 . indeed , clark and parasuraman ( 2014 ) in an editorial on enhancing brain function explicitly state that tms and related brain stimulation methodologies can be used to improve attention , perception , memory and other forms of cognition in healthy individuals nelson , mckinley , golob , warm , and parasuraman ( 2014 ) also showed recently that transcranial direct current stimulation could be used to enhance vigilance in neurologically unimpaired participants . at this stage is it acceptable in our society for someone to seek a performance advantage by replacing healthy parts of their bodies or enhance brain function with technological constructs ? camporesi ( 2008 ) raised a related question : what are the ethics of enhancement using assistive device technology ( as could be possible with many current neuroprosthetics ) or genetic intervention ( as we soon will be able to do ) ? even a decade ago these questions had relevance , but given the pace of advances , they are now critical . they are all the more timely , given that the parts of human function that we are able to modify and restore or enhance continues to expand rapidly ( stieglitz , 2007 ) . the diagram in figure 2 ( inspired by that of stieglitz ) , illustrates some head to toe examples of research or commercially available technological devices that can be implanted or worn by humans now . neural enhancement technologies for rehabilitation that could also be used to exceed the range of typical human functional ability . inspired by stieglitz ( 2007 ) . neural enhancement technologies for rehabilitation that could also be used to exceed the range of typical human functional ability . inspired by stieglitz ( 2007 ) . at initial development , most technologies in these applications were described in terms of restoration and rehabilitation . some , such as the google glass , were initially launched as enhancement or entertainment tools , which are now being viewed with an eye to medical applications ( glauser , 2013 ) . regardless , many if not all of these could be used as functional enhancements that take us beyond the accepted range indicated at the far right of the performance continuum in figure 1 . of course it becomes much more difficult to evaluate what is the range of normal or natural human ability when technology enters the fray . cybernetics is another term that has been used and more directly suggests a level of control systems involved in combining artificial intelligence and machine - biological interfaces . from cybernetics it is a very short jump to the term cyborg . all of these jumps , of course , take us further and further away from the human range of ability . they do , though , provide a frame of reference for what society has come to accept . doctor who the longest running science fiction show in tv history , according to the guinness book of world records in the form of cybermen . the cybermen and recently cyberwomen are discussed in entertaining detail along with everything else in the doctor who universe by paul parsons in the science of doctor who ( parsons , 2010 ) . the cybermen and cyberwomen of doctor who represent the extremes of biological and machine connection . they have a significant biological base , even including an artificial nervous system , surrounded by an iron robotic exoskeleton . the rise of the cybermen is described as a parallel humanoid species that began implanting technology and artificial parts into their bodies until they one day crossed from humanoid species to cyborgs . these are admittedly examples culled from popular culture and clearly we are not that far down that road with current approaches in neurorehabilitation . just as clearly , the london science museum unveiled robotic exoskeleton ( rex ) , a completely manufactured cyborg consisting of organs and organ systems from laboratories and companies around the world , including artificial eye and kidney ( university of california , los angeles , ca ) , ear ( macquarie university , sydney , australia ) , trachea ( royal free hospital , london , england ) , heart ( syncardia , tucson , az ) , spleen ( yale university , new haven , ct ) , pancreas ( de montfort university , leicester , england ) , hand and arm ( touch bionics , livingston , scotland ; johns hopkins university , baltimore , md ) , blood ( sheffield university , sheffield , england ) , and foot and ankle ( mit , cambridge , ma ) . rex stands with the aid of a bilateral leg robotic exoskeleton ( payne , 2013 ) . in truth , i remain stunned by how rapidly research applications are progressing . in inventing iron man ( zehr , 2011 ) i made the argument that for the exoskeleton of marvel 's character to truly function as we see it in movies and in graphic novels ( mangels , 2008 ) , it would have to be a neuroprosthetic controlled by neural commands from the spinal cord and brain . that is , the ultimate brain - machine interface connecting a human to a powered robotic exoskeleton . in framing a technological superhero in this way , i made a number of , what i believed at the time to be speculative and prescient predictions . one was that to truly function seamlessly , a human exoskeleton like that of iron man needed to have sensory feedback . this would come from the suit like a kind of synthetic sensory skin and would feed back into the sensory cortices via the brain machine implant . the second was that connecting a neurological implant in the brain to control an exoskeletal system means a possibility for bidirectional information flow . namely , that if the controllers for the exoskeleton were hacked ( as happens routinely in comic books and science fiction movies ! ) someone would be able to control the human user through the same interface . these were predictions i thought would take some years to achieve , but i was unable to bask in the glory of these predictions for very long . in 2011 before inventing iron man was released by johns hopkins university press , odoherty et al . ( 2011 ) published an article in nature demonstrating enhanced learning of brain machine interface control in the mouse when sensory stimulation was included in the design . this was followed by the same group demonstrating real - time sharing of behaviourally relevant sensory information between the brains of two rats located in separate geographical regions ( natal , brazil ; durham , nc ; pais - vieira , lebedev , kunicki , wang , & nicolelis , 2013 ) . while this design did not actually use the hack of remote controlling one rat from a distance , this was proof principle of exactly this concept . of course a major challenge for neuroprosthetics is also the power supply issue for any controllers , devices , or effectors that might be implanted or worn . just early in 2014 , the laboratory of john rogers ( dagdeviren et al . , 2014 ) provided a proof of principle demonstration of a complete , flexible , and integrated system that is capable of harvesting and storing energy from the natural contractile and relaxation motions of the heart , lung , and diaphragm at levels that meet requirements for practical applications i must admit that in all reality we are left with more questions than answers . the critical piece , though , is that we continue to ask these questions and strive for answers while we continue to advance the field of neurorehabilitation . to return to the quote from the novel amped ( wilson , 2012 ) that led off this essay , the slow invasion of implants and assistive devices wilson 's science fiction novel takes place in a dystopian future where neural implants ( most notably something called the neural - autofocus that is used to sharpen concentration and intelligence ) become widely available . in the beginning , these devices were introduced for use in those with cognitive disabilities , mental challenges , or health risks ( e.g. , to control epilepsy ) , but they eventually see widespread application throughout the population . subsequently a two - tiered class of humans emerges those who are amped and those who are not . shortly many of the technologies may no longer be fictional and many of the ethical issues will require solutions . as we move forward in the fields of neuroprosthetics and neural enhancement that truly are still in their infancy , i suggest it is critical that we allocate some of our attention and some mindfulness on what the future may hold . through our use of technology we are potentially at the threshold of scientific advances that could fundamentally change who we are as a species . will our species of homo sapiens ( literally wise men ) use our technology to transform our species into technical man homo sapiens technologicus a new subspecies deliberately modified and tailored by its own hand ? currently our technologies are still relatively nascent and typically applied in the case of neurorestoration . as the field matures , however , to what ends will these technologies be applied , and using what means ? i suggest that presently we are on the path toward the superman of nietzsche by overcoming the limits of our species through application and internalization of our swiftly increasing technological ability .

abstract . neural enhancement represents recovery of function that has been lost due to injury or disease pathology . restoration of functional ability is the objective . for example , a neuroprosthetic to replace a forearm and hand lost to the ravages of war or industrial accident . however , the same basic constructs used for neural enhancement after injury could amplify abilities that are already in the natural normal range . that is , neural enhancement technologies to restore function and improve daily abilities for independent living could be used to improve so - called normal function to ultimate function . approaching that functional level by use and integration of technology takes us toward the concept of a new species . this new subspecies homo sapiens technologicus is one that uses technology not just to assist but to change its own inherent biological function . the author uses examples from prosthetics and neuroprosthetics to address the issue of the limitations of constructs on the accepted range of human performance ability and aims to provide a cautionary view toward reflection on where our science may take the entire species .

Test CaseDo Oscar Pistorius's Prosthetic Blades Restore or Enhance Human Ability? The Untested CaseNeural Enhancement Beyond the Natural Functional Level in Intact, Uninjured Humans Welcome Superhero or a Bridge Too Far?

PMC3039412

in line with previous studies recording from ventral striatum ( leung and yim , 1993 ; kasanetz et al . , 2002 ; berke et al . , 2004 ; berke ( 2009 ) , van der meer and redish ( 2009b ) , and kalenscher et al . however , they noted distinct power bands , with one centered at around 50 hz ( gamma-50 ) , and another , broader band from 70100 hz ( gamma-80 ) . gamma oscillations in both bands tended to occur in bursts of 150250 ms ( figure 1a ) ; this pattern is consistent with power modulation by the phase of slower rhythms such as theta / alpha ( in dorsal striatum , tort et al . , 2008 ; hippocampus , colgin et al . , 2009 ; human ventral striatum , cohen et al . , 2009 ) . oscillations in the two bands had a tendency to alternate spontaneously , rather than co - occur ( figure 1a ) , suggesting a degree of mutually exclusive coordination between the two . berke ( 2009 ) showed further that switching between gamma-50 and gamma-80 was influenced by systemic application of dopaminergic drugs , shifting away from gamma-50 toward gamma-80 ( figure 2c ) . as ventral striatum receives a strong dopaminergic projection from the midbrain ( mogenson et al . , 1980 ; swanson , 1982 ) and synaptic and intrinsic membrane properties of ventral striatal neurons are modulated by dopamine ( da ) receptor activity ( bracci et al . , 2003 ; kreitzer and malenka , 2008 ) it is possible that this effect is mediated by processes in ventral striatum . switching between ventral striatal gamma-50 and gamma-80 is also present in anesthetized animals ( sharott et al . , 2009 ) and appears similar to gamma - band switching found in a number of other areas ( hippocampal ca1 , colgin et al . , 2009 ; piriform cortex , kay et al . , 2009 ) , although the effects of da in those cases is not known . in any case , the structure of ventral striatal gamma activity , with its distinct gamma-50 and gamma-80 power bands , provides a basis for investigating their behavioral and neural correlates separately . hot colors indicate high power ; figure from van der meer and redish ( 2009b ) . ( b ) simultaneous recording from ventral striatum and piriform cortex ( pir ) shows that lfp gamma oscillations can be nearly identical in both . figure adapted from berke ( 2005 , 2009 ; vmstr is ventromedial striatum , vlstr ventrolateral ) . ( c ) simultaneously recorded local field potential ( lfp ) traces from sites within ventral striatum suggest that gamma power can show heterogeneity between different recording sites . ( a ) gamma-50 ( left ) and gamma-80 power ( right ) were distributed differently over a track where rats ran for food reward ( provided at reward sites f1 and f2 ) . plot shows average across four subjects ; because rats ran a lap - based task , data were linearized for display ( see figure 4 for task details ) . ( b ) example session in which gamma power differentiated between distinct reward sites ( with different appetitive rewards ) on a triangular track in kalenscher et al . this particular lfp trace had almost no gamma power at the left reward site , but strong power at the bottom site . ( c ) systemic application of the dopaminergic agonist apomorphine ( at time 0 ; black arrow ) caused a switch from gamma-50 to gamma-80 . example traces ( 1 ) and ( 2 ) were taken from the times indicated in the spectrogram , before and after drug administration respectively . it is important to establish whether the recorded lfp oscillations are related to neural activity at the recording site , and not simply volume - conducted from nearby but distinct structures without any relationship to ventral striatal processing . for instance , theta - band oscillations ( 610 hz ) generated in the hippocampus can be detected several millimeters from their generation site , with power decreasing only about 20% per millimeter depending on the direction of conduction ( sirota et al . , 2008 ) . thus , berke ( 2009 ) , van der meer and redish ( 2009b ) , and kalenscher et al . ( 2010 ) asked whether the firing of single neurons in ventral striatum , recorded simultaneously with lfp oscillations , was related to gamma phase and power . indeed , both putative fast - spiking interneurons ( fsis ; about 90% , kalenscher et al . , 2010 ; over 50% , van der meer and redish , 2009b ; about 15% , berke , 2009 ) , and msns ( about 3% ; kalenscher et al . , 2010 ) had significant phase relationships ( phase locking ) to the gamma lfp , as indicated by spike - triggered averages , phase distributions , and coherence analyses . interestingly , many fsis cohered with either gamma-50 or gamma-80 , but not both , indicating that the different gamma bands are associated with distinct fsi populations ( figure 3 ) . while in vitro studies have shown that ventral striatal fsis show intrinsic resonance in the gamma range ( bracci et al . , 2003 ; taverna et al . , 2007 ) , those studies did not report fsi groups specifically tuned to gamma-50 or gamma-80 ; it is possible that the observed fsi subclasses result from differences in the source of their inputs , or different responses to neuromodulators . the observation that fsis were preferentially entrained to gamma oscillations is consistent with their integral role in the generation of gamma oscillations in other brain regions , as assessed by dynamic , in vivo control using optogenetic tools ( fuchs et al . , 2007 2009 ) , though usually as part of recurrent local excitatory inhibitory loops ( bartos et al . , 2007 ; fries et al . , 2007 ) striatal fsis can show task - modulated entrainment to gamma oscillations even without changes in firing rate ( berke , 2009 ) , part of a set of recent findings suggesting that fsi spike timing , and fsi spike rate , may be controlled by distinct aspects of their connectivity ( berke , 2008 ; gage et al . , 2010 ; thus , not only do such spiking relationships indicate that gamma oscillations are relevant to local processing , they also provide novel insights into the organization of ventral striatal circuitry . neurons in ventral striatum show firing relationships to gamma band local field potentials . ( a ) putative fast - spiking interneurons ( fsis ) tended to show strong relationships to either gamma-50 or gamma-80 . shown is the average spike waveform ( left ) , spike - triggered lfp average ( middle ) and spike - field coherence ( right ) of two example putative fsis in berke ( 2009 ) . note how the top neuron is coherent with gamma-50 but not gamma-80 , while the bottom neuron is more coherent at gamma-80 ( arrows ) . van der meer and redish ( 2009b ) similarly examined the coherence of a population of putative fsis ( b ) finding subgroups of gamma-50 and gamma-80 coherent neurons . plot shows the z - scored difference of the observed spike - field coherence relative to a set of interspike - interval - shuffled controls ; high z - scores ( hot colors ) indicate high coherence . note the two distinct populations at the top ( gamma-80 ) and bottom ( gamma-50 ; arrows ) . ( c ) phase histograms for two example putative medium - sized spiny neurons ( msns ) in kalenscher et al . ( 2010 ) , showing a tendency to fire at a particular phase of the gamma cycle . schematic representation of the tasks used ( top ) in berke ( 2009 , left ) , van der meer and redish ( 2009b , middle ) , and kalenscher et al . ( 2010 , right ) , and summary of the main findings in each ( bottom ) . in each study , rats ran to obtain appetitive rewards at reward sites ( filled circles ) . in berke ( 2009 ) the rewarded arm was indicated by cue lights visible from the central choice point . in van der meer and redish ( 2009b ) only one side of the maze was rewarded , but the rewarded side was changed between recording sessions . in kalenscher et al . note that , in the summary of the findings presented here ( as in the main text ) , a distinction is made between reward delivery , which is assessed by comparing rewarded and unrewarded trials , and reward sites which may include components ( such as an anticipatory ramp ) independent of actual reward delivery . ecog , electrocorticogram ; da , dopamine ; fsi , fast - spiking interneuron ; msn , medium - sized spiny neuron . all three studies ( berke , 2009 ; van der meer and redish , 2009b ; kalenscher et al . , 2010 ) found changes in gamma oscillations associated with reward sites and delivery . in one manifestation of such reward relationships , van der meer and redish ( 2009b ) found strikingly different distributions of gamma-50 and gamma-80 power across the running track , with gamma-50 increasing abruptly at the reward sites before trailing off gradually , whereas gamma-80 ramped up gradually to the reward sites ( figure 2a ) . zooming in on the time of reward delivery , gamma-80 power showed a complex pattern , ramping up gradually before falling sharply at the time of reward receipt , but then rising again before returning to baseline ( note that this refers to fast - timescale changes beyond those seen in figure 2a ; see figure 9 in van der meer and redish 2009b ) . interestingly , the initial ramping effect was independent of whether reward was actually received or not , while the post - arrival increase was only seen when reward was actually delivered . in apparent contrast , berke ( 2009 ) found that gamma-50 power was virtually abolished following reward receipt , while gamma-80 power showed a transient increase . thus , both berke ( 2009 ) and van der meer and redish ( 2009b ) found an increase in gamma-80 specific to reward delivery , but in van der meer and redish ( 2009b ) gamma-80 additionally ramped up to the reward sites , reminiscent of firing patterns found in ventral striatal neurons , which have been widely shown to anticipate reward delivery ( schultz et al . , 1992 ; lavoie and mizumori , 1994 ; miyazaki et al . , 1998 ; , 2008 ; van der meer and redish , 2009a ) . in further contrast to berke ( 2009 ) , van der meer and redish ( 2009b ) reported that gamma-50 power , like gamma-80 , showed a multiphasic relationship to reward delivery , increasing abruptly immediately following reward receipt , before decreasing and then rebounding , persisting well past initiation of the following lap . the more immediate post - arrival increase in gamma-50 power was dependent on actual reward delivery , while the later one was not . ( 2010 ) similarly found that gamma-50 power tended to be higher on rewarded ( compared to unrewarded ) trials , and in line with van der meer and redish ( 2009b ) , that gamma-80 power was increased prior to reward delivery . in these analyses , it should be noted that behavioral differences ( such as grooming , consumption , frustration , initiation of movement to the next reward site ) could be different between rewarded and non - rewarded trials . however , in van der meer and redish ( 2009b ) the early components of the gamma-50 and gamma-80 differentiated between rewarded and non - rewarded trials even though movement speeds were similar . ( 2010 ) showed that especially gamma-50 ( and to a lesser degree gamma-80 power ) differentiated between visits to different reward sites with distinct appetitive rewards ( figure 2b ) . since behavioral differences such as grooming and consumption are not site - specific and should hence influence gamma power at all reward locations equally , differential effects of site on gamma power suggest that gamma power modulation is at least in part related to reward - specific processing . thus , while the three studies differ in the precise pattern of gamma-50 and gamma-80 power changes relative to reward , taken together they support the notion that ventral striatal gamma oscillations are affected by reward anticipation and receipt . beyond relationships to the reward sites and reward delivery , van der meer and redish ( 2009b ) noted a number of further differences in the task- and behavioral correlates of gamma-50 and gamma-80 . gamma-50 and gamma-80 differed in their development as animals learned to choose the correct maze arm , with gamma-80 power being highest early in a session , while gamma-50 power was almost absent early and developed later ( within the first 5 min or 10 laps , even when rats were already very familiar with the environment ) . gamma-80 power was increased specifically at the maze 's choice point early in sessions , similar to the time course and location of both forward representations ( sweeps ) in the hippocampus , and covert activation of ventral striatal reward - responsive neurons ( johnson and redish , 2007 ; van der meer and redish , 2009a , 2010 ) . van der meer and redish ( 2009b ) also found , consistent with an earlier report ( masimore et al . , 2005 ) , that gamma-50 but not gamma-80 power was transiently increased before movement initiation ( starting to run from having been previously stationary ) , even when they only considered movements away from the reward sites to exclude any potential contribution of reward delivery . in apparent contrast , kalenscher et al . ( 2010 ) found only very weak correlations between gamma power and movement parameters , even when the analysis was restricted to test the effect of positive acceleration on gamma power . however , in addition to the fact that these diverging results may be attributed to differences in the analyses used , this set of observations may not in fact be incompatible , but rather result from gamma-50 power being increased before but not at the time of , positive acceleration accompanying movement initiation . in sum , it appears that the full complement of task- and behavioral correlates of ventral striatal gamma - band activity , which includes not only the complex multiphasic pattern at reward sites , but also movement and other correlates , is not easily captured by a unitary explanation whether in the case of gamma-50 or gamma-80 . this observation raises the question to what extent these different correlates are generated by the same neural process or circuit , or whether they are generated by different ( topographically segregated ) circuits , but mixed together by volume conduction . for instance , in the case of gamma-80 ( in van der meer and redish 2009b at least ) , is the ramping up to reward ( which is independent of reward delivery ) and the transient response to actual reward delivery , linked at the neural level within ventral striatum ? or , alternatively , are there separate circuits , one generating the ramp and another generating the transient reward response , which merely appear linked because both components appear in lfps that reflect a wide area ? as discussed above , several lines of evidence suggest that at least part of ventral striatal gamma activity is locally generated . these include relationships between spiking activity and gamma oscillation phase , reports of intrinsic membrane oscillations in the gamma range in striatal fsis , and the role of similar interneurons in generating gamma oscillations in other structures . these could include synaptic inputs from afferent areas , such as hippocampus , amygdala , prefrontal cortex and thalamus ( which are known to exhibit gamma oscillations : e.g. , colgin et al . 2009 ) , synaptic currents resulting from local projections within the striatum ( both fsis and msns make local connections ) , local intrinsic membrane potentials , and multi - unit spiking activity . these possibilities thus raise the question : how much of ventral striatal gamma is due to structure already present in its inputs , and what ( if any ) is the contribution of local ventral striatal processing ? to address this , berke ( 2009 ) recorded ventral striatal lfps simultaneously with piriform and frontal cortical ( skull screw electrocorticogram ) lfps , finding that ventral striatal gamma-50 appeared similar to piriform gamma ( figure 1b ) . while piriform gamma oscillations can include both gamma-50 and a higher frequency , 70 hz component ( kay et al . , 2009 ) , berke ( 2009 ) found that gamma-80 in ventral striatum could be coherent with frontal cortical gamma-80 . these findings suggest that gamma-50 recorded from rat ventral striatum contains a contribution from piriform gamma ; however , because ventral striatal neuronal firing can cohere with gamma-50 ( figure 3 ) , this is unlikely to be due to volume conduction alone . ventral striatal gamma-80 may reflect increased inputs from frontal cortical areas , again relevant to processing within ventral striatum as indicated by fsi firing relationships . the above results suggest a possible conception of ventral striatal gamma as resulting from synaptic currents from excitatory afferents that already possess oscillatory structure : the major sources of afferent input into ventral striatum ( amygdala , hippocampal formation , prefrontal cortex , piriform cortex , thalamus ) are all known to contain gamma oscillations ( chrobak and buzski , 1998 ; lee et al . , 2003 ; colgin et al . , 2009 ; kay et al . , 2009 ) , . in this scenario , ventral striatal gamma oscillations simply result from synaptic currents generated by afferent inputs to fsis and msns . on the one hand , this would be consistent with the observation that ventral striatum lacks the recurrent inhibitory excitatory loops thought to be responsible for de novo generation ( that is , not present in inputs ) of gamma oscillations ( bartos et al . , 2007 ; fries et al . , 2007 ; tiesinga and sejnowski , 2009 ) and central to computational models of gamma synchronization ( e.g. , lytton and sejnowski , 1991 ; vreeswijk et al . on the other hand , in such a scenario it is not clear how coordinated switching between gamma-50 and gamma-80 ( as in figure 1a ) can arise from distinct inputs ; these would already have to be coordinated in order to generate a consistent switching pattern . an alternative scenario is that the intrinsic membrane properties of fsis allow them to convert excitatory inputs without oscillatory structure into local gamma frequency inhibition . in particular , the subthreshold gamma oscillations observed in fsis in response to non - oscillatory depolarization in vitro ( bracci et al . , 2003 ; may contribute to lfp gamma oscillations directly , but also generate rhythmic , potentially widespread inhibition of msns , even for non - oscillatory input barrages . while fsis are thought to be relatively rare in ventral striatum , and may not act in a coordinated manner ( berke , 2008 ) , they do have a high probability of being connected with surrounding msns via powerful inhibitory synapses ( kos and tepper , 1999 ; taverna et al . , 2007 ) . further experiments may be able to decide between these two proposals for how ventral striatal gamma is generated . for instance , nasal occlusions are known to abolish piriform gamma ( vanderwolf , 2000 ) , an effect that might be exploited to exclude contributions of piriform gamma to ventral striatum . a different , powerful approach would be to excite or inhibit ventral striatal fsis directly , as has been done with optogenetic techniques in different brain areas ( cardin et al . this could test whether ventral striatal fsis are causally involved in the generation of ventral striatal gamma , as well as open the door to studies of their functional significance ( tiesinga and sejnowski , 2009 ) . whatever the precise source of ventral striatal gamma inherited from afferents or generated by local circuitry , with potentially different sources for gamma-50 and gamma-80 it is possible that even oscillations within these separate bands are themselves not unitary phenomena . in particular , different components within the bands , such as the ramp and reward receipt response for gamma-80 , the reward site selectivity and movement initiation correlates for gamma-50 , may well arise from distinct circuits or inputs within ventral striatum . an important point for future research would be to investigate to what extent single neurons ( especially msns , which make up the vast majority of neurons in ventral striatum ) actually participate in these different components ; are these multiple correlates also linked at the single neuron level , or do they become mixed together at the lfp level ? there are many known subdomains of ventral striatum , such as the distinction between core / shell , patch / matrix , and the variety of interacting input gradients ( for cortical and hippocampal formation inputs ) and spots ( in the case of amygdala ) which could in principle support such a separation . in support of the idea that different ventral striatal gamma correlates may arise from spatially distinct sources , kalenscher et al . ( 2010 ) found that the gamma power time series in a substantial number of simultaneously recorded lfps were remarkably poorly correlated ( figure 1c ) . this was the case even when all estimated recording locations were in the ventral striatum core , and poor correlations could not be attributed to differences in net gamma power , or generally low gamma power , suggesting that distinct gamma patterns stemmed from spatially and functionally distinct compartments within ventral striatum . in contrast , berke ( 2005 , 2009 ) found that the time course of gamma oscillations appeared extremely similar across widely separated areas of striatum . ( 2005 ; see also masimore , 2008 ) gamma-50 oscillatory events tended to occur at similar times across recording sites . a possible explanation for this divergence may lie in a subtle difference in the analysis methods used : while kalenscher et al . ( 2010 ) generated a single power time series for a relatively broad frequency band ( 30100 hz ) to correlate between recording sites , berke ( 2009 ) correlated power between recording sites for a range of narrow frequency bands ( i.e. , without mixing frequencies across the gamma band ) . thus , it may be that while the time course of single ( narrow ) gamma frequencies shows relatively little regional differentiation ( berke , 2009 ) , more complex relationships across different frequencies could give rise to heterogeneity only detectable at wider frequency ranges ( kalenscher et al . , 2010 ) . another possibility is that particular , spatially restricted micro - domains within ventral striatum exhibit different gamma oscillations . further studies and analyses , such as those that can reveal cross - frequency correlations between sites ( masimore et al . , 2004 ) , as well as higher - density recordings made possible by silicon recording probes , will be important in investigating these possibilities . the anatomical location of the ventral striatum as a site of convergence with inputs from frontal cortex , hippocampus , and amygdala , modulated by da and providing outputs with motor influence inspired the classical notion of a pathway from motivation to action that of a switchboard ( redgrave et al . , 1999 ; goto and grace , 2008 ; gruber et al . , these proposals imply that rather than simply being a passive recipient of incoming signals , ventral striatum contains a mechanism for switching between them . in such a switch scenario , ventral striatal activity is not dependent on inputs alone , but instead emphasizes ( selects ) a dominant input depending on the state of the switch realized in some internal property of ventral striatum , which may itself be controlled by a distinct switching input . such a mechanism is consistent with a popular proposal about the function of gamma oscillations in the brain : that they serve to bind together aspects of a common representation by functionally linking neural ensembles or brain areas when they synchronize at the same ( gamma ) frequency ( fries et al . , 2007 ; uhlhaas et al . , 2009 ) . applied to ventral striatum , this suggests a possible implementation of the switchboard idea in that during ventral striatal gamma-50 , areas exhibiting 50 hz activity such as the piriform cortex or amygdala are functionally linked to ventral striatum and in control of its influence on behavior , while during gamma-80 , frontal cortical areas control ventral striatal output . while it is currently unknown to what extent ventral striatal gamma oscillations are locally generated ( as discussed above ) , the ability of ventral striatal neurons to dynamically phase - lock to gamma oscillations in a task - dependent manner ( berke , 2009 ) provides one possible switching mechanism . the observation that dopaminergic drugs induce gamma-80 ( berke , 2009 ) suggests that switching can also occur on a more coordinated scale , consistent with in vitro findings on the effects of da agonists on local inhibitory mechanisms within ventral striatum ( bracci et al . switch whose only function is to pass on the selected input verbatim ; in particular , it does not exclude the possibility of further computation taking place . for instance , ventral striatal neurons encode predicted outcomes ( setlow et al . , 2003 ; roitman et al . , 2005 ) , specific action values ( ito and doya , 2009 ; roesch et al . , 2009 ) , and entrain to hippocampal activity patterns ( berke et al . , 2004 , 2009 ; , 2009 ) . when these findings are taken together with the effects of ventral striatum lesions on behaviors dependent on associative learning ( pennartz et al . , 1994 ; schoenbaum and setlow , 2003 ; humphries and prescott , 2010 ) they suggest ventral striatum is not simply a ( switching ) waystation but contains sufficient intrinsic circuitry for operations such as the association of spatial information ( where can a reward be found ) with information about a rewards motivating properties ( how good is the reward ; lansink et al . , 2009 ) or output selection by recurrent inhibition amongst msns ( czubayko and plenz , 2002 ; gurney et al . , 2004 ) . from this perspective , future work might address the relationship between ventral striatal processing during gamma-50 and gamma-80 : is ventral striatum performing the same , or different , operations in those two states ? more generally , the results reviewed here are part of a broad recognition that basal ganglia operations involve specific patterns of synchrony and rhythmicity , rather than firing rates changes alone ( e.g. , bevan et al . , 2002 ) . the study of lfps can be a valuable tool for the exploration of how such dynamics contribute to information processing underlying choices , reinforcement learning , and rewards . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

a vast literature implicates the ventral striatum in the processing of reward - related information and in mediating the impact of such information on behavior . it is characterized by heterogeneity at the local circuit , connectivity , and functional levels . a tool for dissecting this complex structure that has received relatively little attention until recently is the analysis of ventral striatal local field potential oscillations , which are more prominent in the gamma band compared to the dorsal striatum . here we review recent results on gamma oscillations recorded from freely moving rats . ventral striatal gamma separates into distinct frequency bands ( gamma-50 and gamma-80 ) with distinct behavioral correlates , relationships to different inputs , and separate populations of phase - locked putative fast - spiking interneurons . fast switching between gamma-50 and gamma-80 occurs spontaneously but is influenced by reward delivery as well as the application of dopaminergic drugs . these results provide novel insights into ventral striatal processing and highlight the importance of considering fast - timescale dynamics of ventral striatal activity .

Ventral Striatal Gamma Oscillations Consist of Distinct, Switching Rhythms Task- and Reward-Related Modulation of Gamma-50 and Gamma-80 Origins of Ventral Striatal Gamma Where Next? Functional Relevance of Ventral Striatal Gamma Oscillations Conflict of Interest Statement

PMC3634162

sleep disorders have been linked to a number of generalized health and behavioral disorders , including reduced efficiency while operating a motor vehicle . in recent years , a number of studies have shown consistent linkages between lack of sleep , circadian rhythm disorders ( crsds ) , and fatigue , on the one hand , and psychosocial problems , behavioral disorders , and motor vehicle accidents on the other . furthermore , reduced or inadequate sleep is per se considered as a risk factor for several chronic disorders , including hypertension , obesity , type ii diabetes , coronary disorders , cancer , and premature death . a number of specific health and sleep problems have now been shown to have adverse consequences during waking hours . these problems include : ( a ) sleep deprivation or chronic sleep restriction ; ( b ) excessive daytime somnolence ; ( c ) tiredness and/or fatigue;[1315 ] ( d ) high body mass index ( bmi ) ; ( e ) obstructive sleep apnea ( osa ) associated with alcohol consumption ; and f ) other sleep disorders . globally , thousands of accidents occur due to lack of sleep , tiredness , and fatigue . an awareness of this linkage has increasingly prompted public health concerns regarding the possible role of inadequate sleep in road accidents and fatalities . these concerns have in turn resulted in recommendations for specific preventive steps and public education . in several countries , various contributors to traffic accidents have been discussed and considered closely . in 2004 and 2010 , the un general assembly and world health organization ( who ) reviewed the costs of traffic accidents , and unanimously approved a policy statement urging that in the years 2011 to 2020 increased attention be given to the issue of road safety , with the aim of halting or reversing the trend of increasing accidents and deaths worldwide . member states were invited to perform activities in the areas of road safety , infrastructure , vehicle safety , road users behavior , reduction of accidents , traffic education and clinical care post - crash . the recommended activities were to conform to the following five target areas of concern : 1 . road safety management ; 2 . the purpose of these efforts was to change public behavior concerning road safety and to influence the attitudes of government policy makers . more specifically , these activities aimed to increase local government enforcement of road laws , to involve public health sectors , to increase awareness of vehicle manufacturers and to educate local communities . studies have shown that a large proportion of traffic accidents around the world are related to inadequate or disordered sleep , with its resulting decrease in driver alertness being associated with serious injuries and deaths . in view of the direct implications of these associations for public health and safety , the present review sought to examine them more closely . the aim of this review was hopefully to promote greater understanding of driver sleepiness and to suggest possible strategies for dealing with the problem . the seriousness of traffic accidents for public health and safety has been pointed out by a number of surveys and governmental statistics . these have shown that a large number of injuries and deaths have been caused by automobile accidents . in a worldwide survey , who tallied the road traffic injury fatality rates ( per 10.000 population ) of major global regions : south - east asia region ( 16,6 ) ; african region ( 32,2 ) ; eastern mediterranean region ( 32,2 ) ; region of the americas ( 15,8 ) ; european region ( 13,4 ) and western pacific region ( 15,6 ) . india and china have the highest traffic accident rates in the world : 105,725 and 89 , 455 people died in 2006 , respectively . in 2008 , 39,000 people in the european union lost their lives in traffic related accidents ; the european countries with the highest rates of traffic fatalities were mostly eastern countries , namely , lithuania ( 14.8/100,000 inhabitants ) , poland ( 14.3/100,000 inhabitants ) and romania ( 14.2/100,000 ) . in 2008 , out of all deaths in argentina , 3,988 were found to be due to traffic accidents . in the us , 37,261 deaths were the result of car accidents . in a study by seaht et al . traffic accidents in tehran accounted for 17.3% of all accidents and mortality was 26.6 per 1000 people per year . in brazil , there were 158,893 traffic accidents in 2009 , with 93,851 injuries and 7,376 deaths . the brazilian national health system ( sistema nico de sade - sus ) found that , out of nine million hospital admissions , 8.1% were due to external causes , and of these external causes , 15% were due to motor vehicle accidents . several studies have underscored the need for urgency in developing methods to prevent errors associated with traffic accidents and for designing practical tests of driver fatigue , which is the root cause of these accidents . various authors have emphasized the vital importance of directing the attention of society and governments worldwide to the issue of sleepy driving ( falling asleep while driving ) and the threat it represents to public safety . it has been concluded that the need for adequate legislation in this regard is absolutely essential.[2931 ] fatigue as a result of sleep disorders , excessive workload and lack of physical and mental rest , are known to be major contributors to motor vehicle accidents . it has been estimated that in the usa 56,000 accidents a year are related to inadequate sleep . in the uk , 20% of highway accidents and up to one quarter of fatal and serious accidents are known to have occurred because of driver fatigue . in australia , it has been shown that driver fatigue is responsible for 17% of all accidents , 30% of which resulted in deaths . in 2010 , 41% of a sample of u.s . drivers who responded to a telephone survey admitted to having fallen asleep or nodded off at the wheel . in general , fatigue is reported by brazilian drivers to be the cause of approximately 20% of the accidents and over 30% of the 24,000 deaths that occur annually on brazilian highways . in 2008 , the brazilian federal highway police reported that nearly 2,400 accidents had been caused by sleep problems . souza et al . concluded that brazilian truck drivers in mato grosso do sul had increased alcohol consumption , bad sleeping habits and sleep restriction , excessive driving duration and poor quality of life ; these behavioural factors were reflected in the poor scores of epworth sleepiness scale ( ess ) and the pittsburgh sleep quality index ( psqi ) scales . silva - junior et al . interviewed brazilian truck drivers and found that 18.3% stated that they snored , 48.3% consumed alcohol while driving , and that 35% used stimulants , further , the investigations found that and that these factors were involved in 34% of the reported accidents . vakulin et al . surveyed 38 untreated individuals with osa and found that , compared to a control group , the osa respondents were more likely to have had at least one accident resulting from falling asleep at the wheel if they additionally experienced in combination with sleep restriction ( or = 4.0 [ 1.8 - 8.8 ] ) or had consumed alcohol ( or = 2.3 [ 1.0 - 5.1 ] ) . dawson et al . compared the effects of blood alcohol concentration with sleep deprivation and found that 17 - 19 h of sleep deprivation resulted in task performance decrements that were equivalent to the effects of a 0.05% blood alcohol concentration , while further increases in sleep deprivation ( 20 - 25 h of wakefulness ) were as damaging to driving performance as a blood alcohol concentration of 0.10% . carried out telephone interviews with 4,774 french drivers and found that 11.8% of the sample had ess scores of 11 , 28.6% reported experiencing sleepiness at the wheel severe enough to require stopping , feeling sleepy during night - time ( 46.8% ) and daytime of driving ( 39.4% ) , having a near - miss accident during the previous year ( 10.7% ) ( of which 46% of the incidents were reportedly sleep - related ) , and of having a driving accident ( 5.8% ) ( of which 5.2% of were sleep related ) . the authors also found that among the factors linked to motor vehicle accidents or near - miss accidents , the ones having the strongest associations were male gender ( 1.22 - 1.87 , p < 0.001 ) , being a younger driver ( 1.41 - 2.44 , p < 0.001 ) , frequently reporting feeling sleepy ( or 1.67 , 1.29 - 2.15 , p < 0.001 ) , having a sensitivity to caffeine , experiencing episodes of anxiety - depression ( or 1.82 , 1.27 - 2.62 , p < 0.001 ) and having at least one episode of severe sleepiness at the wheel in the previous year ( 5.20 - 8.12 , p < 0.001 ) . an elevated risk of having a motor vehicle accident were being unmarried ( 0.99 - 1.96 , p < 0.05 ) , being in the age range of 18 to 30 ( 1.51 - 3.00 , p < 0.001 ) , being a professional driver ( or 1.52 , 1.08 - 2.13 , p < 0.05 ) , having a sensitivity to caffeine ( 1.11 - 1.85 , p < 0.01 ) and at least one episode of severe sleepiness at the wheel in the previous year ( 1.57 - 2.64 , p < 0.001 ) . found that values of apnea hypopnea index ( ahi 40 ) and ess ( ess 11 and ess 16 ) of 616 japanese drivers were correlated with an increased risk of dozing off at the wheel , as well as a higher risk of being involved in motor vehicle accidents . studies have shown that , compared to normal individuals , individuals with osa have a higher risk of falling asleep while driving and are three times more likely to cause accidents . and george reported that , among sleep disorders , osa represented the highest risk factor for sleepiness and accidents . the partial or total obstruction of the upper airway that is associated with severe oxyhemoglobin desaturation is a cause of hypoxemia , hypercapnia , sleep fragmentation , es , and cognitive impairments . among the impairments caused by osa are those which involve frontal lobe executive functions , essential for decision making while driving . it has been recently demonstrated that these critical functions do not improve following cpap treatment . in one study it was found that , compared to a group without osa ( ahi = 0 to 5 ) , patients with osa had a greater relative risk of having an accident ( 1.6 to 2.9 ) , and a greater rate of having an accident in which there was a personal injury ( 3.0 to 4.8 ) . it was found further that the increase in the risks and rates of accidents was proportional to the seriousness of sleep apnea . vakulin et al . showed that osa patients had performance impairments in a driving simulator when compared with the control group . the authors also reported that after three months of cpap treatment , the osa group had improved their driving performance . a systematic review and meta - analysis of the relationships among osa , cpap and accidents found that cpap use reduced the accident risk of drivers with moderate to severe osa ( rr = 0.278 , 95% ci : 0:22 to 0:35 , p < 0.001 ) , and , additionally , decreased self reported sleepiness , one of the symptoms of osa . interviewed 677 drivers and found that 12% reported having osa symptoms and having fallen asleep at the wheel , and that among these 7% were involved in accidents . tippin et al . compared normal drivers with those who had osa and demonstrated that the osa group had reduced peripheral vision ( 80.7 14.8% vs. 86.7 8.8% , p = 0.03 ) and reported feeling greater sleepiness at the wheel ( stanford sleep scale = 4.2 1.2 vs. 3.6 1.2 , p = 0.03 ) . the authors also showed that an association existed between increased sleepiness and decreased heart rate in osa patients ( r = -0.49 , p = 0.01 ) . philip et al . surveyed 35,004 drivers , and found that 5.2% had osa , 2.3% had restless legs syndrome , 9.3% insomnia , 0.1% narcolepsy and hypersomnia , and 4.4% had multiple disorders . of these drivers , 7.2% reported having had at least one accident in the previous year , 5.8% of which were related to sleep . these results are reflected in related findings that the highest risk of having a motor vehicle accident was associated with narcolepsy or hypersomnia [ or = 8.78 ( 1.97 - 39.06 ) ] , osa [ or = 2.09 ( 1.06 - 4.13 ) ] , insomnia [ ( or = 1.78 ( 1.01 - 3.14 ) ] and others multiple sleep disorders [ or = 1.46 1.20 - 1.78 ) ] . accidents caused by sleepiness occurred primarily on highways ( 77.6% ) and on long trips ( 63.8% ) and more frequently at night ( 40.9% ) than during the day ( 29.4% ) . found an association between sleep disorders ( narcolepsy , insomnia , and osa ) and accidents caused by sleepiness , as well as an association between sleepiness and near miss accidents . concluded that untreated narcolepsy can impair driving performance and increase the risk of traffic accidents . evaluated 750 drivers and found that 14.5% reported sleepiness while driving , and , further , that 2% of the respondents had been involved in accidents caused by sleepy driving . a study conducted by prez - chada et al . of 770 argentinian truck drivers found that 80% were overweight , 71.9% snored ( of those , 4.7% reported apnea ) , 37.8% were hypertensive , 86.9% had insomnia , 13.9% had ess scores above 10 , and 43.8% became sleepy while driving . those suffering from apnea had higher scores on the ess , were more likely to be overweight and a higher prevalence of hypertension . in this population , snoring , ess scores > 10 , and driving for long hours were associated with sleepiness at the wheel , as well as an increased likelihood of having accidents or near misses . out of the total 11% had already had at least one accident because of sleepiness while driving , 33% snored during sleep , 8% reported having respiratory disorders , and more than 50% were obese . de pinho et al . found that out of 300 truck drivers evaluated , 46.3% experienced poor sleep quality , 46% had excessive sleepiness , and 40% were chronically sleep - deprived . snoring was present in 18.3% of the drivers . excessive sleepiness ( es ) was the cause of the majority of accidents reported by the drivers ( 26.5% ) while the reported frequency of es was even greater among drivers who had a history of accidents . a number of studies of shift workers , especially professional drivers , have documented the occurrence of changes in sleep architecture over time . the associated symptoms , including poor quality of sleep , fragmentation due to osa , and sleep deprivation and/or restriction , were thought to explain the es , fatigue , health deficits , possible driving errors , and serious accidents . most european countries now have specific traffic laws for sleep disorders and diseases . several u.s . states , as well as canada and australia , have established laws that prohibit drivers with osa or narcolepsy from driving a car and/or obtaining a drivers license unless their condition is medically controlled . in 2007 , 128,467 traffic accidents were recorded in brazil , with 42,508 being caused by inattention and 3,367 being caused by sleepy driving . in 2008 there were 60,370 confirmed deaths due to accidents , with 36,666 deaths resulting from traffic accidents . in brazil , due to the high rate of traffic accidents caused by sleepiness , a resolution was adopted by departamento nacional de trnsito ( denatran ) in 2008 ( resolution contran / denatran 267/2008 ) to reduce the number of accidents caused by sleepiness , fatigue , sleep respiratory disorders and changes in biological rhythms . some studies have shown that individuals with insomnia and those who were taking various sleep medications had an increased risk of accidents due to fatigue symptoms , reduced alertness and sleepiness at the wheel.[6264 ] over the past 30 years , on - road driving research conducted in the netherlands examined the effects on driving of various medicinal drugs . they were instructed to drive with a steady lateral position and constant speed ( 95 km / h ) . the standard deviation of lateral position ( sdlp ) , i.e. the weaving of the car , was the primary outcome measure of the test . a study of unmedicated healthy volunteers concluded that prolonged nighttime highway driving is associated with increased driver sleepiness , and that this was further associated with progressively increasing sdlp values . after 2 to 3 h of driving , performance impairments are comparable to those seen in drivers who have consumed alcohol and have a blood alcohol concentration ( bac ) or 0.05% , i.e. the legal limit for driving in many countries . as a result of these findings it has been shown effective if during these breaks drivers consume caffeinated beverages such as coffee or energy drinks in order to increase their alertness . the results of these studies showed that benzodiazepine hypnotics and zopiclone significantly impair driving performance , whereas zolpidem and zaleplon seem relatively safe.[6971 ] ramelteon , a melatonin receptor agonist , also significantly impaired next morning driving performance . the findings of these studies are in line with epidemiological evidence showing increased accident risks among those who use benzodiazepine hypnotics and zopiclone and are also generally consistent with other findings cited in this paper showing that a number of sleep disorders can reduce daytime alertness , and thus have adverse effects on driving performance . the various types of evaluations and their respective instruments that are commonly used to study sleep disorders , sleepiness , and wakefulness - rest activity are listed below [ table 1 ] : optalert ( system of infrared reflectance oculography ) : this is a new method for monitoring eye and eyelid movements by infrared reflectance oculography . the method uses transducers attached to a glass frame to measure drivers drowsiness continuously on a new scale ( johns drowsiness scale - jds ) during driving task.the pittsburgh sleep quality index ( psqi ) , includes issues related to sleepiness during daily activities and daily life , sleep , and sleep disorders;the multiple sleep latency test ( mlst),[7779 ] assesses the propensity to sleep in a comfortable position , a factor that is commonly used in the diagnosis of narcolepsy;the oxford sleep resistance test ( osler ) , determines the onset of sleep;the maintenance of wakefulness test ( mwt ) , assesses the individual 's ability to stay awake;pupillography , which evaluates the diameter and variability of the pupil and the relationship of these variables to subjective sleepiness complaints;actigraphy , assesses the activity - rest periods and circadian rhythm disorders of shift workers and may be used to infer hypersomnia and/or fatigue;polysomnography ( psg ) ( the gold standard for diagnosing osa ) is the most commonly used test to evaluate sleep and its disorders . it includes measures based on the electroencephalogram ( eeg ) , mentonian and lower limb electromyography ( emg ) , electrooculogram ( eog ) , electrocardiogram ( ecg ) , pulse oximetry , oro - nasal airflow , respiratory effort measurements , blood gas , snoring , and body position . ( it has been noted however that while psg can contribute importantly to an overall assessment of sleep quality , it is not a specific instrument for assessing sleepiness).[8587 ] the clinical diagnosis of osa is made with protocols used by a physician who specializes in sleep disorders . optalert ( system of infrared reflectance oculography ) : this is a new method for monitoring eye and eyelid movements by infrared reflectance oculography . the method uses transducers attached to a glass frame to measure drivers drowsiness continuously on a new scale ( johns drowsiness scale - jds ) during driving task . the pittsburgh sleep quality index ( psqi ) , includes issues related to sleepiness during daily activities and daily life , sleep , and sleep disorders ; the multiple sleep latency test ( mlst),[7779 ] assesses the propensity to sleep in a comfortable position , a factor that is commonly used in the diagnosis of narcolepsy ; the oxford sleep resistance test ( osler ) , determines the onset of sleep ; the maintenance of wakefulness test ( mwt ) , assesses the individual 's ability to stay awake ; pupillography , which evaluates the diameter and variability of the pupil and the relationship of these variables to subjective sleepiness complaints ; actigraphy , assesses the activity - rest periods and circadian rhythm disorders of shift workers and may be used to infer hypersomnia and/or fatigue ; polysomnography ( psg ) ( the gold standard for diagnosing osa ) is the most commonly used test to evaluate sleep and its disorders . it includes measures based on the electroencephalogram ( eeg ) , mentonian and lower limb electromyography ( emg ) , electrooculogram ( eog ) , electrocardiogram ( ecg ) , pulse oximetry , oro - nasal airflow , respiratory effort measurements , blood gas , snoring , and body position . ( it has been noted however that while psg can contribute importantly to an overall assessment of sleep quality , it is not a specific instrument for assessing sleepiness).[8587 ] the clinical diagnosis of osa is made with protocols used by a physician who specializes in sleep disorders . various methods and instruments for evaluating drowsiness and daytime sleepiness these survey instruments and physiological tests are very important for accurately diagnosing sleep disorders and their symptoms so that driver errors and accidents , which may affect public health , can be avoided . rodenstein summarized several studies dealing with the financial consequences of a - related accidents and concluded that the resulting cost to the public was $ 16 billion per year . it has been projected that cpap treatment could reduce these costs to $ 11 billion per year . further , it has been calculated that 567,000 crashes and 980 traffic deaths could be prevented with osa treatment by cpap . these projections underscore the urgency of the need to develop policies for preventing accidents and promoting the health of drivers who are affected by sleep disorders or other conditions that restrict sleep . the preventive or therapeutic interventions should be emphasized to minimize the deleterious effects of drowsy driving and to educate drivers about the risks of economic loss , poor quality of life and death . figure 1 summarizes of the causes and preventing strategies of sleepiness during driving . due to the high prevalence of osa and sleepiness among drivers , some studies have recommended that educational activities related to drowsy driving and how osa may be an important cause of traffic accidents be made a requirement for the licensure of professional drivers . a summary of the causes and preventing strategies of sleepiness during driving the literature highlights the importance of restorative sleep and the need for drivers to examine the external environment , to recognize the presence of fatigue symptoms , and to determine when to stop to sleep . a number of prevention strategies have been proposed . awareness programs based on educational lectures the aim of these is to promote behavioral change , sleep hygiene strategies , healthy nutritional habits , exercise at certain times , changes in behavior , drugs and alcohol abuse , time of driving and signs of fatigue , stop driving to doze and scheduled naps . some therapeutic interventions include phototherapy to promote alertness and vigilance , with different intensities for each individual , substances or drugs to promote either sleepiness or alertness ( i.e. , caffeine , modafinil , melatonin and others ) , as well as specific sleep disorders treatment . several studies report that naps , phototherapy and caffeine can be helpful for increasing awareness , alertness and memory consolidation . phototherapy using bright light is recommended for night shift workers with insomnia , excessive sleepiness and fatigue . mets et al . demonstrated that 80 mg of caffeine , comparable to a regular cup of coffee or a 250 ml can of red bull energy drink , reduces sleepiness and improves driver performance . similarly , reyner and horne found that 200 mg of caffeine reduced subjective sleepiness in the morning as well decreased accidents . reported that japanese non - professional drivers used naps and coffee intake as strategies for preventing sleepiness at the wheel and possible accidents . lic and summala reported that the most common strategy used by argentinian drivers for avoiding sleepiness was to take a nap for 10 min ( 66.2% ) followed by ingestion of yerba mate infusion with caffeine ( 53.1% ) . the national sleep foundation has recommended that for maintaining alertness and optimal driving performance that sleep be adequate to meet biological needs , should not be fragmented , that the sleep cycle should be synchronized , and that the sleeping environment should be free of disturbances.[104107 ] another strategy reported in the scientific literature is physical exercise in the evening or during the workday to increase the alertness and vigilance of drivers who do shift work or who spend long periods of time behind the wheel . exercise has been shown to increase alertness , reduce sleepiness and accidents by increasing body temperature , as well as to promote restorative sleep . the quality of sleep that an individual experiences , not only its quantity , is an important factor for enhancing ambient , physical and social functioning . sleep that is refreshing , restorative sleep , is important for promoting homeostasis and thermoregulation , and is basic in memory consolidation and cognition , energy restoration and the cerebral energy metabolism . it is known that many individuals experience and adequate total sleep time ( 6 to 8 h ) but do not have enough quality sleep due to repeated awakenings , and/or sleep disorders.[108111 ] abe et al . reported that short duration ( < 6 h ) of nocturnal sleep is associated with an increased risk of rear - end collisions and single - car accidents . it is known that among the numerous types of sleep disorders , excessive sleepiness is more prevalent among drivers with osa . thus , cpap has been recommended as a gold standard therapeutic strategy for remediating osa . yamamoto et al . , sassani et al . , barbe et al . and komada et al . reported that osa treatment with cpap in particular reduces the number and risk of accidents and deaths , as well as improving mood , and reducing excessive sleepiness . fatigue as a result of sleep disorders , excessive workload and lack of physical and mental rest , are known to be major contributors to motor vehicle accidents . it has been estimated that in the usa 56,000 accidents a year are related to inadequate sleep . in the uk , 20% of highway accidents and up to one quarter of fatal and serious accidents are known to have occurred because of driver fatigue . in australia , it has been shown that driver fatigue is responsible for 17% of all accidents , 30% of which resulted in deaths . in 2010 , 41% of a sample of u.s . drivers who responded to a telephone survey admitted to having fallen asleep or nodded off at the wheel . in general , fatigue is reported by brazilian drivers to be the cause of approximately 20% of the accidents and over 30% of the 24,000 deaths that occur annually on brazilian highways . in 2008 , the brazilian federal highway police reported that nearly 2,400 accidents had been caused by sleep problems . souza et al . concluded that brazilian truck drivers in mato grosso do sul had increased alcohol consumption , bad sleeping habits and sleep restriction , excessive driving duration and poor quality of life ; these behavioural factors were reflected in the poor scores of epworth sleepiness scale ( ess ) and the pittsburgh sleep quality index ( psqi ) scales . silva - junior et al . interviewed brazilian truck drivers and found that 18.3% stated that they snored , 48.3% consumed alcohol while driving , and that 35% used stimulants , further , the investigations found that and that these factors were involved in 34% of the reported accidents . vakulin et al . surveyed 38 untreated individuals with osa and found that , compared to a control group , the osa respondents were more likely to have had at least one accident resulting from falling asleep at the wheel if they additionally experienced in combination with sleep restriction ( or = 4.0 [ 1.8 - 8.8 ] ) or had consumed alcohol ( or = 2.3 [ 1.0 - 5.1 ] ) . dawson et al . compared the effects of blood alcohol concentration with sleep deprivation and found that 17 - 19 h of sleep deprivation resulted in task performance decrements that were equivalent to the effects of a 0.05% blood alcohol concentration , while further increases in sleep deprivation ( 20 - 25 h of wakefulness ) were as damaging to driving performance as a blood alcohol concentration of 0.10% . carried out telephone interviews with 4,774 french drivers and found that 11.8% of the sample had ess scores of 11 , 28.6% reported experiencing sleepiness at the wheel severe enough to require stopping , feeling sleepy during night - time ( 46.8% ) and daytime of driving ( 39.4% ) , having a near - miss accident during the previous year ( 10.7% ) ( of which 46% of the incidents were reportedly sleep - related ) , and of having a driving accident ( 5.8% ) ( of which 5.2% of were sleep related ) . the authors also found that among the factors linked to motor vehicle accidents or near - miss accidents , the ones having the strongest associations were male gender ( 1.22 - 1.87 , p < 0.001 ) , being a younger driver ( 1.41 - 2.44 , p < 0.001 ) , frequently reporting feeling sleepy ( or 1.67 , 1.29 - 2.15 , p < 0.001 ) , having a sensitivity to caffeine , experiencing episodes of anxiety - depression ( or 1.82 , 1.27 - 2.62 , p < 0.001 ) and having at least one episode of severe sleepiness at the wheel in the previous year ( 5.20 - 8.12 , p < 0.001 ) . other factors linked to an elevated risk of having a motor vehicle accident were being unmarried ( 0.99 - 1.96 , p < 0.05 ) , being in the age range of 18 to 30 ( 1.51 - 3.00 , p < 0.001 ) , being a professional driver ( or 1.52 , 1.08 - 2.13 , p < 0.05 ) , having a sensitivity to caffeine ( 1.11 - 1.85 , p < 0.01 ) and at least one episode of severe sleepiness at the wheel in the previous year ( 1.57 - 2.64 , p < 0.001 ) . found that values of apnea hypopnea index ( ahi 40 ) and ess ( ess 11 and ess 16 ) of 616 japanese drivers were correlated with an increased risk of dozing off at the wheel , as well as a higher risk of being involved in motor vehicle accidents . studies have shown that , compared to normal individuals , individuals with osa have a higher risk of falling asleep while driving and are three times more likely to cause accidents . and george reported that , among sleep disorders , osa represented the highest risk factor for sleepiness and accidents . the partial or total obstruction of the upper airway that is associated with severe oxyhemoglobin desaturation is a cause of hypoxemia , hypercapnia , sleep fragmentation , es , and cognitive impairments . among the impairments caused by osa are those which involve frontal lobe executive functions , essential for decision making while driving . it has been recently demonstrated that these critical functions do not improve following cpap treatment . in one study it was found that , compared to a group without osa ( ahi = 0 to 5 ) , patients with osa had a greater relative risk of having an accident ( 1.6 to 2.9 ) , and a greater rate of having an accident in which there was a personal injury ( 3.0 to 4.8 ) . it was found further that the increase in the risks and rates of accidents was proportional to the seriousness of sleep apnea . vakulin et al . showed that osa patients had performance impairments in a driving simulator when compared with the control group . the authors also reported that after three months of cpap treatment , the osa group had improved their driving performance . a systematic review and meta - analysis of the relationships among osa , cpap and accidents found that cpap use reduced the accident risk of drivers with moderate to severe osa ( rr = 0.278 , 95% ci : 0:22 to 0:35 , p < 0.001 ) , and , additionally , decreased self reported sleepiness , one of the symptoms of osa . interviewed 677 drivers and found that 12% reported having osa symptoms and having fallen asleep at the wheel , and that among these 7% were involved in accidents . tippin et al . compared normal drivers with those who had osa and demonstrated that the osa group had reduced peripheral vision ( 80.7 14.8% vs. 86.7 8.8% , p = 0.03 ) and reported feeling greater sleepiness at the wheel ( stanford sleep scale = 4.2 1.2 vs. 3.6 1.2 , p = 0.03 ) . the authors also showed that an association existed between increased sleepiness and decreased heart rate in osa patients ( r = -0.49 , p = 0.01 ) . philip et al . surveyed 35,004 drivers , and found that 5.2% had osa , 2.3% had restless legs syndrome , 9.3% insomnia , 0.1% narcolepsy and hypersomnia , and 4.4% had multiple disorders . of these drivers , 7.2% reported having had at least one accident in the previous year , 5.8% of which were related to sleep . these results are reflected in related findings that the highest risk of having a motor vehicle accident was associated with narcolepsy or hypersomnia [ or = 8.78 ( 1.97 - 39.06 ) ] , osa [ or = 2.09 ( 1.06 - 4.13 ) ] , insomnia [ ( or = 1.78 ( 1.01 - 3.14 ) ] and others multiple sleep disorders [ or = 1.46 1.20 - 1.78 ) ] . accidents caused by sleepiness occurred primarily on highways ( 77.6% ) and on long trips ( 63.8% ) and more frequently at night ( 40.9% ) than during the day ( 29.4% ) . found an association between sleep disorders ( narcolepsy , insomnia , and osa ) and accidents caused by sleepiness , as well as an association between sleepiness and near miss accidents . concluded that untreated narcolepsy can impair driving performance and increase the risk of traffic accidents . evaluated 750 drivers and found that 14.5% reported sleepiness while driving , and , further , that 2% of the respondents had been involved in accidents caused by sleepy driving . a study conducted by prez - chada et al . of 770 argentinian truck drivers found that 80% were overweight , 71.9% snored ( of those , 4.7% reported apnea ) , 37.8% were hypertensive , 86.9% had insomnia , 13.9% had ess scores above 10 , and 43.8% became sleepy while driving . those suffering from apnea had higher scores on the ess , were more likely to be overweight and a higher prevalence of hypertension . in this population , snoring , ess scores > 10 , and driving for long hours were associated with sleepiness at the wheel , as well as an increased likelihood of having accidents or near misses . out of the total 11% had already had at least one accident because of sleepiness while driving , 33% snored during sleep , 8% reported having respiratory disorders , and more than 50% were obese . de pinho et al . found that out of 300 truck drivers evaluated , 46.3% experienced poor sleep quality , 46% had excessive sleepiness , and 40% were chronically sleep - deprived . snoring was present in 18.3% of the drivers . excessive sleepiness ( es ) was the cause of the majority of accidents reported by the drivers ( 26.5% ) while the reported frequency of es was even greater among drivers who had a history of accidents . a number of studies of shift workers , especially professional drivers , have documented the occurrence of changes in sleep architecture over time . the associated symptoms , including poor quality of sleep , fragmentation due to osa , and sleep deprivation and/or restriction , were thought to explain the es , fatigue , health deficits , possible driving errors , and serious accidents . most european countries now have specific traffic laws for sleep disorders and diseases . several u.s . states , as well as canada and australia , have established laws that prohibit drivers with osa or narcolepsy from driving a car and/or obtaining a drivers license unless their condition is medically controlled . in 2007 , 128,467 traffic accidents were recorded in brazil , with 42,508 being caused by inattention and 3,367 being caused by sleepy driving . in 2008 there were 60,370 confirmed deaths due to accidents , with 36,666 deaths resulting from traffic accidents . in brazil , due to the high rate of traffic accidents caused by sleepiness , a resolution was adopted by departamento nacional de trnsito ( denatran ) in 2008 ( resolution contran / denatran 267/2008 ) to reduce the number of accidents caused by sleepiness , fatigue , sleep respiratory disorders and changes in biological rhythms . some studies have shown that individuals with insomnia and those who were taking various sleep medications had an increased risk of accidents due to fatigue symptoms , reduced alertness and sleepiness at the wheel.[6264 ] over the past 30 years , on - road driving research conducted in the netherlands examined the effects on driving of various medicinal drugs . they were instructed to drive with a steady lateral position and constant speed ( 95 km / h ) . the standard deviation of lateral position ( sdlp ) , i.e. the weaving of the car , was the primary outcome measure of the test . a study of unmedicated healthy volunteers concluded that prolonged nighttime highway driving is associated with increased driver sleepiness , and that this was further associated with progressively increasing sdlp values . after 2 to 3 h of driving , performance impairments are comparable to those seen in drivers who have consumed alcohol and have a blood alcohol concentration ( bac ) or 0.05% , i.e. the legal limit for driving in many countries . as a result of these findings it has been shown effective if during these breaks drivers consume caffeinated beverages such as coffee or energy drinks in order to increase their alertness . the results of these studies showed that benzodiazepine hypnotics and zopiclone significantly impair driving performance , whereas zolpidem and zaleplon seem relatively safe.[6971 ] ramelteon , a melatonin receptor agonist , also significantly impaired next morning driving performance . the findings of these studies are in line with epidemiological evidence showing increased accident risks among those who use benzodiazepine hypnotics and zopiclone and are also generally consistent with other findings cited in this paper showing that a number of sleep disorders can reduce daytime alertness , and thus have adverse effects on driving performance . the various types of evaluations and their respective instruments that are commonly used to study sleep disorders , sleepiness , and wakefulness - rest activity are listed below [ table 1 ] : optalert ( system of infrared reflectance oculography ) : this is a new method for monitoring eye and eyelid movements by infrared reflectance oculography . the method uses transducers attached to a glass frame to measure drivers drowsiness continuously on a new scale ( johns drowsiness scale - jds ) during driving task.the pittsburgh sleep quality index ( psqi ) , includes issues related to sleepiness during daily activities and daily life , sleep , and sleep disorders;the multiple sleep latency test ( mlst),[7779 ] assesses the propensity to sleep in a comfortable position , a factor that is commonly used in the diagnosis of narcolepsy;the oxford sleep resistance test ( osler ) , determines the onset of sleep;the maintenance of wakefulness test ( mwt ) , assesses the individual 's ability to stay awake;pupillography , which evaluates the diameter and variability of the pupil and the relationship of these variables to subjective sleepiness complaints;actigraphy , assesses the activity - rest periods and circadian rhythm disorders of shift workers and may be used to infer hypersomnia and/or fatigue;polysomnography ( psg ) ( the gold standard for diagnosing osa ) is the most commonly used test to evaluate sleep and its disorders . it includes measures based on the electroencephalogram ( eeg ) , mentonian and lower limb electromyography ( emg ) , electrooculogram ( eog ) , electrocardiogram ( ecg ) , pulse oximetry , oro - nasal airflow , respiratory effort measurements , blood gas , snoring , and body position . ( it has been noted however that while psg can contribute importantly to an overall assessment of sleep quality , it is not a specific instrument for assessing sleepiness).[8587 ] the clinical diagnosis of osa is made with protocols used by a physician who specializes in sleep disorders . optalert ( system of infrared reflectance oculography ) : this is a new method for monitoring eye and eyelid movements by infrared reflectance oculography . the method uses transducers attached to a glass frame to measure drivers drowsiness continuously on a new scale ( johns drowsiness scale - jds ) during driving task . the pittsburgh sleep quality index ( psqi ) , includes issues related to sleepiness during daily activities and daily life , sleep , and sleep disorders ; the multiple sleep latency test ( mlst),[7779 ] assesses the propensity to sleep in a comfortable position , a factor that is commonly used in the diagnosis of narcolepsy ; the oxford sleep resistance test ( osler ) , determines the onset of sleep ; the maintenance of wakefulness test ( mwt ) , assesses the individual 's ability to stay awake ; pupillography , which evaluates the diameter and variability of the pupil and the relationship of these variables to subjective sleepiness complaints ; actigraphy , assesses the activity - rest periods and circadian rhythm disorders of shift workers and may be used to infer hypersomnia and/or fatigue ; polysomnography ( psg ) ( the gold standard for diagnosing osa ) is the most commonly used test to evaluate sleep and its disorders . it includes measures based on the electroencephalogram ( eeg ) , mentonian and lower limb electromyography ( emg ) , electrooculogram ( eog ) , electrocardiogram ( ecg ) , pulse oximetry , oro - nasal airflow , respiratory effort measurements , blood gas , snoring , and body position . ( it has been noted however that while psg can contribute importantly to an overall assessment of sleep quality , it is not a specific instrument for assessing sleepiness).[8587 ] the clinical diagnosis of osa is made with protocols used by a physician who specializes in sleep disorders . various methods and instruments for evaluating drowsiness and daytime sleepiness these survey instruments and physiological tests are very important for accurately diagnosing sleep disorders and their symptoms so that driver errors and accidents , which may affect public health , can be avoided . rodenstein summarized several studies dealing with the financial consequences of a - related accidents and concluded that the resulting cost to the public was $ 16 billion per year . it has been projected that cpap treatment could reduce these costs to $ 11 billion per year . further , it has been calculated that 567,000 crashes and 980 traffic deaths could be prevented with osa treatment by cpap . these projections underscore the urgency of the need to develop policies for preventing accidents and promoting the health of drivers who are affected by sleep disorders or other conditions that restrict sleep . the preventive or therapeutic interventions should be emphasized to minimize the deleterious effects of drowsy driving and to educate drivers about the risks of economic loss , poor quality of life and death . figure 1 summarizes of the causes and preventing strategies of sleepiness during driving . due to the high prevalence of osa and sleepiness among drivers , some studies have recommended that educational activities related to drowsy driving and how osa may be an important cause of traffic accidents be made a requirement for the licensure of professional drivers . a summary of the causes and preventing strategies of sleepiness during driving the literature highlights the importance of restorative sleep and the need for drivers to examine the external environment , to recognize the presence of fatigue symptoms , and to determine when to stop to sleep . the aim of these is to promote behavioral change , sleep hygiene strategies , healthy nutritional habits , exercise at certain times , changes in behavior , drugs and alcohol abuse , time of driving and signs of fatigue , stop driving to doze and scheduled naps . some therapeutic interventions include phototherapy to promote alertness and vigilance , with different intensities for each individual , substances or drugs to promote either sleepiness or alertness ( i.e. , caffeine , modafinil , melatonin and others ) , as well as specific sleep disorders treatment . several studies report that naps , phototherapy and caffeine can be helpful for increasing awareness , alertness and memory consolidation . phototherapy using bright light is recommended for night shift workers with insomnia , excessive sleepiness and fatigue . mets et al . demonstrated that 80 mg of caffeine , comparable to a regular cup of coffee or a 250 ml can of red bull energy drink , reduces sleepiness and improves driver performance . similarly , reyner and horne found that 200 mg of caffeine reduced subjective sleepiness in the morning as well decreased accidents . reported that japanese non - professional drivers used naps and coffee intake as strategies for preventing sleepiness at the wheel and possible accidents . lic and summala reported that the most common strategy used by argentinian drivers for avoiding sleepiness was to take a nap for 10 min ( 66.2% ) followed by ingestion of yerba mate infusion with caffeine ( 53.1% ) . the national sleep foundation has recommended that for maintaining alertness and optimal driving performance that sleep be adequate to meet biological needs , should not be fragmented , that the sleep cycle should be synchronized , and that the sleeping environment should be free of disturbances.[104107 ] another strategy reported in the scientific literature is physical exercise in the evening or during the workday to increase the alertness and vigilance of drivers who do shift work or who spend long periods of time behind the wheel . exercise has been shown to increase alertness , reduce sleepiness and accidents by increasing body temperature , as well as to promote restorative sleep . the quality of sleep that an individual experiences , not only its quantity , is an important factor for enhancing ambient , physical and social functioning . sleep that is refreshing , restorative sleep , is important for promoting homeostasis and thermoregulation , and is basic in memory consolidation and cognition , energy restoration and the cerebral energy metabolism . it is known that many individuals experience and adequate total sleep time ( 6 to 8 h ) but do not have enough quality sleep due to repeated awakenings , and/or sleep disorders.[108111 ] abe et al . reported that short duration ( < 6 h ) of nocturnal sleep is associated with an increased risk of rear - end collisions and single - car accidents . it is known that among the numerous types of sleep disorders , excessive sleepiness is more prevalent among drivers with osa . thus , cpap has been recommended as a gold standard therapeutic strategy for remediating osa . reported that osa treatment with cpap in particular reduces the number and risk of accidents and deaths , as well as improving mood , and reducing excessive sleepiness . this review has cited studies showing that sleep disorders such as narcolepsy , insomnia , periodic leg movement disorder , osa , and a number of other common diseases are all associationed with excessive sleepiness , fatigue symptoms such as reduced driving skills , and cognitive deficits . these in turn have been linked to an increased risk of accidents and fatalities on the highway . although , pharmacological treatment for insomnia is meant to improve sleep and thereby reducing daytime sleepiness , driving studies show that many sleep drugs may actually contribute to daytime sleepiness and the increased risk of accidents . the evidence summarized in this paper supports the conclusion that excessive sleepiness is a serious public health concern that directly affects the safety of all who drive motor vehicles . this evidence further supports the recommendation that all disorders which produce excessive sleepiness should be investigated and monitored so that highway accidents and associated injuries and loss of lives can be reduced .

studies have shown that a large proportion of traffic accidents around the world are related to inadequate or disordered sleep . recent surveys have linked driver fatigue to 16% to 20% of serious highway accidents in the uk , australia , and brazil . fatigue as a result of sleep disorders ( especially obstructive sleep apnea ) , excessive workload and lack of physical and mental rest , have been shown to be major contributing factors in motor vehicle accidents . a number of behavioral , physiological , and psychometric tests are being used increasingly to evaluate the impact of fatigue on driver performance . these include the oculography , polysomnography , actigraphy , the maintenance of wakefulness test , and others . various strategies have been proposed for preventing or reducing the impact of fatigue on motor vehicle accidents . these have included : educational programs emphasizing the importance of restorative sleep and the need for drivers to recognize the presence of fatigue symptoms , and to determine when to stop to sleep ; the use of exercise to increase alertness and to promote restorative sleep ; the use of substances or drugs to promote sleep or alertness ( i.e. caffeine , modafinil , melatonin and others ) , as well as specific sleep disorders treatment ; the use of cpap therapy for reducing excessive sleepiness among drivers who have been diagnosed with obstructive sleep apnea . the evidence cited in this review justifies the call for all efforts to be undertaken that may increase awareness of inadequate sleep as a cause of traffic accidents . it is strongly recommended that , for the purpose of promoting highway safety and saving lives , all disorders that cause excessive sleepiness should be investigated and monitored .

INTRODUCTION Fatigue, sleep disorders, excessive sleepiness and accidents Sleep medication Sleep, sleepiness and sleep disorders monitoring Prevention and therapeutic strategies CONCLUSIONS

PMC4633771

a number of stressors have been implicated in honeybee losses in many parts of the world , including habitat loss ; viral diseases ; parasites such as varroa destructor , which can be a disease vector ; and use of pesticides . because these stressors may interact , it is difficult to predict how they change the colony dynamics separately and in combination . moreover , because of the many feedback mechanisms in honeybee colonies , understanding the relationship between the effects on individuals and the colony level effects is not straightforward . ecological modeling enables us to disentangle these interactions and explore them both separately and in combination , in fully controlled simulations . an innate difficulty in studying the effect of pesticides on honeybee colonies is the level of replication needed to capture low - level effects at the field scale . the european food safety authority ( efsa ) has described specific protection goals for honeybee colonies , stating , the magnitude of effects on colonies should not exceed 7% reduction in colony size . to assess whether this level of impact is occurring a minimum of 60 pairs ( control and treatment ) of colonies and fields are needed for each study . if multiple stressors are to be studied even higher numbers would be needed . ecological models can help in designing and targeting empirical studies , generating specific hypotheses that later may be tested experimentally , and can be used to assess the risk of environmental chemicals to honeybees . there have been many laboratory , semifield , and field studies showing both acute and chronic effects of pesticides on adult honeybees and bee larvae . for example , pesticides have been shown to affect foraging via acute mortality , or alternatively from sublethal effects . other effects , such as reduced learning acquisition , decreased rate of learning from olfactory cues , and reduced communication for recruitment to foraging have also been shown to occur , but the realism of these exposures is unclear . in this study , we will concentrate mainly on hypothetical direct lethal effects on different life stages and reduced egg laying rate that could result from sublethal effects on the queen . this does not capture the complexity of real exposure events , but it is important to compare the sensitivity of the colony to mortality of different cohorts at different times of the year in a controlled way . as pesticides can affect individuals in a number of ways , determining the colony level impact of an individual effect feedback loops may compensate for moderate stresses ( e.g. , earlier onset of foraging if food stores are low ) or exacerbate other processes ; for instance , less comprehensive care of brood as a result of high in - hive worker mortalities . the beehave model is a suitable tool to investigate this complexity because it integrates in - hive processes and foraging activities to simulate interactions between colony and environment . the model consists of four modules : ( 1 ) a landscape module , allowing the user to define a landscape of nectar and pollen in food patches ; ( 2 ) a colony module , an age - based cohort model including processes such as nursing and care of brood ; ( 3 ) a foraging module , an individual - based model calculating the foraging activities on a particular day and the quantity of both nectar and pollen brought back into the hive ; and ( 4 ) a varroa and virus module simulating the population dynamics of the varroa mite and the transmission of viruses . the large number of procedures and feedback loops allow a comprehensive view of the impacts of stressors on the honeybee colony . here , we report simulations using the beehave model to explore the colony - level impact of altering the mortality of a number of honeybee life - stages and reducing the egg - laying rate of the queen at different times of the year . because such simulations enable the user to examine a whole variety of stressors on individual bees , and the effects on the colony , finding a standard way of comparing the responses of the colony would be useful in risk assessment . for environmental chemicals the ld50 is the standard index used to describe the median lethal dose of a toxin i.e. that resulting in 50% subject mortality . here , we present an index to compare the impact of different imposed stresses on colony survival , the lis50 , describing the lethal imposed stress level resulting in a 50% colony mortality as predicted using the beehave model . we also present the lis10 , which predicts 10% colony failure from an imposed stress . we argue that these indices will be useful for comparing the impact of imposed stressors at colony level and could also inform the setting of pesticide protection goals in the future , once the indices have been applied to a wider variety of stressors and their variability has been quantified . the beehave model ( beehave - model version 2014 - 03 - 04 , free to download at www.beehave-model.net ) was modified to increase the daily mortality of different life stages of bees in the colony from a defined day for a defined period to simulate potential effects of an exposure event ( where exposure is defined as the period when toxic effects are imposed ) . we used the default setting as described in becher et al . , altering the landscape as explained below . modifications to the model for the treatments below are outlined in the supporting information ( appendix 2 ) . the simulations started from first january with 10 000 worker bees in a colony and ran for 3 years , and each year had an identical annual weather cycle ( based on maximal temperature and hours of sunlight at rothamsted research , hertfordshire , u.k . in 2009 ) . the colony was free from varroa and disease , as the purpose of the simulations was to look at effects of singular events increasing mortality in isolation . on the last day of each year , if fewer than 4000 adult bees were present , the colony is assumed to die due to winter mortality . at the end of each three - year simulation the number of bees alive in the colony , or alternatively whether the colony had failed , was recorded . beehave allows users to define a dynamic landscape , giving values for the distance of each food patch to the colony , and the nectar quantity ( l ) , nectar quality ( sucrose concentration ( mol / l ) ) and pollen quantity ( kg ) for each food patch on each day of the year . the simulations were set up in a very simplified and stylized modeled landscape : a single food patch was 1 km away from the hive offering 20 l of nectar and 1 kg of pollen each day of the year ( although not representing the complexity of real landscapes , this enables tests of potential exposure in each month of the year in a controlled manner ) . we ran simulations to contrast the effects of five different imposed stresses : reduced egg laying rate ( elr ) of the queen , increased daily larval mortality , increased daily in - hive worker mortality , and increased forager mortality , applied daily or applied on each foraging trip . in reality , an exposure event may affect a combination of life stages over varying timeframes via different routes ( nectar , pollen , honey , wax ) , but to specifically examine the sensitivity of different life stages , we chose a simplified set of simulations : examining increased mortality of individual life stages , during single exposure periods , when that exposure is assumed to be direct via consumption of nectar and pollen . we also ran the simulations for one combination of daily life stage mortalities ( larvae , in - hive workers , foragers ) . pupal mortality was not tested , as the pupae are in capped cells and do not receive food and therefore are unlikely to be exposed directly via nectar and pollen . in each simulation , a single stressor was applied for a continuous 30 day period each year . duration of bloom of different crops differ widely as do persistence of different pesticides , but we here chose a 30 day exposure period as typical . timing - dependent effects were investigated by running scenarios with the 30 day exposure period beginning on the first day of each month of the year . imposed mortalities were applied as both multiples of the control value in the model and as set percent daily mortalities , while reduced egg - laying rate was only applied as a percent reduction . testing a multiple of the control reflects the typical procedure of pesticide risk assessments . we also ran simulations with set percent daily mortalities to determine the actual percentage of increased mortality that the colony could withstand . the daily egg - laying rate varies seasonally depending on the day of the year ( for distribution , see becher et al . ) , and eggs are lost at a rate of 3% per day by default . for the simulations , the egg laying rate ( number of eggs produced on a particular day ) was reduced by 25 , 50 , 75 , and 90% for a period of 30 days with zero reduction applied as a control . the daily mortalities of the larvae , in - hive workers , and foragers were altered in two ways : ( 1 ) control daily background mortality was multiplied by a factor of 1 , 1.5 , 2 , or 3 . ( 2 ) daily background mortality was set to a set percentage during the treatment period : one of 1 , 5 , 10 , 25 , and 50% each day . the control daily background mortality is typically low : 0.4% for the in - hive workers and foragers and 1% for the larvae . for the larvae , this mortality does not include the chance of dying from lack of food or brood care . increased foraging trip mortality was simulated in two ways , similar to the stage - specific daily mortality simulations : ( 1 ) control value was multiplied by 1 ( control ) , 1.5 , 2 or 3 ; and ( 2 ) set values of 1 , 2.5 , 5 , 7.5 , and 10% mortality per trip . lower values than for the daily mortalities were used because foragers take multiple trips on a single day , so the majority of foragers may die if a forager had a 25 or 50% chance of dying on each trip , reducing the impact of higher mortalities as each bee can only die once . these settings were used to simulate pesticide exposure at levels high enough to cause death in the foragers through either immediate acute mortality , gradual weakening during the return flight , or through behavioral changes leading to impaired orientation and consequent homing failure . foraging mortality in the model depends on the duration of a foraging trip and is applied before an individual forager returns to the colony . for the single food patch present in the simulations , the mortality is 1.5% for nectar foragers and 0.9% for pollen foragers under control conditions ( values taken from beehave model during control simulations ) . although these values can vary during a day ( as handling time of a food patch is increased when the patch is depleted ) , enough nectar and pollen are provided at the patch that this variation is negligible . to simulate an event in which several life stages are affected , the mortality of each of the larvae , in - hive workers , and foragers were all modified simultaneously in two ways : ( 1 ) control daily background mortalities of larvae , in - hive bees and foragers were multiplied by a factor of 1 , 1.5 , 2 , or 3 . ( 2 ) daily background mortalities of larvae , in - hive bees , and foragers were set to a set percentage during the treatment period : one of 1 , 5 , 10 , 25 , and 50% each day . while it is unrealistic that different life stages are affected with identical effect levels , this scenario demonstrated the colony s sensitivity to multiple effect types . each scenario was run for 30 replicates , with the mean number of live bees at the end of three years as output . for each combination of mortality type and 30 day exposure period , a linear regression was carried out between either the factor increase of the control or the percentage imposed mortality per bee and the mean number of bees alive per colony at the end of the 3 year simulation . the slopes of these regressions were plotted ( figure 1 ) , showing how sensitive the colony is to increased mortality of each life stage . calculated sensitivity of colonies to each stage mortality imposed for 30 days , calculated as the slope of the linear regression for the simulation data shown in figures s1s3 . for each combination of imposed stress and treatment month , a linear regression was performed with the colony population at the end of the third year against the magnitude of the imposed stress . the graphs show the reduction in colony size ( a ) per percent decrease in egg - laying rate ( elr ) ; ( b ) per multiple of the control background daily mortality for larvae , in - hive bees , and foragers ; ( c ) per multiple of control background per - trip mortality ; ( d ) per percent daily mortality of larvae , in - hive bees , and foragers ; and ( e ) per percent daily per - trip mortality . ( ) in these months , all levels of the combined mortality except 1% mortality lead to all colonies being lost ; therefore , it was not possible to fit a linear regression . ( ) in these months , all levels of both the forager and the combined mortalities except 1% mortality lead to all colonies being lost ; therefore , it was not possible to fit a linear regression . to compare between the effects of these imposed stresses at colony level , we calculated a new index analogous to the ld50 : the lis50 was calculated as the level of imposed stress at the individual level that led , statistically , to 50% of the colonies dying in the beehave simulations within 3 years ( using a threshold for survival of at least 4000 bees alive on the last day of each year ) . in these simulations , the level of imposed stress was the percentage stage - specific daily mortality or percentage chance of dying during a foraging trip . dose.p function in r s mass library on a generalized linear model ( glm ) built using data on the number of colonies alive after increased mortalities were imposed . for each of the imposed stresses in question , the mortality was applied from 0 to 100% ( in 5% increments ) for each month of the year ( separately ) with 50 replicates . for foraging mortality per trip , preliminary runs showed that colony death occurred when foraging mortality was 40% for all tested months , so higher mortalities were not tested . the lis50 was chosen for its theoretical parallel to the ld50 , but a lisx could be calculated for any percentage of colony failure ( x ) that is of interest , for example , lis10 figures are also presented , predicting the level of stress resulting in 10% colony deaths . reducing the elr for 30 days had only a moderate impact with none of the colonies dying in any of the simulations ( figure s1 ) . a reduction of the daily egg laying rate by 90% ( i.e. , to 10% of the control ) in june led to the average colony size at the end of three years being reduced by 35% of the initial population ( figure s1 ) . between april and august , each percent reduction in elr led to only 50 fewer bees per colony after three years ( figure 1a ) . nevertheless , colony dynamics were affected to varying degrees depending on the season and the reduction in the egg laying rate ( figure s1 ) . the colony was not highly sensitive to an increase in daily mortality of the larvae or the in - hive workers within the tested range ( figure 1b ; figure s2a , b ) . for the larvae , the control background mortality was already low ( 1% ) and the majority of larvae that died in the control simulations did so from a lack of resources ( food or brood care ) . therefore , small larval losses from increased background mortality could be compensated in the model by allowing resources to be spread among remaining larvae , reducing mortality from a lack of these resources . as with background larval mortality , the control value of daily in - hive worker mortality in the model is small ( 0.4% ) , such that trebling it equates to 1.2% daily mortality and does not result in large losses over the course of the month . the impact of increasing the control daily forager mortality was low when imposed in january to august , but the colony was sensitive to increased mortality imposed in september to december ( figure 1b ; figure s2c ) . the critical threshold for colony survival in the beehave model was applied on the last day of december ; therefore the colony had the whole year to recover from increased individual mortality applied in january , before winter survival was calculated . when the increased foraging mortality was applied to foragers at the food patch on each successful foraging trip , the impact on the colony was much larger ( colony reduced down to almost 4,000 bees in june ) ( figure 1c ; figure s2 d ) , as the mortality was applied many times per day and background mortality is higher than for in - hive life stages . this impact on the colony was likely due to the decreased food stores in the colony . these effects were particularly strong if the stress was imposed during the summer months when foragers were making the most foraging trips . for simulations multiplying the stage specific control mortalities , per - trip foraging mortality was the only single imposed stress to lead to colony failure with 3x mortality in june leading to 77% colony survival . when the mortality of larvae , in - hive workers and foragers were applied simultaneously as a multiple of the control mortalities , the impact on the colony was similar to the worst case equivalent single mortality ( figure 1b , figure s4 ) , the single life - stage daily mortality to which the colony is most sensitive when applied as a multiple of default . the largest impact on the colony from larval mortality came when the effect was applied in one of the months between april and august ( figure 1d , e ; figure s3a ) . during this period , the colony has a lot of larvae as it is building to peak numbers , and increased mortality reduced or delayed this peak ( figure s5 ) . very high larval mortality in may and june led to the colony population being reduced to between 2000 and 3000 individuals , and winter mortality was high ( figure s3a ) . when larval mortality was increased ( illustrated in figure s5 for a level of 25% daily mortality ) , the resulting loss of larval numbers had the effect of reducing deaths due to lack of food or care during the treatment period , as these became more readily available for the surviving larvae . however , high larval mortalities during summer led to a reduction in the worker population , which in turn led to a further peak in larval mortality 1 week after the end of the treatment period ( figure s5 ) . the modeled colony was sensitive to losses of adult bees in most months ( figure 1d , e ; figures s3b d , s6 , and s7 ) . with respect to daily mortalities , the colony was more sensitive to losses of the younger in - hive workers than to the older foragers . during the period of april september , the same period in which brood mortalities had a noticeable impact ( figure 1d ; figure s3 ) , a daily in - hive worker mortality of over 25% led to all colonies being lost between may and july ( table s2 ) and a 5% daily mortality led to up to 40 000 more in - hive worker deaths over the course of the month ( figure s7 ) . loss of in - hive bees led to a large increase in brood loss from lack of care or food over the rest of the year ( figure s6c ) and reduced the honey stores in the hive ( figure s6e ) . outside spring and summer , high daily forager mortality was devastating to the colony ( figure 1d ; figure s3c ) . this is because during the autumn and winter only few eggs are laid , so , the colony consists primarily of older bees still termed foragers ( even though they rarely exited the colony ) . the results of these simulations also highlighted the potential sensitivity of the colony to patch - specific forager mortalities , experienced on each foraging trip ( figure 1e ) . there was little effect at the very beginning or end of the year due to the lack of foraging activity at these times . between may and october , there was a large impact on the colony from increasing this foraging mortality ( june was the most sensitive month , as there was more time to forage in june than other months ) . a 5% mortality at the food patch applied in june led to an average colony size of 1000 bees at the end of the 3rd year ( figure s3d ) with only 5 of 30 replicate colonies surviving ( 17% table s2 ) . when the mortalities were applied as a set percent to several life stages simultaneously , this had a consistently higher impact than the worst case individual daily life stage mortality in each particular month ( forager mortality in winter and in - hive mortality in summer ) ( figure 1d ) . when colonies were subjected to combined daily mortalities of over 10% at any time of the year , then no colonies survived ( table s2 ) . the lis50 values represent the statistical likelihood that a certain imposed stress will lead to 50% colony failure for the specific control conditions used in the model ( in this case calculated after 3 years ) . with agreement on appropriate control settings , the lis50 could be standardized for use over any number of months or years , depending on the sensitivity required . table 1 contains lis50 values for four stressors imposed for 30 day exposures in 4 different months . the months were chosen to be those when foragers are active and crops flower and the colony is therefore most likely to be exposed . a low value indicates that a low daily percentage mortality imposed on individuals led to high colony failure and , therefore , identifies stressors to which the colonies are most sensitive . no values for the reduction in egg - laying rate were given , as no 30 day reduction of egg - laying rate led to colonies dying for any of the chosen months . values of > 100% imply that in all of the simulations , 50% colony loss was not reached . the colonies were most sensitive to in - hive worker daily mortality and to per - trip foraging mortality ( table 1 , figure 2 ) . the in - hive worker mortality started to become very influential in the period between april and may , when the respective lis50 went from over 100% in april to 11% , in may and 7% in june ( table 1 , figure 2 ) as the brood nest was growing exponentially , requiring a large workforce of nursing bees , and the colony structure moved from mostly foragers ( the overwinter bees ) to more younger in - hive workers . the lis50 for the forager mortality per trip has a relatively low value during the summer , with a value of just 4% in june . such mortality was applied many times a day to foragers , especially those that were particularly active within the treated patch so the cumulative daily mortality was higher . the colonies were also sensitive , but to a lesser extent , to daily forager and larval mortality . daily forager mortality had the largest impact in june and july , larval mortality in may and june . imposed stress response curves for the four individual mortalities investigated : ( a ) daily larval mortality , ( b ) daily in - hive worker mortality , ( c ) daily forager mortality , and ( d ) forager mortality per foraging trip . these show the % survival of 30 colonies for each of the varying mortalities , at the end of three years . different colored lines are shown for these mortalities applied for 30 days in ( yellow ) april , ( orange ) may , ( red ) june , or ( black ) july . the intercept between a response curve and the solid horizontal line indicates its lis50 ( table 1 ) , while the intercept with the dashed horizontal indicates its lis10 . importantly , the different imposed stresses had their greatest impact ( smallest lis50 ) at different times of the year . specifically , larval mortality had the lowest lis50 in may , whereas the two forager mortalities and the in - hive mortalities were at their lowest in june . the reason was that increased larval mortality led to a reduction of the adult in - hive population later in the year ( figure s5a ) therefore reducing larval population in may could lead to reduced in - hive worker population in june ( and all impacts which arise from that ) . alternative thresholds of colony failure may be explored with lisx : so lis10 values have also been included in table 1 showing the level of stress causing 10% colony failure . although the lis10 is likely to be quite variable , it gives useful information when used in conjunction with lis50 : for certain life stages in certain months ( in - hive bees and foraging trip mortality ) , lis10 and lis50 were remarkably similar ( figure 2b , d ) where the stress response curve was so steep that there were effectively tipping points when any increase in daily mortality rates ( imposed for this period of 30 days ) led to all colonies failing . using a set of simplified scenarios , our results showed a large variation in the impacts of imposed stress on the honeybee colony depending on both the demographic stage targeted by the imposed stress and the time of year in which the stress is applied . imposed stress on all stages , except for daily forager mortality , led to highest colony losses from april to august . imposed stress to the adult workers was , most often , more damaging to the colony than effects on the brood ; and imposed stress applied to in - hive workers had a larger impact in late summer than the rest of the year . this was partly a consequence of the large proportion of in - hive workers , and that individuals were in - hive bees for a relatively long time such that cumulative stage mortality was higher , even if daily mortality was the same as for other life stages . in - hive bees ( table s1 ) would have consumed a lot of resources in their development but as yet not started generating them for the colony by foraging for nectar and pollen . when the stress was imposed as a multiple of the default , there was little impact on the colony , except in the case of mortality calculated per foraging trip . this suggests that similar fluctuations in daily mortality are insufficient to cause colony loss in isolation , whereas foraging mortality calculated on a per trip basis carries a higher risk to the colony because a forager can perform many trips per day . when mortality of a life stage is a set percentage , > 10% daily ( or per - trip ) mortality for 30 days is very damaging in all cases . the impact of decreased egg laying rate was sizable but not lethal to the colony and other models show similar results . ( using the pc beepop model ) find that eggs are the least damaging of the life stages to lose and adults are the most . similarly , schmickl and crailsheim ( using the hopomo model ) find that reducing the elr to 60% reduced the number of bees in the colony on day 360 down by 40% from control . in real colonies , that may experience swarming events , there may be periods of around 3 weeks with no egg laying . even when egg production is high , as during the summer , these require no food and only a small amount of nursing , so the investment lost with an egg is minimal . large increases of larval mortality at particular times of year ( figure 1 ) can have a significant impact on the colony . individuals only spend 6 days as larvae so , as the stress was applied daily , an individual s chance of being affected during their larval period is lower for the same daily mortality than for other life stages with longer developmental periods ( table s1 ) . however , the colony invests honey and pollen in feeding larvae and has invested care from in - hive workers , and so , losing larvae will represent a net loss of effort to the colony . figure s5 shows the feedback effect of high larval mortalities , at a sensitive time of year , leading to far fewer bees in the colony as a result of reduced potential worker population . for newly emerged workers , a large amount of resources have been used to raise them to adulthood and they have not yet contributed to the colony . depending on the state of the colony and the time of year , in - hive bees spend around 20 days before becoming a forager , meaning there is a long period over which the imposed stress can have an effect . daily mortalities will build up quickly ; a 5% daily in - hive worker mortality in august can lead to overall stage mortality of 65% and approximately 40 000 more in - hive workers dying ( figure s7 ) and further impacts from a 5% daily in - hive worker mortality ( e.g. , increase in larval loss and reduced food stores ) are shown in figure s6 . figure 1 shows that the colony was more sensitive to in - hive worker losses than forager losses during summer , and the colony was more sensitive to forager mortality toward the end of the year . at the beginning and end of the year , the modeled colony contains mostly foragers ; no new workers were emerging , there was little or no foraging taking place , and in beehave , existing workers are classified as foragers once reaching a certain age . therefore , at these times of year , impacts from forager mortality should be seen as impacts from general adult mortality . hence , high daily adult mortalities can heavily reduce either the colony s ability to survive over winter or the colony s ability to build resources early the following year . high foraging mortality will also trigger in - hive workers to become foragers at an earlier age , reducing the age of first foraging . in reality , such precocious foragers may not be as successful as older foragers , resulting in further stress to the colony . the colony is very sensitive to high percentages of this combined mortality throughout the year ( figure 1d ) but , in many months , not much more sensitive than the worst - case daily life - stage mortality at any one time period . an explanation could be that the loss of certain life stages can lead to the loss of other life stages , and hence , removing , for example , both in - hive bees and larvae will not necessarily cause more damage than only removing in - hive bees , as the larvae would have died anyway due to a lack of brood care . at many time points in the year , there is one life stage in the model that the colony is highly sensitive to losing , but at other times , the colony is more sensitive to losing multiple life stages ( e.g. , april and september ) , and these dynamics need further investigation . the set of simulations described here use a precisely defined exposure period , effects on single life stages , and a stylized landscape . in reality , the heterogeneity of the cropped landscape over time and space and the relative toxicity and persistence of different pesticides in the landscape , and in the hive , may lead to a diverse range of sublethal and lethal impacts on individual bees at different life stages . the next steps in using beehave to examine more realistic scenarios will involve using detailed empirical evidence to capture those exposure routes and timeframes , for specific chemicals in precise locations , and a specific module for this is in development . there are many empirical studies showing how stressors affect individuals or , in some cases , the colony in the short term . long - term , multiyear studies are available , yet uncommon , so the impact of imposed stresses over multiple years is not fully understood . find that 1.5 month exposure to two neonicotinoid insecticides through pollen patties starting in may leads to a 28% reduction in worker population in the following april , along with effects on brood size and food stores . dively et al . also find effects on colony strength and overwintering success after 12 weeks ( may august ) exposure to diet patties with high ( 20100 ppb ) levels of imidacloprid . in contrast , three studies of honeybee colony growth and survival in the field , when exposed via natural foraging on flowering crops treated with neonicotinoids , have shown no significant impact of the pesticide exposure on the colonies ( for clothianidin and for thiamethoxam ) . carreck and ratnieks suggest that the levels of pesticide encountered by foraging honeybees are lower in the field than used in many lab experiments . in large - scale field studies , in which the bees are placed near treated crops to forage the bees may have lower and more variable pesticide exposure than in studies where bees are fed with an artificial feed , with pesticide added at field realistic levels and this may explain why the former studies often find less damaging effects . in addition field studies offering the colony a known amount of pesticide ( such as sandrock et al . and dively et al . ) find that the impact upon the hive from the pesticide can appear sometime after exposure . the simulations presented here show how this can occur within the model : figure s5b shows how the number of larvae in the colony is affected by a 25% larval mortality in june . it is clear that there is an additional delayed impact likely due to a reduction in workers providing brood care . these contrasts also highlight the difficulties of scaling from individual level effects to those at the colony level . the beehave model contains a large number of feedback loops , allowing in - depth investigation into how multiple stressors can disturb the colony dynamics in terms of mechanism , and which particular stresses are more damaging to the homeostasis of the colony . for example , with high forager mortality , worker bees become foragers earlier to compensate ; this in turn may reduce the nursing force , increasing larval mortality . a small increase in larval mortality can reduce the mortality of the surviving larvae from other causes such as lack of food or brood care , and reduces further losses , i.e. show a compensatory effect ( figure s6 ) . the beehave model is a useful tool in the risk assessment of stressors to bees as many potential stressors can be assessed simultaneously , and testable hypotheses can be developed . indeed efsa have recently published a review to suggest , with further development , beehave could be the model of choice for regulatory pesticide risk assessment . several models have been used to explore the impact of pesticides on bee colonies . however , the models of khoury et al . and bryden et al . focus on limited portions of colony dynamics , and lack key processes required to accurately predict how a bee colony reacts to numerous stressors . we have shown that the time at which a stress is applied greatly affects the colonies response . a stress imposed in april has little effect , while the same stress imposed in june will devastate the colony . pcbeepop is a model that includes colony dynamics similar to beehave ; although , beehave also includes a number of factors , such as the landscape and foraging dynamics ( including the flow of energy in the form of honey stores ) integrated with the colony module , which may be key to understanding how pesticides can impact the colony . the beehave model is the only tool to date that also includes a dynamic landscape module and weather providing the potential for climate or location - specific simulations , as well as integration with the foraging and varroa and virus modules , to apply many stressors to the colony at any one time , as would be happening to real colonies in the field . further development of beehave , with a pesticide module , to ensure correct implementation of exposure routes from flower , via forager , into the colony is underway . to quantify the impact of a pesticide on a hive , the efsa guidance classifies the magnitude of an effect by the % change in colony size . efsa considers a change between 3.5 and 7% negligible ; and a change larger than 35% to be large . 2011 model to estimate what forager loss would be permissible for negligible change and find that forager losses of 1.5 control for 6 days ; or 2 control for 3 days or 3 control for 2 days would be permissible . we have shown that effects on fecundity or brood mortality are not as impactful on the colony as adult loss , so worker loss is a conservative measure of the damage possible from a pesticide to the colony . the modeled colony has a certain capacity for compensation , which varies with the life stage affected and time of year and durations , but once the compensation threshold is exceeded the colony is likely to fail . levels of background mortality in the absence of pesticide exposure , depending on weather , forage quality and other stressors present , are likely to influence compensation capacity . due to this compensation capacity and how it may vary with the health of the colony , a percent reduction of bees in the colony could have highly variable results on the health of the colony . modeling , such as with beehave , could , therefore , help supplement the risk assessment procedure by teasing apart such dynamics . lis50 and lis10 provide a tool to compare the effects of a variety of imposed stresses on the colony using beehave , treating the colony as an individual super organism by using the percent chance of colony mortality as a measure of sensitivity to imposed stress . colony failure as a result of an introduced chemical is not an acceptable end point , but these indices ( calculated from simulations ) could be used to provide theoretical comparisons of the effects of different stressors on the colony , which may be informative in discussions of future regulatory risk assessment procedures , and protection goals . this study was designed with impacts of pesticides in mind , but impacts on the colony driven by varroa destructor , related diseases , nosema sp also , comparing lis10 and lis50 provides hypothetical evidence of which stressors , at which levels , may lead to colony tipping points , with the caveat that the tipping point will depend on the control scenario ( e.g. , forage availability and weather will all affect the colony s capacity for compensation ) . the beehave model , together with the use of lis50 , allows consistent investigation into the impact of multiple stressors on the honeybee colony , and could be key for future risk evaluation .

to simulate effects of pesticides on different honeybee ( apis mellifera l. ) life stages , we used the beehave model to explore how increased mortalities of larvae , in - hive workers , and foragers , as well as reduced egg - laying rate , could impact colony dynamics over multiple years . stresses were applied for 30 days , both as multiples of the modeled control mortality and as set percentage daily mortalities to assess the sensitivity of the modeled colony both to small fluctuations in mortality and periods of low to very high daily mortality . these stresses simulate stylized exposure of the different life stages to nectar and pollen contaminated with pesticide for 30 days . increasing adult bee mortality had a much greater impact on colony survival than mortality of bee larvae or reduction in egg laying rate . importantly , the seasonal timing of the imposed mortality affected the magnitude of the impact at colony level . in line with the ld50 , we propose a new index of lethal imposed stress : the lis50 which indicates the level of stress on individuals that results in 50% colony mortality . this ( or any lisx ) is a comparative index for exploring the effects of different stressors at colony level in model simulations . while colony failure is not an acceptable protection goal , this index could be used to inform the setting of future regulatory protection goals .

Introduction Methods Results Discussion

PMC3321559

multiple endocrine neoplasia type 2 ( men2 ) is a rare autosomal dominant inherited disorder with a high lifetime risk of medullary thyroid carcinoma ( mtc ) [ 1 , 2 ] . men2 consists of three syndromes : familial medullary thyroid carcinoma ( fmtc ) , men2a , and men2b [ 1 , 3 ] . men2a families have mtc , with at least one individual developing pheochromocytomas , parathyroid hyperplasia , or both . men2b patients have mtc ( with or without pheochromocytoma ) and other characteristic clinical features : mucosal ganglioneuromas , gi ganglioneuromas , eye abnormalities , and skeletal abnormalities including marfanoid body habitus [ 47 ] . men2 is caused by pathogenic mutations found exclusively within the ret proto - oncogene ( rearranged during transfection ) . these are gain - of - function dominant mutations which are commonly heterozygous missense mutations found at specific codons within ret exons 10 , 11 , and 1316 and rarely found within exons 5 and 8 [ 1 , 810 ] . the medical management for the patient and potentially their family members is based on the familial ret variation , which is usually determined by sanger sequencing . discovery of a known men2 pathogenic ret mutation within a family leads to screening for mtc , pheochromocytomas , or parathyroid hyperplasia , and potentially prophylactic thyroidectomy to increase survival rate for the intractable , aggressive mtc . approximately 7580% of mtc patients have the sporadic form of mtc ( i.e. , isolated , nonfamilial mtc ) , not men2 . patients with apparent sporadic mtc are always tested for an ret germline mutation , in case they actually have men2 and require different medical management . although there are many well - known pathogenic ret mutations causative of men2 , it may be difficult to know if a rare or novel germline ret variant is a pathogenic mutation ( patient has men2 ) or nonpathogenic polymorphism ( patient has sporadic mtc ) . interpretation of rare and novel variants will increase in importance as more people are sequenced at the exome , whole genome , or targeted gene levels . many new changes will be found with unknown clinical significance and their presence and allele frequency within the general population is of importance to help determine pathogenicity status of a variant . consortiums like the 1000 genome and other large sequencing projects are making great progress in understanding population sequence variation . yet more direct studies on single genes or gene panels can yield higher sequencing read coverage and more cost - effective sequencing over a smaller genetic area . also , a particular chosen cohort can be sequenced for a particular locus , such as in the case of this study , where a cohort that was self - reported to have no personal or family history of men2 or mtc was sequenced for a section of the ret protooncogene where most pathogenic men2 causative mutations are located . ret sequence variation detected in this men2 unaffected population can then be added to the men2 ret database . this data could be used for several reasons : ( 1 ) to help interpret the pathogenicity of clinically detected ret sequence variation ; ( 2 ) as a reference for any future men2 case studies ( variant was not found in those unaffected by men2 disease ) ; ( 3 ) for improved genetic test design , to avoid or minimize designing probes or primers over known ret sequence variation . to further reduce costs of sequencing large numbers of individuals , multiple samples can be pooled ( without indexing ) before next - generation sequencing ( ngs ) . this was the focus of several studies that analyzed the ability to detect true variants within nonindexed pooled sample sets [ 1116 ] . thirty samples ( 60 alleles ) were the maximum pooling number indicated by our prior studies and in other reports [ 12 , 14 , 17 , 18 ] , for reproducible and accurate singleton allele detection within the pool ( a singleton is a unique allele within the pool ) . a pool of this size was expected to a have a singleton allele read frequency of 1.67% , and with consideration of sequencing error rates and potential variance in ngs determined variant read frequencies , singleton variants are expected to be detected above a cutoff of > 1% variant reads [ 17 , 18 ] . in this study , 136 individuals of an men2 unaffected cohort were sequenced on the illumina genome analyzer utilizing laboratory and bioinformatics protocols from our previous studies for nonindexed , multiple sample pooling . the pool size was limited to less than 30 , which is the previously determined optimal pooling size for accurate singleton variant detection [ 12 , 14 , 17 , 18 ] . in total , 61 variants were detected within the men2 unaffected cohort , which included 20 novel variants . peripheral blood samples from 136 adult volunteers ( 113 caucasian and 23 non - caucasians for ethnic diversity ) were collected and deidentified using university of utah irb protocol no . 7740 . the donors for this unaffected cohort were self - described as not having a personal or family history of neither medullary thyroid carcinoma nor multiple endocrine neoplasia type 2 ( men2 ) . the 51 samples used as controls were deidentified according to irb no.7275 and were sanger sequenced for ret exons 10 , 11 , and 1316 , including exon / intron boundaries . the single - sample control did not have ret mutations causative of men2 , while the 50 pool the 50 pool control was sequenced on the illumina genome analyzer several times previously [ 17 , 18 ] . dna samples were amplified from ret intron 9 to intron 16 using long - range pcr technology . amplicons were normalized by sequalprep ( invitrogen corp , carlsbad , ca ) , quantified using quant - it picogreen dsdna kit ( invitrogen corp ) , and equimolar pooled before illumina library prep , utilizing previously described protocols . between 27 and 29 caucasian samples ' amplicons were combined into four separate pools ( p1 , p2 , p3 , and p4 ) before illumina library prep and ngs . the non - caucasian cohort 's 23 samples were sequenced in a separate pool ( ethnic pool ) . the pcr - amplified ret positions 19180 are positions 4360869143617870 in reference sequence nc_000010.10 ( table 1 ) . two controls were also included in this study , a single sample and also a pool of 50 samples . each pool and each control were sequenced in a separate flow cell lane on the illumina genome analyzer , using single - end read chemistry . sequencing image files were processed and reads aligned to the ret reference sequence with seqman ngen version 2.1 software ( dnastar , madison , wi ) , as described previously [ 17 , 18 ] . reads used were of 67 base lengths since the 3 end read positions of longer reads can have an increase in sequencing background errors , as shown in previous studies [ 17 , 19 , 20 ] . as previously described , several base quality score screening thresholds ( q - threshold ) evaluated for read coverage , errors ( especially for outlier errors , which could be mistaken for false positives in a pool ) , variant read percentage , and base quality score statistics to determine the 30 q - threshold should be used for analysis of all data sets , which minimized errors while maintaining adequate target read coverage ( data not shown ) [ 17 , 18 ] . excluded from analysis was a region of repeats and homopolymers that caused misalignment errors in all data sets ( designated repeat region , amplicon positions 7686 to 7720 ) . changes from the reference sequence were designated variants , and the variant read percentage is the ngs - determined allele frequency . the previously developed subtractive correction method of variant detection was applied wherein the control 's variant read percentages ( at every position and possible variant change ) are subtracted from the pooled data 's variant read percentages , to yield a pooled data set without background sequencing error [ 17 , 18 ] . a subset of the ngs - detected ret sequence variants were validated by either high - resolution melting analysis ( hrm ) and/or sanger sequencing . ret intron 9 used hrm analysis primers ( 5 to 3 ) : forward aca ctg caa tgt gcg ggt ca and reverse gtc ccc caa caa tgc tgc cc . sample dna ( ~5 to 15 ng / ul final concentration ) was amplified and analyzed as described previously , except the lightscanner 32 instrument ( idaho technology , inc . , salt lake city , ut ) which was used for both pcr and hrm analysis . the lightscanner parameters included uracil - dna glycosylase step ( 50c for 10 min ) ; polymerase activation ( 95c for 10 min ) ; 40 pcr cycles ( denaturation at 95c for 1 s , annealing at 62c for 1 s , extension at 72c for 4 s ) ; formation of amplicon heteroduplexes ( 95c for 1 s , then cool rapidly to 40c for 10 s with ramp rate of 20c / s ) ; high - resolution melting protocol ( 70 to 96c with ramp rate of 0.3c / s ) . in order to detect samples with a homozygous ret variant that could not be distinguished from homozygous wild - type samples during hrm analysis , the same procedure was performed , except wild - type dna ( ~5 ng / ul final concentration ) which was spiked into the pcr reaction . the sequence of the single - sample control used in this study exactly matched the reference sequence and therefore had no true variant changes from the reference sequence , only background sequencing error ( table 1 ) . this sample was an ideal control for error rates since any variant reads from the ret reference at each sequence position reflects the background ngs error rates , and also illumina genome analyzer sequencing has demonstrated reproducible , nonuniform , sequence - specific background error rates , read coverage , and base quality scores between lanes and runs using the same version chemistry [ 11 , 12 , 1720 , 23 , 24 ] . this single sample controls for the sequence - specific error rates within the pooled data sets by using the subtractive correction method , as described in our previous studies [ 17 , 18 ] . for subtractive correction , the single - sample control 's variant reads at every possible sequence position , and change from the reference sequence is subtracted from the pool 's variant read percentages . this yields an estimation of the pooled data without background sequencing error rates contributing to the variant read percentages ( examples in figure 1 ) . the single - sample control and 50-pool data were also used for selection of the 30 q - threshold used for quality screening of the data before analysis ( data not shown ) [ 17 , 18 ] . the 50-pool control demonstrated sensitivity to detect known variants with low read percentages for this ngs run and for using the subtractive correction method ( as shown in figure 1 ) . the 50-pool contained 100 alleles and had an expected 1% singleton variant read frequency ( singleton is unique within the pool ) . all 59 variants previously detected in the 50-pool were present at > 0.5% variant reads and at similar percentage variant read values as determined in a previous ngs run with the same library ( r = 0.9991 , figure 1(a ) ) . the 50-pool data had some potential false positives around the cutoff of 0.5% variant reads but after subtractive correction with the single - sample control data , and all true variants were readily detected from the background error ( figure 1(b ) ) . based on our previous work and other studies , sample pools were restricted to 30 or less samples within each pool to result in a variant read percentage above 1% , the chosen cutoff for the most accurate singleton variant detection [ 12 , 14 , 17 ] . caucasian pool p1 had 27 samples , p2 had 29 , p3 had 28 , and p4 had 29 samples , with an expected 1.85% , 1.72% , 1.77% , and 1.72% singleton variant read frequency , respectively . all pool data sets were evaluated with and without subtractive correction of sequencing background error rates using the single - sample control ( figure 1(c ) and data not shown ) . a total of 51 variants were detected in the caucasian men2 unaffected cohort with > 1% variant read values , of which 23 were not found in the non - caucasian men2 unaffected cohort ( ethnic pool ) ( table 1 ) . the lowest singleton variant in the caucasian data sets was in p2 with 1.12% variant read frequency . the 23 non - caucasian samples were in one pool ( ethnic pool ) , with an expected 2.17% singleton allele read frequency . a total of 38 variants were detected in this ethnically diverse men2 unaffected cohort with > 1% variant read values , of which 10 were not found in the caucasian data sets . the variant read percentages for each detected variant is shown in table 1 per pool and also summarized for the four caucasian pools . for comparison , the ncbi dbsnp allele frequency values for detected variants all variants detected were intronic changes , except the expected common polymorphisms found in exons 11 , 13 , 14 , and 15 . of the total 61 variants found in the men2 unaffected cohort , 20 variants were novel changes , not seen in the 50-pool control or in ncbi dbsnp132 . since the 136 unaffected cohort samples had not been sequenced previously by either sanger or ngs methods , several variant locations within three pools ( total of 79 samples ) were chosen for validation . the high - resolution melting ( hrm ) analysis method , which is a rapid , closed - tube mutation scanning assay , was chosen to genotype each individual sample for validation of ngs variant detection and the ngs determined variant allele frequency ( table 2 ) . high - resolution melting analysis detects sequence variation within the pcr amplicon using a saturating dsdna dye and in many cases can uniquely identify each variant based on differential melting profiles ( figure 2(a ) ) [ 2528 ] . hrm assay states 100% specificity and sensitivity for detection of heterozygous variants within small amplicons ( < 300 bp ) . hrm analysis was used to detect sequence variations within a section of ret intron 9 and exons 13 and 15 ( figure 2 and data not shown ) . ret exons 13 and 15 were chosen since they each contain a common polymorphism present in all pools that could be detected using the previously developed hrm assay . exon 13 contains c.2307g > t variant and exon 15 contains c.2712c > g variant ( ret amplicon positions 5153 and 6943 , respectively , tables 1 and 2 ) . intron 9 was chosen since it contains three ngs - detected variants in close proximity at ret amplicon positions 117 , 156 , and 174 ( c.1760197g > t , c.1760158c > g , and c.1760140c > g , resp . ) , and also to verify the novel c.1760158c > g variant detected in caucasian pool p2 which had the lowest variant read percentage of 1.12% ( expected 1.72% singleton read frequency within that pool of 29 samples ) . since some homozygous variants can have similar melting profiles as the wild - type sample , a technique that spikes wild - type dna into the pcr reaction to allow distinction of homozygous variants was performed on any sample that appeared wild - type after testing in the first hrm assay . this technique identified four homozygous variants in ret exon 15 and one homozygous variant in ret intron 9 ( figure 2(b ) and data not shown ) . the hrm determined allele frequency correlated well with the ngs variant read percentage for each variant in each pool ( table 2 ) . the variant with the lowest read percentage ( position 156 in p2 , 1.12% ) was verified as present and heterozygous in one sample within caucasian pool p2 . this paper describes ret proto - oncogene sequence variation detected in an men2 unaffected cohort of 136 individuals . the previous genome analyzer sequenced 50-pool library [ 17 , 18 ] was used to control for the detection of variations with low read frequency , and all known variants were detected > 0.5% variant reads . with similar error rates between genome analyzer lanes of the same run [ 11 , 12 , 17 , 19 , 20 , 23 , 24 ] , the singleton variants in a less than or equal to 30-sample pool should be accurately detected above background error using our previously determined cutoff value of > 1% variant read frequency . the single sample controlled for background sequencing error rates across each ret sequence position and was used for the subtractive correction method of variant detection for pools [ 17 , 18 ] . the majority of the men2 unaffected cohort were of caucasian ethnicity , while 23 samples were non - caucasian ( ethnic pool ) and were used to identify ret variants within a more ethnically diverse sample set . the 136 samples were distributed into five nonindexed pools and were sequenced in five separate flow cell lanes . using previously described protocols for bioinformatics , subtractive correction , and variant read cutoff value of > 1% [ 17 , 18 ] , a total of 61 ret variants were detected within the men2 unaffected cohort . twenty of these variants were novel , not in ncbi dbsnp 132 ( which includes 1000 genome data ) or found in our previous sample pooling studies on the ret proto oncogene [ 17 , 18 ] . many of these novel changes were specific to either the caucasian or ethnic samples and were of low variant read frequency , so they were likely to be singleton or doubleton variants within the pools . several variants were verified by hrm and sanger sequencing , including the novel change ( c.1760158c > g ) with the lowest variant read percentage ( 1.12% ) . the ret men2 database developed by the author so far has 147 entries , of which 74 are known pathogenic mutations and 62 are variants of uncertain significance . this database has been used as a model for predictions of phenotypic severity of variants of unknown clinical significance within the ret proto oncogene . ret sequence variation data for these men2 unaffected cohorts will be added to the men2 ret database . this variant data will help in medical interpretation of variations found in this ret proto - oncogene region using methods such as sanger sequencing or ngs ( targeted to the ret gene , the whole exome , or whole genome sequencing ) . any ret sequence change detected in individuals with a family history of men2 symptoms or where men2 is suspected ( patient with apparent sporadic mtc or pheochromocytoma ) can be compared to the available ret men2 database , and also to the benign ret sequence variation present in the large cohort of unaffected individuals that was generated in this paper . this highlights the importance of clinically relevant databases with not only known pathogenic changes , but also the inclusion of known benign changes for clinical test interpretation . the men2 unaffected cohort 's variant results can also be used in comparison to variants detected in suspected men2 patients for case reports . a potential problem for genetic test design is unknown variants present in the location of the pcr primers , sanger sequencing primers , or melting analysis probes . results from this unaffected population will help with genetic test design , so that primers , and probes will not be designed over the known ret sequence changes . this paper presents sequence variation detection methods that could be used for other genes and analysis for specific cohorts ( unaffected versus affected , by different ethnicities , or those with specific symptoms of disease ) . the resulting data can be added to locus - specific databases to help interpret the pathogenicity of clinically detected sequence variation . these validated methods can also apply to other pooled samples ( such as genetic locations for gwas followup or for testing - specific populations ) and natural pools ( such as mitochondrial heteroplasmy or mixed tumor populations ) .

multisample , nonindexed pooling combined with next - generation sequencing ( ngs ) was used to discover ret proto - oncogene sequence variation within a cohort known to be unaffected by multiple endocrine neoplasia type 2 ( men2 ) . dna samples ( 113 caucasians , 23 persons of other ethnicities ) were amplified for ret intron 9 to intron 16 and then divided into 5 pools of < 30 samples each before library prep and ngs . two controls were included in this study , a single sample and a pool of 50 samples that had been previously sequenced by the same ngs methods . all 59 variants previously detected in the 50-pool control were present . of the 61 variants detected in the unaffected cohort , 20 variants were novel changes . several variants were validated by high - resolution melting analysis and sanger sequencing , and their allelic frequencies correlated well with those determined by ngs . the results from this unaffected cohort will be added to the ret men2 database .

1. Introduction 2. Materials and Method 3. Results 4. Discussion

PMC4782628

liver fibrosis is an important cause of mortality and morbidity and contributes substantially to increase health care costs in patient with chronic liver diseases . fibrosis can lead to cirrhosis , for which the complications such as hepatic decompensation , hepatocellular carcinoma , and portal hypertension involve growing public health concerns . cirrhosis and chronic liver disease were the 10th leading cause of death for men and the 12th for women in the united states in 2001 , leading to the death of about 27,000 people each year . cirrhosis was first considered as an irreversible process , but , with the growing understanding of hepatic fibrogenesis mechanisms , more effective treatments have been developed [ 3 , 4 ] . however , the latter must be initiated at a specific and early stage in fibrous development , and their administration requires regular clinical followup . while histological analysis after liver biopsy is the gold standard for the diagnosis , inherent risk of a recognized morbidity and mortality renders this method unsuitable for clinical monitoring [ 5 , 6 ] . it has been demonstrated that perfusion imaging has the potential to detect and assess vascular modifications associated with liver fibrosis . several studies , using magnetic resonance dynamic contrast - enhanced imaging ( mr - dce ) to quantify liver perfusion , have shown that some perfusion parameters were relevant indicators for liver fibrosis assessment [ 1012 ] . in a previous work , an mri protocol associated to a dedicated processing step to quantify liver perfusion was developed [ 12 , 13 ] . results demonstrated that mr perfusion imaging could be a noninvasive method for the clinical followup in patient with chronic liver diseases . nevertheless , the evaluation was restricted to an roi , and regional variations often met in diffuse liver diseases could not be observed . roi - based perfusion quantification already requires heavy processing methods such as image registration , denoising , and data fitting . processing time drastically increases and becomes really prohibitive for clinical application for 2d or 3d mapping . in this context , parallel computing on distributed infrastructures such as clusters , grids , or clouds proves to be an interesting solution . such infrastructures can bring significant speedup for a large spectrum of applications from various scientific domains . they have already been used for medical imaging as described in [ 14 , 15 ] but never before for 3d - liver perfusion mapping . nevertheless , new applications still require extra work for adapting ( porting ) them on the considered infrastructure . this work describes an mr acquisition protocol and a processing method using distributed computing on the european grid infrastructure ( egi ) to allow 3d liver perfusion parametric mapping after mr - dce imaging with the ms-325 blood pool agent . processing speed , reproducibility , and accuracy were assessed and adequate acquisition requirements were defined . the study protocol was approved by the local experimentation ethics committee , and informed consent was obtained from each patient . seven subjects ( 4 women , 3 men ; average age , 40 12 years ; mean weight , 75 8 kg ) were enrolled . among this group , one healthy subject was used as control and six patients with chronic liver diseases were prospectively enrolled ( maximum prospective period of one month ) after having had a liver biopsy . tissue samples were fixed in buffered formalin and embedded in paraffin . 4 m - thick sections were stained with hematoxylin - eosin - saffron , iron stain , and masson trichrome reagents and evaluated by two pathologists . fibrosis was evaluated on trichrome - stained slides according to the metavir classification ( score f0 : absence of fibrosis ; score f1 : portal fibrosis ; score f2 : portal fibrosis with isolated bridges score f3 : fibrosis with numerous bridges without cirrhosis ; score f4 : cirrhosis ) . acquisitions were performed using a siemens magnetom symphony maestro class 1.5 t imaging system ( siemens medical solutions , erlangen , germany ) . a t1-weighted vibe 3d sequence with a parallel imaging technique was used ( grappa , r - factor = 2 ) . the sequence parameters were as follows : te / tr/ , 1.22/2.87 ms/12 ; k - space partial filling , 6/8th according to slice and phase direction ; reduction of the slice and phase encoding step , 63 and 50% , respectively . the plane was coronal oblique with a rectangular fov ( 400 300 mm ) for a rebuild matrix of 256 192 pixels with right / left phase - encoding direction . the rationale behind the use of coronal imaging was to minimize the flow - related enhancement of the aorta . the exploratory volume was acquired with a 1-sec temporal resolution with 6.4 cm slab thickness ( 16 slices of 4 mm ) . the signal was collected using two circularly polarized phased array coils ( cp body array and cp spine array ) with a bandwidth of 650 hzpixel . acquisition has begun at the time of injection of the contrast medium ( ms-325;epix pharmaceutical , inc . , lexington , ma , usa ) , and it continued for 2 minutes [ 12 , 13 ] . injection was performed with an injection rate of 1 mls , a posology of 0.03 mmkg , and flushed with 25 ml of physiologic saline injected at the same rate . finally , sixteen 2d + t volumes with t = 120 were acquired leading to 1920 images per examination . images were first imported on a personal computer running an in - house developed application written in matlab r2010a ( the mathworks , natick , ma , usa ) . due to free - breathing acquisition , spatial shifts linked to motion had to be corrected . this method consisted in the estimation of the transformation vector needed to register each moving images in relation to a static reference image . for each image from 2d + t volumes , a pixel - based method was used ( iconic approach ) to control the transformation of an input image . an error measure was used to measure the registration error between the moving and static image . the reduced - memory broyden - fletcher - goldfarb - shanno ( bfgs ) quasi - newton algorithm was used to move the control points to achieve affine rigid registration between both images with a minimal registration error native 2d + t volumes were converted in to 2d + t ms-325 mass concentration maps from a pixel - by - pixel operation based on the relationship between signal intensity and ms-325 concentration . thirdly , native arterial and portal input functions , ca(t ) and cp(t ) , were measured using squared rois of 25 pixels placed by an experienced radiologist ( f.p . 12 years of postgraduate experience in digestive imaging ) at the level of the abdominal aorta close to the cliac trunk and the main portal vein . finally , definitive arterial and portal input functions were converted into continuous form ( function of the time ) instead of vectorial form ( discrete form ) , by an interpolation using spline curves . hepatic capillary system was modeled by a 3-parameter one - compartment pharmacokinetic model adapted to hepatic dual supply ( portal and arterial ) . the leakage of tracer through venous washout is carried out exponentially over time and the inverse of the constant of elimination is the mean transit time ( mtt ) . the equation describing this model is as follows : ( 1)c(t)=[ca(ta)a+cp(tp)p]et / mtt , where designates the convolution product and the volemic mass considered to be equal to 1 gml . the parameters , a and p , are the arterial and portal perfusion , respectively , expressed as ml100 gmin . ca and cp , are respectively , the arterial and portal input functions . the two delays , a and p , take into account the temporal offset between central compartment input and measured input from arterial and portal rois . while a , p , and mtt are model parameters , delays are independent of the fit procedure . the hepatic perfusion index ( hpi ) , defined as the arterial perfusion to total perfusion ( arterial + portal perfusion ) ratio , was also calculated . for each part of the image , pixel - by - pixel tissular time activity curves were obtained and a nonlinear least - square fit was performed according to the model previously described(1 ) using the levenberg - marquard algorithm . because some coefficients are closely connected , in particular portal perfusion and arterial perfusion with arterial and portal delay , the results of optimization were strongly influenced by the choice of starting coefficients , and algorithm may converge to local minima . in order to improve the robustness and reliability of optimization , but also to avoid any convergence to local minima , the algorithm needed to be started with a grid of pseudorandom starting points generated within two bounds ( multistart technique ) . so , each fit procedure was done two - hundred - fold , with two - hundred different initializations . for each fit procedure , delays were determined as the time between the beginning of tissular enhancement and the beginning of arterial enhancement in celiac trunk . these starting points are chosen as the maximum of second - order derivative of tissue time activity curve and arterial input function . from this step , three perfusion parametric maps were obtained , one for each parameter of the model used . the processing step was parallelized and executed on egi within the biomed virtual organization ( vo ) . the parallelization was handled at the input data level , by splitting each volume into several pieces . each piece was processed by independent jobs running in parallel on multiple grid resources and eventually merged . the whole processing operation was modeled and implemented as a grid workflow using the gwendia language and the moteur workflow engine . the splitting and merging algorithms were developed in c++ , while the processing algorithm was developed in matlab . all three programs were compiled on a grid compliant operating system ( centos ) and deployed on the fly on the grid nodes . for the matlab code the interface with the grid resources was provided by the vip web platform ( https://vip.creatis.insa-lyon.fr/ ) . a specific cartography workflow was developed for this application and integrated into the vip platform . the user uploaded the input volumes on the grid and launched the processing workflow from a web portal . in order to evaluate the speedup provided by our parallel approach , the total cpu time to make span ratio was determined . the makespan was defined as the time elapsed between the launch and the completion of the workflow , and the total cpu time as the sum of cpu times of all jobs in a workflow . in order to evaluate the reproducibility of our distributed computing algorithm , relative standard variation ( coefficient of variations ) was then mapped for each parametric map for all patients and defined as the standard deviation to arithmetic mean ratio . next , to evaluate the accuracy of our method , results between roi - based quantification method described in and the method presented in this paper were compared . the difference between methods was evaluated using the bland - altman representation for each perfusion parameters , the spearman 's coefficient calculation , and the nonparametric wilcoxon test . among the 6 biopsied patients , histological results were as follows : 2 patients were scored f0 , 3 patients scored f2 , and 1 patient scored f4 . a representative set of 2d parametric maps extracted from 3d volumes on the healthy patient ( metavir f0 ) is shown in figure 1 . then , parameter values were stratified according to the fibrosis severity . results corresponding to the advanced stage ( metavir stage f2 ) and early stage ( metavir stage < f2 ) are presented in table 2 . a significant correlation was observed between roi - based method and distributed method for each parameter . spearman 's coefficients ( ) were 0.86 , 0.92 , and 0.80 ( p < 0.01 ) for arterial perfusion , portal perfusion and mtt , respectively . regarding the wilcoxon test and the bland - altman representations ( figure 3 ) , significant difference was shown between compared methods . however , bland - altman representations showed a systematic decrease of mtts values calculated with distributed method compared to roi - based reference method . the major drawback of the perfusion - based method , its prohibitive computing time , has been overcome with the help of the egi . by using the resources of distributed european infrastructure , 1 cpu year ( corresponding to twenty - one 3d mapping procedures ) the speedup varied among the 21 workflows from 20 to 94 with an average value of 48 . the average error ratio for the experiments presented here was of 18% , with a maximum of 43% for one of the workflows . as shown in figure 2 , the late completion of the last jobs significantly increases the makespan . the workflow needs to wait for the completion of all jobs in order to produce the final result . thus , for the experiments presented here , the makespan was almost tripled ( from roughly five to fifteen hours ) because of these late jobs . similar results have been reported in other studies such as , where the authors propose a dynamic load balancing approach in order to improve performance . nevertheless , the dynamic load balancing approach proposed in only works for monte - carlo - based simulations . the presented method was reproducible and results were correlated with roi - based reference method run locally on a personal computer . the quantified parameters were found to be in the same range as those obtained with the reference roi - based method and those related in the literature[9 , 10 , 12 ] . nevertheless , patient - wise , quantified values were slightly modified even if the shift was not found significant . indeed , compared to the roi - based method , blood flow quantified with the presented 3d method is overestimated whereas ; on the contrary , mtts are underestimated . additionally , for each parameter , standard deviation observed with 3d methods run on egi was found lower compared to the roi - based method . when results are globally stratified according to fibrosis severity , the difference between mean values for each parameter computed with presented method is systematically lower than with roi - based method . these findings confirm the smoothing effect induced by 3d quantification algorithm . indeed , in roi - based estimation method , arterial and portal delays are optimally set by user . however , these delays depend on spatial location and take into account the time shift between the measured input functions and the position where modeling takes place in parenchyma . hence , manual setting is not possible in the 3d case , and an automatic estimate of both delays was mandatory . due to relatively low image signal - to - noise ratio ( snr ) of about eighteen , this step requires hard smoothing filtering , affecting quantification results with the acquisition data currently available . another limitation is the restricted exploration volume . indeed , to keep an acceptable snr acquired with a high temporal resolution of 1 sec , the number of encoding steps in the slice - encoding direction was limited and the whole liver volume was not covered . these restrictions ( snr and coverage ) can be overcome with the latest imaging mr systems with improved acquisition capabilities using 32 receiver channels with multiple element array coils . an snr value of 80 was measured based on preliminary test performed at our institution with a 3 t gehc mr 750 ( gehc , milwaukee , wi , usa ) with 32 ch body coil . the parallelization of the method brings significant speedup and renders it feasible despite its prohibitive computing time . currently the poor scheduling of the last tasks is largely due to platform heterogeneity and multiple task resubmissions caused by high error ratios . data transfers account for most of the errors , while the rest are mostly application failures due to improper grid node configuration . as future work , scheduling will be improved by taking into account these considerations . to conclude , this preliminary study demonstrated that the described method allows 3d liver perfusion quantification within a reasonable processing time . it is now suitable to be used for similar clinical studies in a research context . while the distributed processing method was validated compared to the roi - based quantification , such fully automatic processing requires high - quality images . the required snr , together with a high temporal resolution and large volume exploration , can now be achieved on the latest 3 t mri systems available . further work will have to demonstrate the interest of parametric 3d perfusion maps for fibrosis assessment on a larger number of subjects with chronic liver disease .

an mr acquisition protocol and a processing method using distributed computing on the european grid infrastructure ( egi ) to allow 3d liver perfusion parametric mapping after magnetic resonance dynamic contrast enhanced ( mr - dce ) imaging are presented . seven patients ( one healthy control and six with chronic liver diseases ) were prospectively enrolled after liver biopsy . mr - dynamic acquisition was continuously performed in free - breathing during two minutes after simultaneous intravascular contrast agent ( ms-325 blood pool agent ) injection . hepatic capillary system was modeled by a 3-parameters one - compartment pharmacokinetic model . the processing step was parallelized and executed on the egi . it was modeled and implemented as a grid workflow using the gwendia language and the moteur workflow engine . results showed good reproducibility in repeated processing on the grid . the results obtained from the grid were well correlated with roi - based reference method ran locally on a personal computer . the speed - up range was 71 to 242 with an average value of 126 . in conclusion , distributed computing applied to perfusion mapping brings significant speed - up to quantification step to be used for further clinical studies in a research context . accuracy would be improved with higher image snr accessible on the latest 3 t mr systems available today .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion