The availability and low cost of lignocellulosic biomass has caused tremendous interest in the bioconversion of this feedstock into liquid fuels. One measure of the economic viability of the bioconversion process is the ease with which a particular feedstock is hydrolyzed and fermented. Because monitoring the analytes in hydrolysis and fermentation experiments is time consuming, the objective of this study was to develop a rapid fluorescence-based method to monitor sugar production during biomass hydrolysis, and to demonstrate its application in monitoring corn stover hydrolysis.Escherichia coli strain CA8404 (a hexose and pentose-consuming strain), modified to produce green fluorescent protein (GFP). The combination of hydrolytic enzymes and a sugar-consuming organism minimizes feedback inhibition of the hydrolytic enzymes. We observed that culture growth rate as measured by change in culture turbidity is proportional to GFP fluorescence and total growth and growth rate depends upon how much sugar is present at inoculation. Furthermore, it was possible to monitor the course of enzymatic hydrolysis in near real-time, though there are instrumentation challenges in doing this.Hydrolytic enzymes were used in conjunction with We found that instantaneous fluorescence is proportional to the bacterial growth rate. As growth rate is limited by the availability of sugar, the integral of fluorescence is proportional to the amount of sugar consumed by the microbe. We demonstrate that corn stover varieties can be differentiated based on sugar yields in enzymatic hydrolysis reactions using post-hydrolysis fluorescence measurements. Also, it may be possible to monitor fluorescence in real-time during hydrolysis to compare different hydrolysis protocols. Fermentation reactions are important for the production of many valuable products including pharmaceuticals, beverages, and biofuels. In lignocellulosic ethanol production, biomass feedstocks are chemically pretreated, hydrolyzed by cellulases and hemicellulases, and the resulting sugars are fermented by yeast or bacteria to produce ethanol . In largin vitro gas production [One approach is to complete a simultaneous saccharification and fermentation (SSF) process  and to aoduction . In thisIn contrast to full SSF processes, many sugar detection and quantitation methods can be employed, including chemical reducing sugar assays and enzymatic assays ,7. All oOnce a sugar scavenger has been added to the mixture of hydrolytic enzymes, the problem of product inhibition has been solved, but another problem replaces it: the sugars produced are consumed as they are produced, making measurements of sugar concentrations a poor predictor of total sugar released. For this reason, simply relying on a sugar assay or a sugar biosensor, such as one described by Lidgren et al , will noEscherichia coli strain CA8404 was selected as the microbe because it carries the crp* mutation which reduces catabolite repression and thereby allows both the 5- and 6-carbon sugars produced from corn stover hydrolysis reactions to be metabolized simultaneously [E. coli metabolize the sugars from the hydrolysis reaction, cell mass (X) changes at a rate of dX/dt. This change in culture cell mass can be monitored by light-scattering measurements of culture turbidity. However, certain reactions, including lignocellulosic biomass hydrolysis, produce substances that interfere with light-scattering measurements. In order to more easily monitor change in cell culture mass, the E. coli strain CA8404 was modified to produce a visual marker, green fluorescent protein (GFP) [Our approach was to develop a microbial system that reports sugar levels in a reaction that mimics the NREL lignocellulosic ethanol production process . The preaneously . As the in (GFP) ,11. The in (GFP) .® Site-Directed Mutagenesis kit in order to produce a pPNptGreen plasmid without a functional GFP fluorophore . Approximately 300 bp into the GFP coding sequence, DNA encoding a glutamate residue (GAA) was changed to encode a stop codon (TAA).  The new plasmid was designated pPNptOchre. The original pPNptGreen plasmid and the pPNptOchre plasmid without the functional GFP fluorophore were transformed separately into E. coli strain CA8404 to produce the two strains, crp*-gfp and crp*-gfp-.Site-directed mutagenesis was conducted according to the Stratagene product QuikChange IIAt grain maturity, cobs were removed from the corn plants and all corn stover samples were cut at approximately six inches above the soil by a forage chopper. Approximately 0.8 kg of sample (wet weight) at a moisture content of about 35% was collected from each plot and samples were dried at 55°C for one week. The material from each sample was ground by a hammermill with a 1 mm screen. We did not evaluate the distribution of different botanical tissues in the ground material; however, the ground material appeared uniform. To minimize the effect of a non-uniform distribution of biological material in the sample we used multiple subsamples in each experiment.® A-40 (a cellulase/hemicellulase mixture from Genencor Intl.), we carried out hydrolysis reactions in the presence or absence of 10 mM d-glucose . Each treatment was run with four replicates using 5 mg of a stover sample treated with a 1:20 dilution of enzyme Multifect® A-40 in citrate-phosphate buffer . Hydrolysis was conducted at 60°C for 90 min. Following hydrolysis, the tubes were centrifuged for 1.5 min. at 10,000 × g in a microcentrifuge . An aliquot of the supernatant from the hydrolysis reaction was measured with a hexokinase glucose assay kit . The absorbance was measured at 340 nm (OD340) using the MRXII plate reader by DYNEX . The absorbance value was converted to glucose yield with a standard curve constructed by plotting OD340 values versus glucose concentrations following analysis of a series of solutions with known glucose concentrations.To characterize the product inhibition of the enzyme preparation MultifectE. coli crp*-gfp and crp*-gfp- paired by treatment were grown in modified 1 × M9 minimal media [595) measurements were taken by the MRXII plate reader . The samples from the wells to be measured were then transferred into a black, 96-well cell culture plate , and fluorescence measurements  were taken by the SpectraFluor Plus plate reader . Note that these wavelengths  were used consistently throughout the study. The AirPore™ seal was replaced on the clear 96-well plate and returned to the incubator. To obtain a value for GFP-specific fluorescence for each culture pair, the fluorescence reading of the crp*-gfp- strain was subtracted from the fluorescence reading of the crp*-gfp strain.Cultures of al media . The M9 al media . d-glucoE. coli crp*-gfp and crp*-gfp- were grown in modified 1 × M9 minimal media containing 2, 4, or 8 mg/ml d-glucose. Absorbance and fluorescence were measured every 2 h for 22 h, and the GFP-specific fluorescence was determined.To establish whether it was possible to use GFP to detect differences in changes in culture cell mass in response to sugars, cultures of E. coli crp*-gfp and crp*-gfp- were grown in modified 1 × M9 minimal media containing d-glucose in concentrations ranging from 0.025 mg/ml to 6.0 mg/ml. Absorbance and fluorescence measurements were taken 20 h after inoculation, and the GFP-specific fluorescence was determined.To determine the sensitivity and dynamic range of the microbial system, cultures of E. coli strains crp*-gfp and crp*-gfp- were grown in modified 1 × M9 minimal media containing 2 mg/ml d-glucose for 20 h. After 20 h, half of the cultures (one set) were randomly selected to receive an addition of 8 mg/ml d-glucose for a total of three replications each of spiked cultures and unspiked cultures. Absorbance and fluorescence were measured every 2 h, and the GFP-specific fluorescence was determined.To determine the response time of the microbial system, glucose was added to the reaction when the culture reached stationary phase. Two sets of three replications of both E. coli strains crp*-gfp and crp*-gfp- were grown in modified 1 × M9 minimal media, containing 20 mg/ml d-glucose. Chloramphenicol was added to a random selection of half of the cultures after 13 h for a total of three replications each of cultures with chloramphenicol and without chloramphenicol. Absorbance and fluorescence were measured every hour, and the GFP-specific fluorescence was determined.Another way we examined the response time of the microbial system was by stopping protein production when the culture was in mid-log phase. Six replications of both -. The difference in the fluorescence of these two tubes was used to determine the GFP-specific fluorescence. Then 1150 μl of 0.5% (v/v) sulfuric acid were added to each tube, and the tubes were incubated at 100°C for 1 h [4, 164.2 μl 1 M CaCl2, 1.7 ml thiamin at 10%, 8.3 ml kanamycin at 10 mg/ml, and 33 ml crp*-gfp or crp*-gfp- liquid culture (grown overnight at 37°C in 1 × M9 media to an OD595 of ~0.6). In addition, 25 μl of 1:1 GC220: Multifect® Xylanase (Genencor Intl.) were added to each tube. The tubes were allowed to incubate with shaking at 37°C and 225 rpm. Samples containing 100 μl of 0, 2, 4, 6, 8, 10, 12, 14, 16, or 18 mg/ml sugar at ratios of 37.5 xylose: 62.5 glucose in place of corn stover were included as positive controls. Absorbance and fluorescence were measured after 20 h of incubation by allowing the stover particles to settle in the culture tube and transferring 100 μl of the culture to a 96-well plate.The microbial system described here, referred to as simultaneous saccharification and catabolism (SSC) was used to analyze corn stover samples of five different corn varieties. For each sample to be analyzed, 25.0 ± 0.2 mg of dried and ground corn stover was weighed into two separate 14 ml sterile test tubes . Two tubes were used to control for variations in fluorescence of the corn stover samples: an experimental tube to be inoculated with crp*-gfp and a control tube to be inoculated with crp*-gfp- cultures from crp*-gfp cultures. These values were then analyzed by ANOVA in order to characterize variation in the experiment. When variation was significant, a student's t-test was performed on each pair to compare means of the samples. The coefficient of variance (CV) was also computed in order to determine the variation in measurements for each genotype.The GFP-specific fluorescence values were computed by subtracting the fluorescence from crp*-gfpThe SSC method described above was used to analyze stover samples from five corn varieties. Absorbance and fluorescence were measured every 2 h for 24 h and once at 36 h.® A-40 was strongly inhibited by the relatively low level of glucose (10 mM d-glucose). We observed 10-fold or greater reductions in glucose yield when hydrolyzing corn stover in the presence of added glucose (data not shown). This clearly demonstrated the need for an assay in which the hydrolysis products were removed from the hydrolysis reaction as they were produced.Enzyme MultifectThe objective was to establish whether it was possible to detect differences in changes in culture cell mass using GFP as a reporter and to determine the relationship between these changes in culture cell mass and GFP fluorescence. Three different concentrations of glucose were added to culture media and the absorbance and fluorescence of the cultures were measured over time. The absorbance data were fit to the Gompertz equation ,16 whichdX/dt (measured by monitoring culture turbidity with time) of the culture. To test this hypothesis, these data were fit to the first derivative of the Gompertz equation  between the integrated fluorescence and culture OD. To further characterize the relationship between initial sugar concentration and GFP-specific fluorescence, we regressed GFP-specific fluorescence at 20 h, and integrated fluorescence from 0 to 20 h, on initial glucose concentration, finding R2 values of 0.91 and 0.98 respectively. The integrated fluorescence was the better predictor of initial sugar concentration. The 20 h GFP-specific fluorescence predicted sugar concentration fairly well (R2 of 0.91), so this value would be useful in a high-throughput screening method where it may not be realistic to take measurements throughout the reaction. The absorbance and fluorescence data from the growth curves  against the fluorescence units from either GFP-specific fluorescence or integrated fluorescence at 18 h, the R2 values were 0.86 and 0.99 respectively (data not shown), confirming our hypothesis that integrated fluorescence values are the most accurate predictors of sugar concentration.We conducted two experiments to determine the response time of the microbial system to either a flux in sugar concentration or a sudden limitation in sugar during a hydrolysis reaction. The first objective was to determine how quickly the microbe responded to adding glucose to a reaction where glucose was limiting. To test this, after growing several cultures on 2 mg/ml glucose to stationary phase, we spiked half the cultures with 8 mg/ml sugar then monitored the response of the microbe. This spiking caused the absorbance and fluorescence to increase Figure . As obseIn addition to determining how quickly the microbe can respond to an increase in sugar concentration, it is important to establish the response time to a sudden limitation in sugar. We reasoned that the response to a sudden limitation in sugar would be limited by the rate of decay of existing GFP, so we sought to determine this parameter by halting protein production in mid-log phase and determining the effect on cell density and GFP-fluorescence. This was accomplished by the addition of chloramphenicol  to the bacterial cultures and measuring absorbance and fluorescence over time. Absorbance measurements showed that the bacteria entered stationary phase 1 h after addition of chloramphenicol, and the fluorescence decreased proportionally to the decrease in rate of growth Figure . Within brown midrib allele: W64A × A619 (wild type), W64A × A619 bm1, W64A × A619 bm2, W64A × A619 bm3, and W64A × A619 bm4. The brown midrib mutations either alter the composition or reduce the amount of lignin in the corn stover, making the stover more conducive to hydrolysis [brown midrib lines would yield more sugar upon hydrolysis than the line without the brown midrib phenotype when performing endpoint hydrolysis on this set of corn stover samples. There was a significant difference in the mean GFP-specific fluorescence values for all four brown midrib mutants when compared with the near-isogenic line without the brown midrib mutation , and that the integral of fluorescence would therefore be proportional to the culture cell mass, X, which in turn is proportional to the amount of sugar consumed by the microbe. It was expected that the integrated fluorescence values would be the most accurate predictors of sugar concentrations during SSC because they are not based on a specific growth model but on the total sugar catabolized by the microbe (as shown earlier by the glucose-spiking experiment). Under this assumption, there was no reason to fit a Gompertz equation or the first derivative of the Gompertz equation to the data because neither would be expected to accurately model culture growth when sugars are being produced during culture growth. The absorbance and fluorescence data are shown in Figures -4, Figure For some applications, it may be important to monitor the products of a hydrolysis reaction with our microbial system over time. For example, this may be useful for optimizing mixtures of hydrolytic enzymes. The near-isogenic hybrids W64A × A619 and W64A × A619 bm1 were analyzed using SSC and the culture OD and fluorescence were measured over time. Our hypotheses, based on the previous growth curve observations, were that instantaneous GFP-specific fluorescence would be proportional to the sugar catabolism rate  was proportional to fluorescence at any point in time. We also showed that a higher amount of sugar in solution will cause the cells to stay in the log growth phase, and therefore have higher fluorescence, for a longer period of time than when sugar levels are lower.  These data support our revised hypothesis that instantaneous fluorescence is proportional to the sugar catabolism rate. Based on the growth curves with different glucose concentrations, it is best to measure fluorescence between 16 and 22 h of growth when the E. coli are limited in carbon and the rate of growth is most different. As we observed a high correlation (R2 of 0.98) between sugar concentrations and integrated fluorescence at 20 h, we hypothesize that the integral of fluorescence is proportional to the amount of sugar consumed by the microbe. We verified this in the glucose spiking experiment.Based on modeling by Leveau and Lindow  we expecThe sensitivity and dynamic range of the microbial assay allows for the detection of sugar in the hydrolysis reactions performed. We were able to observe significant differences between the amounts of sugar produced in the corn stover hydrolysis reactions with only 25 mg of sample. The amount of sugar produced by the hydrolysis reaction is within the dynamic range of the microbial assay.The data presented in this paper establish a clear relationship between fluorescence and culture growth rate. To meet the objective of using this system to monitor sugars present in biomass and/or produced in hydrolytic reactions, we must assume that culture cell mass is proportional to the amount of sugar available to the culture. While this was the case in cultures containing purified sugars, this may not be the case when the sugars are provided from biomass. Compounds present in biomass or released by its hydrolysis that support or inhibit microbial growth would interfere with this assay. Further experiments to quantify the magnitude of growth promotion or inhibition effects of biomass would increase the utility of this assay.brown midrib mutants using this approach showed that they yield a significantly higher amount of available sugars per unit mass than a genotype with wild-type lignin levels. This result is consistent with the large body of data suggesting the low lignin genotypes of both corn and sorghum are more readily hydrolyzed and digestible than their normal lignin counterparts [Our data suggest that post hydrolysis monitoring  is feasible. Our evaluation of low lignin terparts -27.dX/dt. After 16 h, the fluorescence appears to be proportional to the cell density. If the values of the absorbance and fluorescence are predicted between 24 and 36 h and the integral of the fluorescence versus absorbance is plotted, there is a clear deviation from a linear trend at an OD595 of ~0.5 where the predicted values cause the slope to increase dramatically (data not shown). This deviation is most likely due to a new and steady supply of sugar to the bacteria provided by the hydrolytic enzymes. Based on the data shown in Figure In the real-time corn stover hydrolysis, shown in Figures From the data presented here, we have hypothesized that during growth the level of sugars starts high and decreases during the course of the reaction until the sugar levels limit growth. In contrast, when the sugar required for the growth of the microbe is provided by a hydrolysis reaction such as in the SSC method, we hypothesize that a certain amount of sugar is present in solution thereby inhibiting enzymatic hydrolysis of the corn stover until the microbes can deplete the sugars enough for the enzymes to regain function. From the point in time when the enzymes regain function, the hydrolysis proceeds in equilibrium with bacterial growth until the feedstock becomes depleted and the hydrolysis reaction slows. For example, a stover sample more conducive to hydrolysis would allow for sugar to be released by the enzyme into solution more rapidly, therefore supporting a higher steady-state level of GFP. This would give a higher amount of steady-state fluorescence. A sample less conducive to hydrolysis would release the sugars more slowly causing lower steady-state GFP levels.It may be possible to develop a method that predicts the suitability of biomass for enzymatic hydrolysis using the microbial system described here. This would involve using the microbial system to monitor a small-scale version of the hydrolytic process of interest. The small-scale method should be designed to emulate the large-scale process as closely as possible. For example, as described here, the results of our process will be influenced by the presence of soluble sugars. If the hydrolytic process of interest does not capture the soluble sugars from the biomass, then it may be appropriate to wash the biomass prior to hydrolysis in the small-scale system.E. coli strain designed to report sugar levels in lignocellulosic biomass hydrolysis reactions. The sugar-sensing system described here overcomes product inhibition of hydrolytic enzymes by removing the sugars from solution as the culture grows. This allows the enzymes to hydrolyze the biomass further and gives us the ability to differentiate samples based on their suitability for hydrolysis. Because the microbe expresses GFP constitutively, it allows the use of fluorescence as a measure of sugar concentration when the solution is too turbid to measure absorbance accurately or when other compounds interfere with this culture density measurement.We characterized growth of an dX/dt, based on growth curves with different glucose concentrations. We hypothesized that the integral of fluorescence is proportional to the amount of sugar consumed by the microbe, and we verified this in a glucose-spiking experiment. By using our fluorescence-based method to monitor sugars released from hydrolysis reactions with corn stover samples, we found that post-hydrolysis monitoring could be used as a high-throughput screening method to determine available sugars and, in turn, to predict ethanol production potential in different feedstocks. Also, it may be possible to use the real-time hydrolysis of corn stover method to compare different hydrolytic enzymes by using the same feedstock and monitoring the release of sugars by measuring fluorescence during the course of the reaction.We found that instantaneous fluorescence is proportional to the change in culture cell mass with time, The authors declare that they have no competing interests.LJH carried out all experiments and analyses in the studies and drafted the manuscript. JGC and AJL selected, produced and collected corn stover samples and prepared them for analysis. DRR and RPA interpreted and modeled the time-sequence data and significantly edited the manuscript. RPA provided project integration and coordination as PI of the 'Integrated Feedstock Supply Systems for Corn Stover Biomas' grant. MPS conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Plasmodium oocyst burden in the midguts of Anopheles spp. is important for many of assays investigating transmission. However, current assays are very time-consuming, manually demanding and patently subject to observer-observer variation.Malaria transmission is now recognized as a key target for intervention. Evaluation of the Plasmodium berghei and a custom-written macro for ImageJ. The counting macro was optimized and found to be fit-for-purpose by performing gametocyte membrane feeds with parasite infected blood. Dissected midguts were counted both manually and using the automated macro, then compared. The optimized settings for the macro were then validated by using it to determine the transmission blocking efficacies of two anti-malarial compounds - dehydroepiandrosterone sulphate and lumefantrine, in comparison to manually determined analysis of the same experiment.This report presents the development of a method to rapidly, accurately and consistently determine oocyst burdens on mosquito midguts using GFP-expressing 2 = 0.973) and reproducible. Estimated transmission blocking efficacies between manual and automated analysis were highly concordant, indicating that dehydroepiandrosterone sulphate has little or no transmission blocking potential, whilst lumefantrine strongly inhibits sporogony.Concurrence of manual and macro counts was very high (RRecognizing a potential five-fold increase in throughput, the resulting reduction in personnel costs, and the absence of inter-operator/laboratory variation possible with this approach, this counting macro may be a benefit to the malaria community. Plasmodium, the causative agent of malaria has a complex life cycle, requiring both vertebrate and mosquito hosts. Merozoites invade mammalian red blood cells (RBCs), wherein they replicate producing schizonts, which rupture the RBCs releasing daughter merozoites that are then free to invade other RBCs. With each such asexual cycle, a small subset of merozoites is committed to form male or female gametocytes, the sexual stages of the parasite that are uniquely infectious to mosquitoes [squitoes . When a squitoes . Followisquitoes .Plasmodium transmission to the mosquito (gametocyte to oocyst transition) is to feed groups of mosquitoes on parasite-infected hosts or infected blood in artificial membrane feeders (the standard membrane feed assay - SMFA), and then count the number of oocysts that develop on the mosquito midgut [The 'gold standard' assay for measuring factors affecting o midgut . This tyo midgut ,6, transo midgut -9, mosquo midgut  and to po midgut ,12. Whilo midgut , allowinAll work involving laboratory animals was performed in accordance with the EU regulations 'EU Directive 86/609/EEC' and within the regulations of the United Kingdom Animals (Scientific Procedures) Act 1986.Plasmodium berghei constitutively expressing GFP (PbGFPCON - derived from the ANKA line) [Female TO mice were treated with phenylhydrazine three days prior to infection by i.p inoculation with KA line) . To ensu2 gas as 30 mM stock solutions. Compounds and solvents were provided blind to the experimenter and only un-blinded when all experiments were complete. All compounds were used in the transmission-blocking assay with no more than a 0.1% final solvent concentration. 0.1% DMSO or DMF were used as 'solvent controls'.Two anti-malarial compounds - dehydroepiandrosterone sulphate (DHEA-S) and lumefantrine (LUM), and a known inhibitor of microgametogenesis and infection (positive control) - cycloheximide (CH) , were seAnopheles stephensi (strain SDA 500) mosquitoes in groups of ~80, which were then allowed to feed on the blood for 30 min [For each of the three replicate experiments, three infected mice (see above) were used per assay. They were anaesthetized, before being rapidly bled by cardiac puncture and their blood pooled. 500 μl of infected blood was mixed with each compound to be tested, or DMSO/DMF as a control, to yield a 10 μM final concentration of compound. The infected, drug-treated blood was then injected into a membrane feeder pre-warmed to 39°C and offered to overnight-starved r 30 min . The nexr 30 min  which waGroups of 20 fixed guts were mounted on glass slides containing a small amount of Vectashield mountant and a coverslip applied. To facilitate imaging, guts were then flattened by gently withdrawing some fluid from the preparation. The slides were observed with a Leica DMR microscope at ×5 magnification using a Leica HCX PL FLUOTAR 5×/0.15 lens  and guts imaged with a Zeiss AxiocamHR camera controlled by AxioVision v4.7.2 software. Images from the same set of feeds were all taken at a resolution of 1388 × 1040 pixels with the same exposure setting and guts were all imaged on the same day under the same conditions. This was done to ensure that there was minimal variation in fluorescence intensity between guts due to the degradation of GFP within the oocysts over the time they were stored.The acquired images of the infected mosquito midguts can be broken down into two components: 1. Oocysts that are small (~7-15 pixels/14-30 μm diameter), round and show high GFP fluorescence; 2. Midgut tissue that is larger than the oocysts (~600 × 400 pixels/1200 × 800 μm), and has fainter variable and non-uniform yellow-green autofluorescence. Even though oocysts are brighter in greyscale than the surrounding midgut tissue, it is impossible to reliably separate them by simply thresholding the image Figure , whereasAn automated oocyst counter must therefore be able to process each image in exactly the same manner to give reproducible data, therefore, background fluorescence must be standardized before an accurate counting analysis can take place. Experimentally this is very difficult to achieve and so a macro for ImageJ was developed that can take any midgut image and process it with a simple set of standard image transformations that remove the background tissue fluorescence whilst preserving the oocyst component of the image Figures  and 1e.The background fluorescence for each image is minimized by taking a copy of the image, displacing it horizontally by 2 pixels , and then subtracting it from the original image. Subtracting a copy of the image slightly out of phase with the original causes large areas of relatively uniform and weak fluorescence intensity to be cancelled out (midgut tissue). Small areas (~7-15 pixels diameter) of bright fluorescence (oocysts), whilst diminishing in size and intensity remain distinct Figure . A stand2). For example, objects of 2 × 2 pixels and 1 × 4 pixels in size are both classified as being 2 pixels2. This value was hypothesized to be critical for the accuracy of counting. To test this, gut images from three gametocyte membrane feeds  were counted by the macro using different minimum particle sizes. To enable the precision of the macro counts to be evaluated, the same midgut images were then recounted and recorded manually by an experienced trained observer. The observer recorded each identified oocyst individually and used their own judgement to discriminate between overlapping oocysts.The ImageJ particle counter function used by the counting macro allows the user to specify the minimum particle size counted. ImageJ defines a particle as an object of undefined shape with an area measured in pixels squared  and 8 pixels2 under-counting   Figure . To inve) Figure . The mac) Figure  and 2d. 2 Figure  was foun2 Figure  and 2d.2 gave a prevalence of 100%. At 3 pixels2 this dropped to 9% and by 6 pixels2 had reduced to 2%. The 2 images that the macro incorrectly identified at this size contained either abnormally high background fluorescence in the midgut tissue or contained a single fluorescent artefact that was the same size and shape as an oocyst.The effect of particle size on calculating the prevalence of infection was also tested by imaging 100 uninfected guts, and then counting them using the macro set at the different minimum particle sizes (Table 2 (recognizing objects in the processed image containing 9 pixels or greater) was selected for use in further experiments with this material as it most closely balanced out the false positives and false negatives produced by the macro , feeds were analysed by expressing the oocyst intensity for each individual replicate as a percentage of its respective solvent control, and then taking the mean of the three replicates (Table ®) for treating malaria [P. berghei  in triplicate experiments. At day 7-9 the guts were dissected, imaged and then counted both by the macro at a minimum particle size of 3 pixels8-58 in tBy manual count, the earlier observations of Toyé et al  were conUnder the conditions described in this report, researchers were able to perform 20 experiments per week on a sustained basis using the macro. This compares very favourably with manual counting, which permits only four experiments per week to be completed on a sustained basis. The counting macro, therefore, as well as saving time, also reduces the cost of performing SMFAs and increases the reproducibility of their analysis.Plasmodium development in the mosquito but remain very low throughput due to the complexity of the experimental set up, and the laborious man-hours of repetitive observations needed for analysis. The oocyst counting macro greatly reduces the time and labour needed to analyse such assays, allowing for greater throughput and massively increased productivity. Despite slightly under-estimating the oocyst intensity and over-estimating the prevalence, it gives highly consistent (R2 = 0.97-0.98) and unbiased counts, well within the range of operator-operator variability and also never suffers from counting fatigue. Whilst automated counting systems have been developed for determining blood-stage parasite burdens [Assays for transmission blocking vaccines/drugs and phenotypic analyses are vital for the study of  burdens ,21, currP. berghei day 7-9 oocysts, recently a P. falciparum transgenic line constitutively expressing GFP throughout its life cycle has been generated . Initial testing has shown that the counting macro is applicable to this parasite line also. It is anticipated that the counting macro will be easily adaptable to any technique that renders oocysts a sufficiently contrasting colour to midgut tissue. The counting macro maximizes the throughput of transmission blocking mosquito-based assays in their current format. There still remain bottlenecks to further improvements in throughput, such as dissecting the mosquitoes and faster imaging. Automated microscopy techniques may reduce the latter. The former would require a more drastic reworking of current assay procedures to perhaps include reporter parasites that can be detected either in situ in the mosquito, or from crude mosquito extract (such as luciferase reporter genes). These approaches however may not give as useful information as they would assess parasite biomass, which certainly does not correlate directly with parasite number.Although the macro settings were optimized to detect This report demonstrates and validates the utility of the macro in accurately determining and reporting the transmission blocking activity of two anti-malarial compounds. With the growing recognition that blocking parasite transmission to the mosquito is likely to play an important role in the eradication of malaria ,23, thisThe authors declare that they have no competing interests.MJD conceived and designed the algorithm, and performed the experiments described here. MJD and RES participated in drafting the manuscript. All authors read and approved the final manuscript.


Until now there is a lack of carefully controlled studies with conventional MR imaging performed exclusively in concussion with short lasting loss of consciousness (LOC).A MR investigation was performed within 24 hours and after 3 months in 20 patients who had suffered a concussion with a verified loss of consciousness of maximally 5 minutes. As a control group, 20 age- and gender matched patients with minor orthopaedic injuries had a MR investigation using the same protocol.In a concussion population with an average LOC duration of 1. 4 minutes no case with unequivocal intracranial traumatic pathology was detected.An ordinary concussion with short lasting LOC does not or only seldom result in a degree of diffuse axonal injury (DAI) that is visualized by conventional MR with field strength of 1.0 Tesla (T). Analysis of earlier MR studies in concussion using field strength of 1.5 T as well as of studies with diffusion tensor MR imaging (MR DTI) reveal methodological shortcomings, in particular use of inadequate control groups. There is, therefore, a need for carefully controlled studies using MR of higher field strength and/or studies with MR DTI exclusively in common concussion with LOC of maximally 5 minutes. Several authors suggest that the symptoms of postconcussion syndrome are due to structural abnormalities and/or organic cerebral dysfunction [After concussion (or mild traumatic brain injury (MTBI)), a significant proportion of subjects report persisting symptoms that include headache, cognitive dysfunction, dizziness, fatigue, and irritability. This cluster of rather non-specific symptoms has been termed as the postconcussion syndrome (PCS), a condition that has been debated since the end of the 19function . Others function ,4.For the concept of PCS being caused by an organic injury it seems necessary to assume that the concussion produces long-lasting morphological and/or functional lesions. This is, however, still equivocal, at least when using neuroimaging procedures such as cerebral computer tomography (CT) or magnetic resonance (MR) imaging. Studies using CT show lesions only in a small minority of cases, whereas conventional MR studies show pathology in the acute state in 10 to 57% of cases . All theSince traumatic white matter lesions may be confused with foci of non-specific high signal intensity that is associated with normal aging ,12, the The patients had no other injuries than the concussion, except for small skin lesions, bruises and other insignificant injuries. Exclusion criteria were diabetes, hypertension, affective disorders, prior history of alcohol or drug abuse, prior history of psychiatric or neurological disorder, prior history of epilepsy or seizure associated with the concussion, earlier concussion and concussion due to assault. There were also no focal neurological signs and normal neurological status at admission except for amnesia and slight and transitory confusion.As controls, individually sex- and age (plus/minus 3 years of age) matched subjects aged 18 to 40 years with the same exclusion criteria as the study group were taken from the records of the traumatology department by identifying patients with minor to moderate non-head injuries who had attended this department in a time period of maximally 2 weeks after the concussion date of the traumatized matching patient. If there were several potential controls within this period, the patient was taken who concerning the time of the attendance to the clinic was nearest that of the head-traumatized individual.After inclusion, the patients with concussion and non-head traumatized controls underwent an MR investigation within 24 hours after the trauma and after 3 months using the same protocol each time. MR imaging was performed on a Gyroscan T10-NT 1.0 T . A scout sequence was used to align the subsequent scans. The matrix in all sequences was 205 × 256. Axial imaging plane and 5 mm slice thickness was used for all patients in all sequences. Three pulse sequences were included: T1W/SE ; T2W/FLAIR ; and to detect hemosiderin deposits T2W/FFE . The MR covered the whole brain. The scan time was 15 minutes and the slice number 22.Final evaluation of MR was made by two neuroradiologists blinded for the diagnostic status of the individual.The study was in compliance with the ethical principles for medical research involving human subjects according to the Helsinki Declaration and was approved by the Ethical committee of the Medical Faculty of the University of Kaunas. All patients gave written informed consent to participate.Of the twenty patients in the study group 10 were females  and 10 males   and the 10 males a mean age of 26.0 years (range 18 to 39 years)  was not significantly different from that in the study group (n = 3) In the present study in which the most common type of concussion was investigated by MR imaging and inclusion of age-and gender matched non-head injured controls, no case was detected with unequivocal intracranial traumatic pathology. There were three male subjects in which high signal intensity foci were found in the white matter. The changes had the same appearance 3 months post injury and were, therefore, likely to be pre-existing findings of non-traumatic origin. In the control group only one individual had a non-traumatic, pre-existing hyperintensive focus. Possibly due to the low power of the study, the difference in prevalence of pre-existing hyperintensive foci was insignificant. Nevertheless, this result may indicate that individuals prone to head injury differ from healthy individuals and individuals prone to orthopaedic injuries in respect to pre-traumatic MR findings.The study had the limitation that it was performed with 1.0 T MR and 5 mm slices and that traumatic lesions such as small sub-cortical hemorrhages shown by high field MR may have escaped detection. On the other hand, diffuse axonal injury can be demonstrated with MR of even lower field strength than in the present study .A general methodological problem for earlier studies on concussion with structural neurodiagnostic techniques performed in the last 25 year is that many of them did not have a control group and in those who had, the control group was mostly inadequate. An adequate control group should be age- and gender-matched individuals with minor non-brain injuries such as in the present study. Presumably, such group would have a socio-economic status as well as a degree of accident-proneness, psychopathology and alcohol consumption that is more similar to individuals with concussion than a control group of healthy individuals. It is known that psychopathology may predispose to injuries ,15 and tThere are few earlier studies (field strengths from 0.064 T up to 1.5 T) that selectively have investigated MR findings in concussion , and no Taken the results and the mentioned methodological shortcomings of these earlier studies into account and in view of the results of the present study it seems justified to conclude that until now there is no reliable documentation that an ordinary concussion with short lasting LOC in a significant number of cases causes a degree of diffuse axonal injury that is visualized by conventional MR with a field strength up to 1.5 T.In recent years, several attempts have been made to detect signs of axonal injury by use of diffusion tensor imaging (DTI) MR, a method that is able to demonstrate white matter microstructure deficitsThe findings in MTBI patients of these studies were usually discrete, inconsistent and diverging -28. In oUsing a rigidly controlled design with non-head injured controls and performing a baseline scan within 24 hours followed up with the same protocol at 3 months, this 1.0 T MR study shows that none of the patients with common concussion developed abnormalities that could be clinically  detected and related to the brain trauma. Hence, no support was found for a potential organic substrate for PCS at this level of detection.Obviously, the study does not rule out subtle traumatic abnormalities below the present detection threshold. In order to demonstrate such discrete traumatic changes  with conventional MR (or MR DTI) one should perform studies in a greater MTBI population than have been done hitherto and investigate only patients with common concussion , preferably using MR with a higher field strength . Such studies should include a carefully matched control group of a sufficient size consisting of persons with a similar risk profile for MR changes, e.g. non-head injured individuals with alcohol consumption comparable to that of the MTBI group. Even if studies with this design should document traumatic lesions, it remains still uncertain whether lesions that are too discrete to be shown with a 1.0 T MR machine can explain the clinical symptoms of PCS.The authors declare that they have no competing interests.HS and DM conceived and designed the study, analyzed the data and drafted the manuscript. RG and SJ analyzed the MR and participated in the design and coordination of the study. DS participated in the study design and coordination and together with DM identified the patients in the study and control group. LJS participated in the design of the study, in analyzing the data and writing of the paper. DO participated in the design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2342/9/11/prepub


Resurfacing of the patella during primary total knee arthroplasty (TKA) is often recommended based on higher revision rates in non-resurfaced knees. As many of these revisions are insertions of a patella component due to pain, and since only patients with a non-resurfaced patella have the option of secondary resurfacing, we do not really know whether these patients have more pain and poorer function. The main purpose of the present paper was therefore to assess pain and function at least 2 years after surgery for unrevised primary non-resurfaced and resurfaced TKA, and secondary among prosthesis brands.ΔEQ-5D) were also used as outcomes. Outcomes were measured on a scale from 0 to 100 units (worst to best). To estimate differences in mean scores, we used multiple linear regression with adjustment for possible confounders.Information needed to calculate subscales from the knee injury and osteoarthritis outcome score (KOOS) was collected in a questionnaire given to 972 osteoarthritis patients with intact primary TKAs that had been reported to the Norwegian Arthroplasty Register. Pain and satisfaction on visual analog scales and improvement in EQ-5D index score We did not observe any differences between resurfacing and non-resurfacing in any outcome, with estimated differences of ≤ 1.4 units and p-values of > 0.4. There was, however, a tendency of better results for the NexGen implant as compared to the reference brand AGC for symptoms , pain (VAS) , and satisfaction (VAS) . However, none of these differences reached the stated level of minimal perceptible clinical difference.Resurfacing of the patella has no clinical effect on pain and function after TKA. Differences between the brands investigated were small and they were assumed to be of minor importance. There is an ongoing discussion regarding whether resurfacing of the patella during primary total knee arthroplasty (TKA) should be recommended or not. This has led to several observational and randomized studies, and eventually to meta-analyses . The metA previous observational study from the Norwegian Arthroplasty Register (NAR) found a 1.3 times higher but not statistically significantly elevated (p = 0.2) overall rate of revisions for non-resurfaced prostheses . There wThe major goal of the present study was therefore to investigate whether the levels of function and pain are different for patella resurfaced and non-resurfaced, unrevised, total knee prostheses, and our secondary aim was to investigate whether function and pain vary with different prosthesis brands.Practically all patients (99%) who receive a primary arthroplasty of the knee are reported to NAR . The regPossible participants were patients registered in the NAR with at least 1 unrevised cemented primary TKA inserted due to gonarthrosis. The individuals should be aged 85 years or less, and the operation should have been performed at least 2 years prior to the survey to ensure that the result of the intervention had stabilized . Only paAfter 2 months, a reminder was sent out to those who failed to respond to the initial questionnaire. In all, 972 patients completed the questionnaire, 305 either declined or did not respond, and 7 patients had died or were unable to be located by the post office.www.koos.nu.The questionnaire consisted of the valid and reliable self-administrated instrument for calculation of the knee-specific knee injury and osteoarthritis outcome score (KOOS) . A NorweTo assess the potential effect of general health factors, the questionnaire also included questions needed to calculate the Charnley category applied to knee arthroplasty patients and the valid and reliable instrument for health quality measurement, the EQ-5D index score . InformaThe study was approved by the Norwegian Data Inspectorate, Norwegian Social Science Data Services, and the Regional Committee for Research Ethics in Western Norway . The patients received the questionnaire together with an information letter, and returned the questionnaire to the register with a signed consent to participate in the study.www.koos.nu.KOOS, which was used as primary outcome on patients' perception of pain and function, consists of 42 individual questions, making up 5 subscales: Pain, other symptoms (Symptoms), activities in daily living (ADL), function in sport and recreation (Sport&Rec) and knee-related quality of life (QOL). Only the previous week was to be considered when answering most of the questions, and each question received a score from 0 to 4. A normalized score (100 indicating no symptoms and 0 indicating extreme symptoms) was calculated for each subscale. Calculation of the scores and treatment of missing data were done in accordance with the description at In addition, we used “pain from the operated knee” (Pain(VAS)) and “satisfaction with the operation” (Satisfaction (VAS)) as outcomes. In the analyses the VAS scores were reversed, with 100 indicating the best possible state and 0 indicating the worst possible state. Finally, improvement in quality of life (ΔEQ-5D), calculated as the difference between the present and preoperative EQ-5D index scores multiplied by 100, was used as outcome.A bias may have been introduced if a non-resurfaced prosthesis was more likely to be revised than resurfaced  and varying numbers of revised non-resurfaced implants. The latter were randomly selected from the 148 revised non-resurfaced knees that met the inclusion criteria. The Mann-Whitney test was used to compare pain and discomfort between the groups using the specific EQ-5D question regarding pain and discomfort . This information related to when the questionnaire was filled in for patients with intact prostheses, while patients with a revised prosthesis gave information in retrospect regarding their situation before the revision.Minimal perceptible clinical difference is 8–10 units for KOOS subscales  and 9–12Differences in response rates were tested with the chi-squared-test. To estimate differences in mean outcome scores for non-resurfaced and resurfaced prostheses, we used multiple linear regression with adjustment for possible confounding by age , sex, preoperative EQ-5D index score , Charnley category , and brand of prosthesis. We also investigated any differences within a particular prosthesis brand. Adjusted differences in mean values between resurfaced and non-resurfaced prostheses are presented with 95% confidence intervals (95% CIs) and p-values.Multiple linear regression was also used to investigate any possible association between prosthesis brand and mean outcome scores. In these analyses, we also adjusted for time since operation. Adjusted differences in mean scores are presented with p-values relative to the AGC prosthesis brand. 9 patients who did not have the AGC Universal design were excluded from this analysis (5 resurfaced and 4 non-resurfaced).In addition, all analyses were performed excluding prostheses with posterior cruciate ligament sacrificing design and also excluding constrained condylar prostheses (18 prostheses: 10 resurfaced and 8 non-resurfaced).In the analyses, p-values less than 0.05 were considered statistically significant. The analyses were performed using SPSS statistical software version 15.0.0.1.We received completed questionnaires from 972 (76%) of the 1,284 individuals selected for the study. Thus, the study included 504 knees with resurfaced TKA and 468 knees with non-resurfaced TKA. The response rate was similar for non-resurfaced prostheses (76%) and resurfaced prostheses (75%) (p = 0.7), but it was lower for female patients (73%) than for male patients (82%) (p = 0.001). It was also less for older patients: 88% for those less than 65 years of age and 67% for those older than 80 (p < 0.001). Response rates for each brand of prosthesis varied between 71% and 79% . Improvement as measured by ΔEQ-5D decreased, however, with increasing age .Exclusion of prostheses with posterior cruciate ligament sacrificing design and of constrained condylar prostheses gave only minor changes to the results above.The mean pain and discomfort score (EQ-5D) was 1.6 both for intact resurfaced and non-resurfaced prostheses . For resurfaced knees, the mean score increased to 1.7 when all revised knees (n = 23) were included. The mean score was also 1.7 for non-resurfaced knees when the same number of revised knees was added . No statWe have studied performance of TKA based on data from the NAR. Using self-reported degree of pain and function as outcome, our analyses did not show any differences between resurfaced and non-resurfaced primary total knee prostheses 2 years or more after surgery. Differences between prosthesis brands were small and did not reach the required level of minimal perceptible clinical difference relative to the reference brand, AGC.The strength of our study is that use of data from a nationwide register with almost complete coverage gives us the opportunity to include several implant designs and to involve large numbers of surgeons and hospitals performing various amounts of surgery. Since this gives us information from a broad spectrum of implants, surgical techniques, surgeon experience, and procedures, the validity of the results may be more global than that from randomized controlled trials (RCTs).Despite having several advantages, observational studies may be affected by limitations that are absent in well-designed RCTs. We have treated the most common confounding factors by using matching procedures and adjustments in the statistical model, but there may still have been variables that were not taken into account. Different criteria in the decision making by the surgeons might possibly lead to confounding. Imbalanced differences between the groups are, however, not very likely in Norway as most surgeons—for specific time periods—have used one of the two treatment options almost exclusively.Selection bias is not very likely since the group of non-responding individuals was acceptably small and there were no statistically significantly different response rates between the most important subgroups. It is difficult to point out important factors that could have characterized the group of non-responding individuals and among those who were not eligible for inclusion, such as patients who had died.Bias may, however, have been introduced if the study included a falsely low proportion of individuals with poor results among the non-resurfaced patients. This might be the case if non-resurfaced knees were revised more often than resurfaced knees, making such patients ineligible for inclusion in the study. This was investigated in a sensitivity analysis where information from varying numbers of revised prostheses were added to the original material. However, we observed no statistically significant difference between the treatment groups until more than 3 times as many revised non-resurfaced prostheses (n > 69) as revised resurfaced prostheses (n = 23) had been added to the material. Such a substantial difference is not supported by the results of large observational studies  were small and less than the stipulated minimal perceptible clinical difference of 8–10 units for KOOS subscales  and 9–12Male patients performed better than female patients on all postoperative outcome measures, but we did not observe any gender differences in improvement based on pre- and postoperative EQ-5D index scores (ΔEQ-5D). While the EQ-5D index score is not necessarily strongly related to having undergone TKA, this finding is in accordance with other studies that have shown that improvement in knee scores is similar for females and males after TKA , but thaEven though studies based on data from registers give a unique opportunity to discover and indicate underlying or hidden mechanisms, their limitations underscore the need for more studies. Further research performed by the use of both observational studies and clinical trials is therefore needed in order to confirm our findings, especially since our results contradict with findings in previous studies .Recent studies not included in the meta-analyses that have focused on outcomes other than revision rates, have differed in their conclusions regarding recommendation to use a patella component. 2 RCTs did not find any differences between the 2 treatment options when the Miller-Galante II system was used . This coFew studies make use of the advantages of data available from arthroplasty registers, but an 8-year-old study from the Swedish Knee Arthroplasty Register showed that resurfacing was the best choice of treatment as measured by rate of patient satisfaction with the result of the intervention, but this advantage decreased with the length of time that had passed since operation .Our study indicates a need to reconsider the widely accepted recommendation of primary resurfacing of the patella. A change in operation procedures towards less use of a patella component during primary TKA might be advisable. This will probably give the advantages of less extensive operation procedures with better preservation of the soft tissue of the patella, lower risk of revision due to infections, lower risk of patella fractures, shorter operation time, and lower cost .The results of our study indicate that resurfacing of the patella has no clinical effect on pain and function after a TKA. The differences between the brands investigated were small and they were assumed to be of little importance clinically.Click here for additional data file.www.actaorthop.org website, identification number 3061/09.Supplementary data. Table 1 and Figure 4 can be found on the


The Conserved Oligomeric Golgi (COG) complex is an eight-subunit assembly that localizes peripherally to Golgi membranes and is involved in retrograde vesicular trafficking. COG subunits are organized in two heterotrimeric groups, Cog2, -3, -4 and Cog5, -6, -7, linked by a dimeric group formed by Cog1 and Cog8. Dysfunction of COG complex in humans has been associated with new forms of Congenital Disorders of Glycosylation (CDG), therefore highlighting its essential role. In the present study, we intended to gain further insights into the evolution of COG subunits in vertebrates, using comparative analyses of all eight COG proteins.dN/dS ratios as a measure of the rate of proteins evolution. The results showed that all COG subunits are evolving under strong purifying selection, although COG1 seems to evolve faster than the remaining proteins. In addition, we also tested the expression of COG genes in 20 human tissues, and demonstrate their ubiquitous nature.We used protein distances and COG complex has a critical role in Golgi structure and function, which, in turn, is involved in protein sorting and glycosylation. The results of this study suggest that COG subunits are evolutionary constrained to maintain the interactions between each other, as well with other partners involved in vesicular trafficking, in order to preserve both the integrity and function of the complex. Most cellular processes are carried out by multiprotein complexes that constitute important functional units in the cell . This faA cellular process in which protein complexes are known to be involved is the transport of proteins between cellular compartments (vesicular trafficking) ,6. ProteSeveral studies have been performed demonstrating the involvement of COG in retrograde vesicular trafficking of Golgi resident proteins -18, inclCOG complex is composed by eight distinct subunits ,13,24-26COG5 to COG8) does not substantially interfere with normal cell growth .In the present study, we used a broad range of comparative sequence analyses to track the evolutionary profile of this complex in vertebrates.-3 and using the reciprocal best-hit approach [orthogues option. Available sequences from the following taxa were considered in this study: mammals , birds , reptiles (Anolis carolinensis), amphibians  and fishes . COG3 from G. aculeatus, COG5 from X. tropicalis and COG6 from D. rerio were assembled based on the genomic sequence through the comparison with the orthologous protein from other species, and through TBLASTN searches on ESTs database. Accession numbers are available in the additional file P. troglodytes COG4, residues 57 to 146 and 436 to 470 from P. pygmaeus COG7 as well as residues 493 to 522 from M. mulatta COG7 were replaced by missing data.Human protein sequences corresponding to each COG subunit COG1-COG8) were retrieved from the National Center for Biotechnology Information (NCBI) . These sapproach . Alternaapproach , release-COG8 werProtein sequences were aligned with MUSCLE  and manuTo reconstruct species phylogeny, the protein-coding sequences were aligned with ClustalW program  implemendS) and the number of nonsynonymous substitutions per nonsynonymous sites (dN) have been estimated with CODEML from the PAML v.4.4 package [cleandata = 0). The input alignments were modified by removing positions that showed evidence to represent true indels, while keeping those that appear to be missing data. Alignments are available in additional file dN/dS ratio (ω) among lineages have been applied: the one-ratio model that assumes the same ω ratio for all branches in the phylogeny; the free-ratios model that allows ω to vary on every lineage; and the two-ratio model, which assumes that the branch of interest has an ω value (ω1) different from the ratio of the other lineages (ω0- background ratio). The above models can be compared using a likelihood ratio test (LRT) to test different hypothesis, as described by Yang [The number of synonymous substitutions per synonymous site . A number of erroneously assigned ESTs for human and mouse COG8 were manually removed. For simplicity, only homologous tissues for which information was available in both organisms were included, resulting in a total of 29 tissues. The clusters on the heatmaps were made by an in house tool, using correlation as the measure of similarity.Expressed sequence tag (EST) profiles from several human and murine tissue samples were extracted for database  as EST cCOG genes have no detectable expression (absence of EST counts), we have further tested the presence of all COG transcripts in 20 different human tissues included in a RNA panel obtained from Ambion . cDNA was synthesized from 1 μg of RNA using the First Strand cDNA synthesis Kit , according to the manufacturer's instructions. Primers specific for COG transcripts, as well as for the positive control GAPDH, were designed to avoid amplification of contaminating genomic DNA, either because they span an intron or the forward primer anneals with an exon/exon boundary .As a first measure of the rate of COG proteins evolution, we calculated the pairwise protein distance for human proteins and each of the 20 orthologs. These values were plotted against the corresponding divergence times for the compared species, and the linear regression trend line was estimated from each group, as shown in Figure dN) to synonymous (dS) substitutions rate ratio (dN/dS or ω).In addition, we used a classical measure of protein evolution based on the nonsynonymous , the rodent's branch length is more similar to all the other branches  have no detectable expression of specific COG genes. In order to assess the presence of COGs transcripts in some of those and other human tissues, we performed reverse transcription-PCR (RT-PCR). Although no quantitative inferences could be made, using this simple methodology we were able to detect the eight COG transcripts in 20 human tissues . CDGs are a genetically heterogeneous group of disorders characterized by a deficient glycosylation of glycoconjugates, such as proteins and lipids. Since 2004, defects in COG1 [COG4 [COG5 [COG7 [COG8 [COG1 gene was detected in two patients with a cerebrocostomandibular-like syndrome [We demonstrate that all COG proteins are evolving under strong evolutionary constraints, as revealed by the low  in COG1 , COG4 [5G1 [COG4 , COG5 [5G4 [COG5 , COG7 [1G5 [COG7 ,54-56 anG7 [COG8 ,58 have syndrome , showingBeing part of a multi-subunit assembly and having such an important functional role in cells must impose strong constraints on the evolution of COG proteins. On one hand, they must be constrained to maintain the structural integrity of the complex, presumably through the conservation of the residues that are involved in the interaction between subunits. This is expected to be true if we assume that COG structure is maintained by the same type of protein-protein interactions in different species. On the other hand, additional interactions with other functional partners  also ndN/dS ratio of the associated genes tends to decrease.In fact, the low rate of evolution of COG proteins is consistent with results from more comprehensive studies showing that evolutionary conservation increases from monomeric proteins to members of transient interactions and finally to components of stable complexes (proteins that are permanently associated with each other) ,62. WongTo evaluate the impact of different selective forces on COG proteins, it would be interesting to compare the rates of substitution for interacting and non-interacting residues, and also structural and functional domains. Unfortunately, the structure of fragments of only two COG subunits have been reported ,63, hampCOG proteins are evolving under strong selective constraints, COG1 seems to be the one with the highest rate of evolution. This subunit, together with COG8, is the bridging subunits of the mammalian COG complex, bringing together COG2-4 and COG5-7 subcomplexes [Despite all omplexes ,29. Inteomplexes . COG1 fromplexes . Interesomplexes .COG genes exhibit a ubiquitous nature. These results can be taken as a starting point for more detailed quantitative expression studies that can bring additional insights into COG subunits interaction and function and, eventually, to the understanding of the phenotypic heterogeneity associated with different COG defects.In this study we have also demonstrated that the expression of In the past years several studies have been focused on the evolution of protein complexes in terms of type of interactions, revealing that proteins in stable complexes are more conserved than those in transient interactions and those with no apparent interacting partners .COG transcripts in 20 human tissues.In this study, in turn, we have investigated the evolution of different subunits belonging to the same protein complex in vertebrates. Our results showed that the eight COG subunits seem to be conserved and evolving under strong purifying selection, in order to maintain the integrity and function of the complex. Finally, we confirm the ubiquitous tissue expression of the eight Homo sapiens).COG: conserved oligomeric Golgi complex; CDG: congenital disorders of glycosylation; EST: expressed sequence tag; Taxa terminology is abbreviated using the first letter of the genus and two letters of the species name  estimated under the free-ratio model. In COG5 phylogeny the branch leading to Euarchontoglires (primates and Glires) presents a ω ratio of 4.70 . A LRT comparing the one-ratio model with the two-ratio model , revealed that the estimated ω ratio for the Euarchontoglires branch  was not significantly higher than the background ratio (0.094). Moreover, the LRT comparing the two-ratio model with and without the constraint ωEuarchontoglires ≤ 1 revealed that this ratio was not significantly higher than 1 as well.Click here for fileCOG2 and COG6 genes phylogeny with branch lengths defined as the estimated nonsynonymous substitutions rate (dN) or the estimated synonymous substitutions rate (Sd).Click here for file


In a previous study 13 identified loci with effects on body-weight, only explained a small proportion of the large variation in the F2 population. Epistatic interaction analysis however, indicated that a network of interacting loci with large effect contributed to the difference in body-weight of the parental lines. This previous analysis was, however, based on a sparse microsatellite linkage map and the limited coverage could have affected the main conclusions. Here we present a revised QTL analysis based on a high-density linkage map that provided a more complete coverage of the chicken genome. Furthermore, we utilized genotype data from ~13,000 SNPs to search the genome for potential selective sweeps that have occurred in the selected lines.We have performed Quantitative Trait Loci (QTL) analysis of an FWe constructed a linkage map comprising 434 genetic markers, covering 31 chromosomes but leaving seven microchromosomes uncovered. The analysis showed that seven regions harbor QTL that influence growth. The pair-wise interaction analysis identified 15 unique QTL pairs and notable is that nine of those involved interactions with a locus on chromosome 7, forming a network of interacting loci. The analysis of ~13,000 SNPs showed that a substantial proportion of the genetic variation present in the founder population has been lost in either of the two selected lines since ~60% of the SNPs polymorphic among lines showed fixation in one of the lines. With the current marker coverage and QTL map resolution we did not observe clear signs of selective sweeps within QTL intervals.The results from the QTL analysis using the new improved linkage map are to a large extent in concordance with our previous analysis of this pedigree. The difference in body-weight between the parental chicken lines is caused by many QTL each with a small individual effect. Although the increased chromosomal marker coverage did not lead to the identification of additional QTL, we were able to refine the localization of QTL. The importance of epistatic interaction as a mechanism contributing significantly to the remarkable selection response was further strengthened because additional pairs of interacting loci were detected with the improved map. The twoanalysis . DespiteGrowth9 QTL on chromosome 7. Growth9 was also detected as the only QTL reaching genome-wide significance in the standard QTL analysis of body-weight at 56 days. The growth-promoting effect of the interacting loci was highly dependent on the genotype at Growth9 as HWS homozygosity at this locus was required for the other interacting loci to have a significant effect on growth. Moreover, LWS line homozygosity at either Growth9 or all three of the loci interacting with Growth9 effectively led to the network having no effect on growth. Hence, the significant marginal effect observed in the one-locus QTL analysis for Growth9 and the suggestive evidence for marginal effects of the three other major loci in the network is manifested solely by the interactions between Growth9 and the other loci in the network. The genetic network was estimated to account for about 45% of parental line difference at 56 days of age [To further study the genetic architecture causing the phenotypic difference between the HWS and LWS lines Carlborg et al.  analyzedThe previous analyses -4 were cHere we report results from a QTL analysis, based on a comprehensive linkage map, for body-weight traits using our intercross between the HWS and LWS lines. First, we performed a standard genome-wide one-dimensional QTL analysis to fit a one-locus model to detect QTL with main effects. Secondly, we performed a genome-wide pair-wise search to detect epistatic interactions between loci. Finally, we used data on about 13,000 SNPs to compare allele frequency distributions between the HWS and LWS lines to investigate if we could detect any molecular footprints in the form of selective sweeps caused by selection for body-weight.2 intercross pedigree was set-up by crossing individuals from the HWS and LWS lines [A large FWS lines . The twoWS lines .2 progeny) we mated 10 HWS line males with 22 LWS line females and eight LWS line males to 19 HWS line females. From the F1 generation four HL males were mated to 37 LH females and, four LH males were mated to 38 HL females to produce an F2 population consisting of 874 chickens. The F1 and F2 animals were housed in the same facilities and fed the same dietary as the parental selection lines. Chickens were provided ad libitum access to water and feed. The F2 generation was produced in a single hatch and all animals were sacrificed at 70 days of age.The development of the intercross and a detailed description of the husbandry have previously been described . Briefly2 bird at hatch, at 14, 28, 42, 56 and at 70 days of age.Table 1 and F2). Methods for SNP selection and the genotyping protocol used to generate the genotypes for ~13,000 SNP markers for 15 individuals from each parental strain have been published elsewhere [DNA was extracted from blood by AGOWA  from chickens included in the intercross pedigree . Sex was included as a fixed effect in the QTL model for all body-weight traits. In contrary to the analysis performed by Park et al. [2 intercross, but consistent with our previous analysis of epistatic interaction [2 full sibling family) as a fixed effect in our QTL model because the number of full-sib families was large  and because maternal effects in chicken are expected to be small. Furthermore, we did not include reciprocal mating in the QTL model because our previous analysis showed that mating type did not have a significant effect on growth [Factors affecting phenotypic trait variation in the Fk et al.  and Jacoeraction  we did nn growth .The QTL analysis of autosomes was performed using interval mapping as first described by Lander and Botstein  and late2 individuals where the genotype for all QTL in the main four locus network affecting body-weight at 56 days of age could be determined with high confidence (n = 727). Only individuals where the genotype probabilities exceeded 0.70 for all four loci in the network were selected. The individuals were then classified according to the most probable genotype  for each locus. We calculated sex-averaged phenotypic means for all nine two-locus genotype classes and plotted the data. The combined effect of the epistatic loci was visualized as follows. First, the dataset was stratified according to the genotype at the main locus Growth9, one group to include the HWS homozygotes, a second group to contain the heterozygotes and a third group including LWS homozygotes individuals . For each of the three groups we then created four more strata with individuals that were HWS homozygous for 0, 1, 2 or 3 of the loci Growth4, Growth6 and Growth12. A one-sided two-sample t-test was used to test for differences in mean body-weight at 56 days of age between the groups.To visualize the effect of individual QTL pairs and also to estimate the total contribution of the epistatic network, we produced a dataset including only FFst values  were calWe designed Illumina assays for parallel genotyping of 384 SNP markers segregating in the HWS/LWS intercross in order to establish a linkage map with an improved genome coverage compared to our previous microsatellite map. Table Twenty out of the 138 microsatellites were excluded from the current map either because of low information content or because they showed inheritance errors that became apparent with the high-density marker map. Further, 35 SNPs were excluded, as they did not give reliable genotype calls, and additionally 33 SNPs out of the 384, were not considered in the current study as they are located on the Z chromosome. This updated sex-average autosomal linkage map now includes 434 genetic markers and spans totally 2954 cM, with an average distance between adjacent markers of 6.8 cM. The total map length is considerably shorter than expected from the old consensus map  but in gThe current linkage map includes markers on chromosome 1–28, and in addition we also have markers on unassigned linkage group E22C19W28E50C23. Two genetic markers  did not show linkage to any other marker in the dataset. It is possible that they are located on microchromosomes lacking other markers, or alternatively the genetic distance to neighboring markers was too far to be detected in the linkage analysis.By calculating the distance between the first and last marker for each linkage group, we have physical coverage over 890.3 Mb corresponding to approximately 93% of the autosomal chicken genome assembly .2 population.Descriptive statistics for phenotypic traits analyzed in this study are presented in Table Growth1-13) given that they mapped to approximately the same genomic position [All QTL with significant marginal effect on body-weight traits are presented in Table position .2 population, ranging from 1.5 – 4.4% (Table Three QTL regions influenced early growth (0–14 days) whereas four regions appeared to primarily affect later growth (14–70 days). For all QTL the allele originating from the HWS line increased growth. Each individual QTL explained only a small portion of the residual phenotypic variation in the F4% Table .Growth1 we cannot exclude the possibility that it represents the same locus although Growth1 affects primarily later growth  for body-weight at 56 days with the new map that was not detected with the old map due to the poor map coverage in the interval. No other chromosomal region lacking markers in the old map showed significant QTL effects. The localization of the major QTL on chromosome 7 is now much more precisely defined after we have added more markers on this chromosome . The genotype-phenotype map relationship for each pair was visualized by plotting mean values for each two-locus QTL genotype in a line graph  at Growth4, Growth6 and Growth12 had no phenotypic effect in a Growth9 H/L or L/L background. In contrast, chickens homozygous H/H at all three loci were 314 grams heavier than those homozygous L/L at all three loci, when the genetic background at the Growth9 locus was H/H. This demonstrates the strong epistatic interaction among these loci. An analysis including all five loci that interacted with Growth9, would have been desirable, however that would require a larger sample size than the ~800 F2 individuals we studied.In a combined analysis we studied the effect of We searched for potential selective sweeps by screening 15 birds from each line using a SNP panel comprising about 13,000 loci evenly distributed throughout the genome. Two birds from the LWS line were excluded from the analysis as results from a phylogenetic analysis indicated that there had been a sample mix up. We then calculated Fst and homozygosity values to identify the regions showing the most pronounced divergence between lines.-13). Hence, the distribution of fixed markers was clearly non-random across the genome as several clusters of fixed loci were detected. There were 36 clusters including two or more SNPs separated by less than 1 Mb between subsequent fixed SNPs and only 23 of the 139 SNPs were not closely linked to another fixed SNP. The QTL regions for body-weight at 56 days of age  provided reliable genotype calls  and 42% of those  showed allelic variation within or between the two lines. The average distance between polymorphic markers was 210 kb. A total of 1,917 SNPs segregated within both lines whereas 1,393 and 1,328 SNPs segregated only in the LWS or the HWS line respectively. Fixation of different alleles between lines was observed for 139 SNPs. The allele frequency changes observed show that considerable within-line fixation occurred during the course of the selection experiment. This is because both lines are derived from a common founder population in which all 4,777 SNPs must have been segregating. To test whether the distribution of the fixed SNPs deviates from a random distribution we computed the distances between consecutive markers in sets of 139 randomly chosen SNPs  and compared the random distribution to the observed one. A Kolmogorov-Smirnov test showed that the observed distribution deviates from the random distribution  for this QTL is expected to be located within this region of high homozygosity. The region with near homozygosity in the LWS line spans from position 173.4 to 176.9 Mb on chicken chromosome 1, a region still too large and containing too many genes to pin-point strong positional candidate genes.An indication of a selective sweep was, however, observed for To be an exhaustive search for QTLs, a genome scan should be based on a marker set that covers the entire genome. The map used in the current study covered 93% of the assembled chicken genome a clear improvement to the ~80% coverage achieved with our previous microsatellite-based map. In particular, we have added markers on six microchromosomes that previously lacked markers. There are, however, still seven microchromosomes lacking markers in the current map. This is because they are missing in the genome assembly and no markers from these chromosomes have yet been reported. These missing microchromosomes are all small, on the order of five Mb or less but are expected to have a higher gene density than the macrochromosomes . It is aGrowth2, Growth3, Growth8, Growth10, Growth11 and Growth13) that did not reach statistical significance in the present study. Our previous analysis included family as a fixed effect in the regression model whereas we decided to not include family as a fixed effect in the statistical model used in the present study. To test whether the new information provided by the improved linkage map or the change in regression model caused this discrepancy we reran the analysis including family as a fixed effect for those growth loci that we could not replicate. With a QTL model including Family as a fixed effect, the F-value associated with Growth2 increased from 5.4 to 6.3 and therefore reached the suggestive significance threshold. For Growth3, the F-value increased from 4.5 to 6.0 with family effect in the model. A more dramatic increase in significance was observed for Growth8 since the F-value increased from not reaching even the suggestive threshold to become genome-wide significant with an F-value of 9.3 when the Family effect was included in the model. It is very likely that these three loci  represents true QTL effects and at each locus the allele from the HWS line is increasing growth as expected from the line differences in growth. In contrast, for Growth10, 11 and 13 there was only a minor difference in statistical significance between the two models and in these cases it is more likely that the previous suggestive evidence for QTL at these positions in fact were false positives.If we compare our current QTL analysis based on an improved linkage map with our previous QTL analysis  there arThe eight-fold difference in body-weight at selection age between two divergently selected chicken lines is determined by many QTL each with a small individual effect. Although no major QTL explaining a large proportion of the residual phenotypic variance was found, a network of interacting loci with a large effect was identified using the current extended linkage map. Furthermore, results from the QTL study was consistent with our analysis of Fst and homozygosity using a marker set including ~13,000 SNP markers. The results from that analysis failed to detect any major selective sweeps within QTL region suggesting that selection has been acting on standing genetic variation present in the founder population rather than recently derived mutations.PW organized the SNP genotyping of the pedigree, constructed the linkage map, contributed to the QTL analysis and the SNP analysis, summarized the results and drafted the manuscript. ÖC supervised and contributed to the QTL analysis. XT performed statistical analysis of SNP data, assisted with the organization of the genotype data. A-CS supervised the genotyping of the pedigree at Uppsala University. MF, ML and IG generated the SNP data used to detect selective sweeps. PBS designed the study together with LA, organized and produced the intercross and supervised the collection of all phenotypic measurements. LA supervised the molecular and the statistical analysis performed in Uppsala, revised and made final adjustments of the manuscript. All authors read and approved the final manuscript.Genetic markers included in the new linkage map. Positions of markers are shown in Kosambi cM and in Mb according to the current genome assembly .Click here for fileGenotype-phenotype interaction maps for all epistatic QTL detected in the intercross for growth traits. Mean values of body-weight (in grams) for each of nine possible allelic combinations are shown in the plots. The genotype classes for one QTL is given on the X-axis  and the curves represent the genotype classes of the other QTL. Error bars represent the standard error of mean (s.e.m.). HWS alleles are abbreviated as "H" and LWS alleles as "L". The figure is arranged so that all traits significant for the same unique interaction pair are displayed together. (A) QTL at Chr1, 316 cM – G6, (B) QTL at Chr1, 565 cM – Chr2, 224 cM, (C-D) Chr1, 473 cM – Chr10, 64 cM, (E-F) G12 – Chr1, 477 cM, (G) G9 – Chr24, 49 cM, (H) G9 – Chr28, 11 cM, (I-K) G9 – G12, (L) G1 – G12, (M-P) G9-G6, (Q – R) G9 – Chr2, 240 cM, (S-T) G9 – G1, (U-V) G9 – Chr3, 0 cM, (W-X) G9 – chr13, 30 cM, (Y) G1 – G6.Click here for file


With the Interferon-γ release assays (IGRA) a new method for the diagnosis of latent tuberculosis infections (LTBI) is available. Due to the lack of a gold standard for the diagnosis of LTBI, the IGRA is compared to the Mantoux Tuberculin Skin Test (TST), which yields discordant results in varying numbers. Therefore we assessed to which extent discordant results can be explained by potential risk factors such as age, BCG vaccination and migration.In this pooled analysis, two German studies evaluating the Quantiferon-Gold In-Tube test (QFT) by comparison with the TST (RT23 of SSI) were combined and logistic regressions for potential risk factors for TST+/QFT− as well as THT−/QFT+ discordance were calculated. The analysis comprises 1,033 participants. Discordant results were observed in 15.4%, most of them being TST+/QFT− combinations. BCG vaccination or migration explained 85.1% of all TST+/QFT− discordance. Age explained 49.1% of all TST−/QFT+ discordance. Agreement between the two tests was 95.6% in German-born persons younger than 40 years and not BCG-vaccinated.After adjustment for potential risk factors for positive or negative TST results, agreement of QFT and TST is excellent with little potential that the TST is more likely to detect old infections than the QFT. In surveillance programs for LTBI in high-income, low TB incidence countries like Germany the QFT is especially suited for persons with BCG vaccination or migrants due to better specificity and in older persons due to its superior sensitivity. The burden of tuberculosis (TB) in healthcare workers (HCW) remains high in low- and middle-income countries Mycobacteria tuberculosis-specific antigens  while the tuberculin of the TST is a mix of about 200 non-specific antigens that are shared with nontuberculous mycobacteria (NTM) as well as with the strains developed from Mycobacterium bovis used for bacilli Calmette-Guérin (BCG) vaccination For the diagnosis of latent tuberculosis infections (LTBI) the recently developed Interferon-γ release assays (IGRA) are good alternatives to the unspecific tuberculin skin test (TST) In a German contact-tracing study only those with a positive IGRA progressed towards active TB, while none of those with a positive TST but negative IGRA (TST+/IGRA−) developed TB in the two years following close contact to an active TB case In the literature little attention is given to TST-negative but IGRA-positive results. Assuming the IGRA to be highly specific it is likely that this combination indicates LTBI So far the extent to which BCG vaccination and NTB explain TST+/IGRA− discordance is not analyzed and the reasons for positive IGRA that are not verified in the TST (TST−/IGRA+) are unknown For this analysis we combined two study populations consisting of 1,040 healthy persons. Due to indeterminate results in the IGRA 7 persons had to be excluded from the analysis. Out of the remaining 1,033 persons , 601 werIn both study populations the TST was performed using 2-TU of PPD RT23 . The test was administered to the volar side of the forearm of the participants and read 72 to 96 hours after the application. The transverse diameter of the induration was measured. The observers were blinded to the IGRA results.Before TST application, the standardized interview was performed and blood for the IGRA was drawn. For the IGRA, a variation of the QuantiFERON-TB Gold assay , the QuantiFERON-TB Gold In-Tube test (QFT), was used. This whole-blood assay uses overlapping peptides corresponding to ESAT-6, CFP-10, and a portion of tuberculosis antigen TB7.7 (Rv2654). Stimulation of the antigenic mixture occurs within the tube used to collect the blood. Tubes were incubated at 37°C overnight before centrifugation, and INF-γ release was measured by ELISA following the protocol of the manufacturer. All the assays performed met the manufacturer's quality-control standards. The test was considered positive when INF-γ was ≥0.35 IU after correction for the negative control. Observers were blinded to the results of the TST.Due to the lack of a gold standard, sensitivity and specificity were not calculated. The Pearson Chi-square test was used to compare frequencies of test results among different groups of participants. For ordered risks, the proportions of positive test results were compared using the chi-square test of trend. P<0.05 was considered to be statistically significant. Agreement and Kappa values were calculated for the two tests with varying cut-offs for a positive TST . Odds ratios (OR) for discordant test results depending on different putative predictive variables were calculated using logistic regression. Model building was performed backwards using the chance criteria for variable selection Data analysis was performed using SPSS, Version 14 . The study protocols of both studies combined for this paper were approved by the ethics committee of the Hamburg Medical Council. All persons gave their written informed consent prior to their inclusion in the studies.Using a diameter of 10 mm as cut-off point for the TST yielded 159 (15.4%) discordant test results ; most ofTST−/QFT+ discordance was observed in 34 (3.3%) participants . Out of 35.9 (3.5%) discordant results (18.6 TST+/QFT− and 17.3 TST−/QFT+) remain unexplained by risk factors which yields a corrected agreement for the two test of 96.5%. This agreement corresponds with the agreement of the two tests in young people (under 40 years), born in Germany and not BCG vaccinated .With our data we were able to analyze migration, BCG vaccination and age as risk factors for 159 discordant results in contacts of TB cases tested with TST and QFT simultaneously. The proportion of discordant results we observed in our pooled analysis was somewhat lower (15% instead of 24%) than that described in a recent meta-analysis As a limitation of our study, we had no information on the age at vaccination, revaccination or exposure to NTM. Therefore we were not able to analyze the influence of these factors on TST. But we believe it to be likely that the observed interaction between being foreign-born and BCG vaccination might be explained by these factors.M. avium) were 5 times more likely in recruits born outside the US Birthplace was a risk factor for a positive TST and QFT as well as for TST+/QFT− discordance in our data. Similar results were observed in US Navy recruits Age is a strong predictor for a positive QFT that is not confirmed by the TST. In young people TST−/QFT+ results are rare. In 856 Navy recruits (mean age 20 years) no TST−/QFT+ combination was found So far two prospective studies investigating the progression to active TB have been published The proportion of discordant results that can not be explained by BCG vaccination, being foreign-born or by age is rather small (4.4%), indicating little potential for false-negative or false-positive QFT results. These findings support the hypothesis that the QFT is the test of choice in populations with a high BCG vaccination rate or with an increased chance of exposure to non-tuberculous mycobacteria (NTM), or in people older than 40 years. In young persons not vaccinated and unlikely to be exposed to NTM, the TST and the QFT are of comparable quality and the agreement between the two tests is above 95%. In older populations the TST is less sensitive than the QFT and in the German population with a migration background and/or a BCG vaccination the TST is far less specific than the QFT. In Germany, HCW with regular contact to TB patients are surveyed and in the general population contacts to TB patients are traced In conclusion, according to our data, it is not likely that the TST is more sensitive to old LTBI than the IGRA. Therefore we would like to suggest the use of an IGRA as the first test after exposure to a patient with active TB and in periodic screening for LTBI among exposed HCW, especially for those of foreign birth.