Exhaled breath condensate (EBC) is increasingly being used as a non-invasive method for disease diagnosis and environmental exposure assessment. By using hydrophobic surface, ice, and droplet scavenging, a simple impaction and condensing based collection method is reported here. Human subjects were recruited to exhale toward the device for 1, 2, 3, and 4 min. The exhaled breath quickly formed into tiny droplets on the hydrophobic surface, which were subsequently scavenged into a 10 µL rolling deionized water droplet. The collected EBC was further analyzed using culturing, DNA stain, Scanning Electron Microscope (SEM), polymerase chain reaction (PCR) and colorimetry (VITEK 2) for bacteria and viruses.3 in exhaled breath, including both viable and dead cells of various types. Sphingomonas paucimobilis and Kocuria variants were found dominant in EBC samples using VITEK 2 system. SEM images revealed that most bacteria in exhaled breath are detected in the size range of 0.5–1.0 µm, which is able to enable them to remain airborne for a longer time, thus presenting a risk for airborne transmission of potential diseases. Using qPCR, influenza A H3N2 viruses were also detected in one EBC sample. Different from other devices restricted solely to condensation, the developed method can be easily achieved both by impaction and condensation in a laboratory and could impact current practice of EBC collection. Nonetheless, the reported work is a proof-of-concept demonstration, and its performance in non-invasive disease diagnosis such as bacterimia and virus infections needs to be further validated including effects of its influencing matrix.Experimental data revealed that bacteria and viruses in EBC can be rapidly collected using the method developed here, with an observed efficiency of 100 µL EBC within 1 min. Culturing, DNA stain, SEM, and qPCR methods all detected high bacterial concentrations up to 7000 CFU/m Bioaerosols are present virtually anywhere in the environment, and their exposure is shown to cause numerous adverse health effects Among many other diseases, respiratory infection accounts for 23.3–42.1% of the total hospital infections For EBC related studies, the first key step is the collection of exhaled breath. Over the years, a variety of devices  of the device was evaluated. In addition, biological analysis and characterization of EBC samples collected from human subjects were conducted using culturing, DNA stain, SEM, qPCR and species identification tool VITEK 2. This work contributes to the effort in applying EBC together with molecular tools as a non-invasive method in rapid disease diagnosis.The collection method and device developed and experiential set up for collecting EBC are shown in For EBC collection, sterile water (DNA and RNA free) was first added into the collection base of the device up to a depth of 5 mm as observed in After the collection, about 10 µl of DNA and RNA free DI water was pipetted onto the hydrophobic film as observed in To investigate the amount of variability in EBC collected by the method developed, six student volunteers were recruited to exhale through the device for 1, 2, 3 and 4 min. The volume of collected EBC was measured by a calibrated pipette . The amount of EBC per unit time collected by the device was determined using averages of EBC samples obtained by the volunteers under each of specific collection time tested. For each EBC collection, a different hydrophobic film and a different exhalation straw were used. In addition, the particle size distributions in the exhaled breath through mouth-breathing were also measured in a particle free bio-safety hood using an Optical Particle Counter (OPC)  at a flow rate of 1.2 L/min. To ensure air stream balance, the OPC was connected to a two-way tubing, which connects to clean air  and the breathing straw, respectively.3  by considering the collection time and an average breathing rate of 12 L/min for an adult. Besides, the culturable bacterial species were identified using VITEK® 2 . In addition, molecular detection of bacteria and virus using qPCR and RT-qPCR, respectively, were performed according to the procedures described in Supporting Information S1. To further confirm the bacterial presence DNA stain of EBC sample by Acridine Orange (AO) was also conducted.In this work, seven patients with onset flu symptoms . A p-value of less than 0.05 indicates a statistically significant difference at a confidence level of 95%. Collection of EBC from human subjects was approved by Peking University Ethnics Committee.Here, a novel EBC collection method and device was developed and evaluated in collecting EBC samples from human subjects using culturing and molecular methods. Compared to those currently available devices shown in As listed in Here, the EBC samples collected by the developed device from seven human subjects recruited from a respiratory unit of Peking University Third Hospital in Beijing were studied using culturing, DNA stain, SEM and molecular methods. In this study, the particle size distributions trends in a typical exhaled breath were also measured and are shown in 3. ANOVA tests indicated that there were statistically significant differences in culturable bacterial aerosol concentrations for EBC samples collected from different subjects . In a recent study, human occupants are also identified as the significant contributors for indoor bacteria, i.e., the emission rate is about 37 million gene copies per person per hour, and a distinct indoor air signature of bacteria was demonstrated to be associated with human skin, hair, and nostrils Sphingomonas paucimobilis and Kocuria rosea were detected using Vitek2 in six EBC samples as shown in 3 and Sphingomonas paucimobilis, a non-fermenting Gram-negative bacillus, were detected. In a previous study, S. paucimobilis was found to cause nosocomia bacteremia outbreak Rhodococcus equi, a bacterium causing pyogranulomatous bronchopneumonia, were detected in the exhaled air from foals in a recent study B. subtilis) had a Ct value of 15 and the negative control had a value of 28. Based on the DNA standards used, the concentrations of bacterial DNA in the EBC samples (Sample 1–7) were in the range of 0.32 µg/µL–3.15 µg/µL. Detection of the bacterial DNA in EBC samples was also confirmed by the melting curve of qPCR amplification as shown in B. subtilis. For a few different peaks observed, they might be the possible primer dimer (PD) from the PCR non-specific amplification process. In addition to the qPCR amplification of bacteria in EBC samples collected, DNA stain (AO method) was also performed and the results are shown in B. subtilis samples, while no cells were detected in the negative control. SEM images with different resolutions and agar plate culturing shown in Sample 1– were in In this study, qPCR was also applied to detecting influenza A H3N2 viruses in EBC samples collected by the device. As observed in Exhaled breath holds great promise for monitoring human health and for the diagnosis of various lung and systemic diseases, but analysis challenges remain due to the complex matrix of the breath Figure S1Experimental procedures used in this study include physical characterization and molecular analysis of the EBC collection efficiencies of the device and its pilot application in a respiratory clinic.(TIF)Click here for additional data file.Figure S2Optical images of EBC samples stained by Acridine Orange (AO): Bacillus subtilis species were used as the positive control and DI water was used as the negative control.(TIF)Click here for additional data file.Figure S3Detection of H3N2 influenza viruses in EBC samples collected from three human subjects with ID: 1, 2, 3 corresponding to those listed in (TIF)Click here for additional data file.Table S1Characteristics of widely used EBC collectors.(DOCX)Click here for additional data file.Table S2Medical conditions of seven human subjects visiting a respiratory clinic whose exhaled breath condensate samples were collected in this study.(DOCX)Click here for additional data file.Information S1PCR test and Acridine Orange stain.(DOC)Click here for additional data file.


P = .026) and dystonia severity lower (P = .033) in the group with complete improvement to sensory tricks compared with the group with partial improvement to sensory tricks. A significant effect for group and modality and a significant interaction between group × modality were found, with lower visuotactile discrimination thresholds in the group with complete improvement to sensory tricks compared with the other groups. In primary cervical dystonia, a complete resolution of dystonia during a sensory trick is associated with better visuotactile discrimination and shorter disease duration compared with patients with less effective sensory tricks, which may reflect progressive loss of adaptive mechanisms to basal ganglia dysfunction. © 2013 Movement Disorder SocietyA characteristic feature of primary cervical dystonia is the presence of “sensory tricks” as well as the impairment of temporal and spatial sensory discrimination on formal testing. The aim of the present study was to test whether the amount of improvement of abnormal head deviation due to a sensory trick is associated with different performance of temporal sensory discrimination in patients with cervical dystonia. We recruited 32 patients with cervical dystonia. Dystonia severity was assessed using the Toronto Western Spasmodic Torticollis Rating Scale. Patients were rated according to clinical improvement to a sensory trick and assigned to 1 of the following groups: (1) no improvement (n = 6), (2) partial improvement (n = 17), (3) complete improvement (n = 9). Temporal discrimination thresholds were assessed for visual, tactile, and visuotactile modalities. Disease duration was shorter ( Dystonia is characterized by sustained involuntary muscle contractions causing twisting and abnormal posturing.13On the basis of the findings that a successful sensory trick requires sensory input to be processed and that sensory discrimination can be impaired in primary dystonia, we hypothesized that the more the sensory processing is impaired, the less successful the sensory trick will be. The aim of this study was to test this hypothesis on patients with adult-onset primary cervical dystonia, by measuring temporal sensory  discrimination.We recruited patients with AOPCD who were attending the movement disorder or botulinum toxin clinics at the National Hospital for Neurology and Neurosurgery, London, as well as healthy controls. After giving written consent, eligible patients were clinically examined by an experienced movement disorders specialist (G.K.). Participants with clinical evidence of polyneuropathy or carpal tunnel syndrome were excluded. Dystonia severity was quantified with the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS), which is divided into 3 subscales. Subscale I assesses motor severity, with a maximum score of 35; subscale II assesses disability, with a maximum score of 30; and subscale III assesses pain severity and its disability, with a maximum score of 20.Temporal discrimination thresholds were examined with pairs of tactile, visual, and tactile/visual  stimuli. Square-wave electrical pulses, which were delivered by a constant current stimulator  through surface skin electrodes (4 mm in diameter) were used for tactile stimulation. The electrodes were placed on the index and middle fingers of both hands. The anode was 1.5 cm distal from the cathode. The intensity of tactile stimulation for each subject and each finger was determined by delivering a series of stimuli with increasing intensity from 2 milliamperes (mA) in steps of 1 mA. The minimal intensity at which electric stimuli were perceived in 10 of 10 stimuli was used in the experimental test. Care was taken that stimuli did not induce pain or discomfort.Visual stimuli were delivered through light-emitting diodes (LEDs) positioned on a black table. Subjects' hands were positioned near the LEDs. Subjects were asked to look at the fixation point in the middle between the LEDs throughout each trial. Both visual and tactile stimuli lasted 5 ms. The experimental test was delivered in 6 combinations of stimulation: 2 tactile (left and right), 2 visual (left and right), and 2 crossmodal (visuotactile left and visuotactile right). The order in which the 6 combinations of stimuli were presented was randomized across the subjects. Each combination of stimuli was performed in 4 separate blocks. In the first trial of each block, pairs of simultaneous stimuli  were delivered. In subsequent trials, ISIs were progressively increased in steps of 10 ms. In each block trials with simultaneous stimuli were interspersed to check for attention.The temporal discrimination threshold was considered the first of 3 consecutive ISIs during which subjects recognized the stimuli as asynchronous. In addition, subjects were asked to judge which stimulus preceded (or followed) the other. The first of 3 consecutive ISIs during which subjects also reported correctly the temporal order in a pair of stimuli was termed temporal order judgment.P < .05 was considered significant. In primary adult-onset primary cervical dystonia, existing literature suggests that sensory discrimination thresholds depend on age but less on disease severity or disease duration. Therefore, Pearson's correlation coefficients were calculated to characterize and test (without adjustment for multiple testing) these associations.Data were analyzed using SPSS Statistical Package for Windows version 14.0 and R statistical software (version 2.9.0). Data are presented as mean and standard deviation of the mean for age, age at disease onset, disease duration, TWSTRS score, TWSTRS subscores I–III, visual-, tactile-, and crossmodal temporal discrimination thresholds, and temporal order judgments. One-way ANOVA with post hoc Bonferroni test was applied with the sensory trick group as factor and age, age of onset, disease duration, TWSTRS score, and subscores I–III as dependent variables. Temporal discrimination thresholds  were first analyzed using a 3-way ANOVA model with time (in milliseconds) as the dependent variable and sensory trick group , modality , and task  as explanatory variables (fixed factors), including an interaction term between sensory trick group and modality. Post hoc pairwise comparisons between the fixed factors were performed using Tukey's procedure. F = 4.6, P = .018), with a significantly (P = .026) shorter disease duration in patients with a complete sensory trick compared with patients with a partial sensory trick in the post hoc analysis and for the TWSTRS score  and subscore III  with significantly lower scores in patients with a complete sensory trick compared with patients with a partial sensory trick in the post hoc analysis . No significant effects were found for age, age of onset, or TWSTRS subscores I and II.Thirty-two patients  with adult-onset primary cervical dystonia were included. The mean age of patients was 56.4 ± 9.9 years , mean age of onset was 44 ± 11.4 years , and mean disease duration was 12.5 ± 8.5 years . Eighty-four percent of our study population was right-handed. The mean TWSTRS score was 24.7 ± 9.3 . An improvement in a sensory maneuver was present in 20 of 24 female patients (83%) versus 6 of 8 male patients (75%). Mean temporal discrimination thresholds were: visual, 53.2 ± 15.7 ms ; tactile, 85.7 ± 38.5 ms ; and crossmodal , 134.5 ± 38.7 ms . Mean temporal order judgments were: visual, 55.3 ± 16.2 ms ; tactile, 101.2 ± 46.6 ms ; and crossmodal , 141.3 ± 39.3 ms . No significant effect was found for the factor sex for either of the evaluated variables. Six patients with adult-onset primary cervical dystonia (19%) were allocated to the no sensory trick group, 17 patients (53%) to the partial sensory trick group, and 9 patients (28%) to the complete sensory trick group. As presented in F = 6.2, P = .002) and modality  but not for task. Therefore, temporal discrimination thresholds and temporal order judgments were averaged to further test the interaction sensory trick group × modality, which was significant . Post hoc pairwise analysis of the interaction between sensory trick group and modality showed significant differences in the crossmodal paradigm between the complete sensory trick and no sensory trick groups (P = .049) as well as between the complete sensory trick and partial sensory trick groups (P = .008), whereas no significant difference was found between the no sensory trick and the partial sensory trick groups. Post hoc analysis did not show significant differences between the sensory trick groups either for the visual or for the tactile paradigms. Pairwise post hoc analysis between different modalities within the same sensory trick group throughout showed significant differences, except for the tactile versus the crossmodal paradigm in the group without sensory trick and the visual versus tactile paradigm in the complete sensory trick group  and with tactile temporal discrimination without reaching the level of significance , but not with crossmodal temporal discrimination. Motor disease severity, expressed by TWSTRS subscore I, and disease duration did not correlate with the temporal discrimination thresholds.Among all patients, age correlated with visual temporal discrimination . The finding that patients with a short disease duration experienced a better effect of a sensory maneuver has been described in different types of focal dystonia, such as cervical dystonia,One interesting result of the present study is that patients with a complete sensory trick performed significantly better in the crossmodal temporal discrimination task, as expressed by lower thresholds, compared to patients with only a partial or absent effect to a sensory trick. It is particularly interesting that only the crossmodal task revealed differences between the groups selected by the effect of a sensory maneuver, whereas the unimodal tasks did not see . This maThe results of a number of other studies have consistently shown a relationship between processing of sensory input and sensory tricks. One study investigated patients with blepharospasm and found an association between the presence of sensory tricks and normal prepulse inhibition of the blink reflex.These observations from functional imaging studies showing that the parietal cortex, as an important center of multimodal sensory integration, is hyperactive during the execution of a sensory maneuver is well in line with our findings of better performance  in patients with a complete sensory trick compared to patients with less effective or absent sensory tricks. This means that parietal function  decreases with longer disease duration. As shown in the prepulse blink reflex inhibition or in the fMRI experiments, the progressive increase in temporal crossmodal discrimination thresholds may reflect progressive loss of cortical adaptive mechanisms to the basal ganglia dysfunction of adult-onset primary cervical dystonia. Our results provide evidence that patient assignment according to their sensory trick represents a different selection from a pathophysiological point of view where the functional integrity of the parietal cortex is important, which is reflected by the crossmodal sensory discrimination thresholds.In summary, this study confirms that in adult-onset primary cervical dystonia, sensory tricks are more prevalent in patients with short disease duration and less severe disease expressed by low TWSTRS score, whereas it does not depend on the patient's age. This could be explained by intact sensory afferents in early disease stages that get impaired the longer disease progresses. On a cortical  level, functionally intact adaptive mechanisms to the basal ganglia dysfunction seem to be a premise for the presence of a good clinical effect to a sensory maneuver, because with its loss, crossmodal (visuo/tactile) temporal discrimination thresholds increase.


This small observational study was motivated by our belief that scaling the tidal volume in mechanically ventilated patients to the size of the injured lung is safer and more 'physiologic' than scaling it to predicted body weight, i.e. its size before it was injured. We defined Total Lung Capacity (TLC) as the thoracic gas volume at an airway pressure of 40 cm H2O and tested if TLC could be inferred from the volume of gas that enters the lungs during a brief 'recruitment' maneuver.Lung volume at relaxed end expiration (Vrel) as well as inspiratory capacity (IC), defined as the volume of gas that enters the lung during a 5 second inflation to 40 cm H2O, were measured in 14 patients with respiratory failure. TLC was defined as the sum of IC and Vrel. The dependence of IC and Vrel on body mass index (BMI), respiratory system elastance and plateau airway pressure was assessed.TLC was reduced to 59 ± 23% of that predicted. Vrel/TLC, which averaged 0.45 ± 0.11, was no different than the 0.47 ± 0.04 predicted during health in the supine posture. The greater than expected variability in observed Vrel/TLC was largely accounted for by BMI. Vrel and IC were correlated (r = 0.76). Taking BMI into account strengthened the correlation (r = 0.92).We conclude that body mass is a powerful determinant of lung volume and plateau airway pressure. Effective lung size can be easily estimated from a recruitment maneuver derived inspiratory capacity measurement and body mass index. However, the use of this threshold as a surrogate for severe lung impairment has yet to be validated and is obviously influenced by the choices of PEEP, VT, respiratory muscle activity, and the mechanical properties of the chest wall [The low tidal volume trial of the ARDS Network , supported by a long list of preclinical and clinical studies, has unequivocally established that mechanical ventilation with large tidal volumes (VTs) can be injurious to the lungs of patients with acute lung injury (ALI) or the acute respiratory distress syndrome (ARDS) . Howeverest wall . Indeed,est wall ,8.2O is sufficient to predict TLC at the bedside. We show that there is a reasonable correlation between the inflation maneuver-derived inspiratory capacity (IC) and the thoracic gas volume (TGV) at relaxed end-expiration (Vrel) and that, in the supine posture, Vrel/TLC is determined in large part by the body mass index (BMI). We also confirm earlier reports that suggested great variability in parenchymal deformation of patients with injured lungs when VT is targeted to PBW as opposed to effective lung size [This small observational study on 14 mechanically ventilated patients was motivated by our belief that scaling VT to the size of the injured lung is safer and more 'physiologic' than scaling it to PBW . Considering this premise, we set out to measure the total lung capacity (TLC) of 14 mechanically ventilated patients with respiratory failure and to test whether measuring the volume of gas that enters the lungs during a brief inflation to 40 cm Hung size  and addr2) concentration of not more than 0.65 and who were sufficiently sedated to tolerate a 5-second lung inflation to an airway pressure of 40 cm H2O without inducing respiratory effort, were studied. The protocol was approved by the Institutional Review Board, and informed consent was obtained from each patient's legally authorized representative.Fourteen hemodynamically stable  patients, who were mechanically ventilated with a fractional inspired oxygen  or 5 cm H2O (subsequent 10 patients) and TGV at relaxed end-expiration (Vrel) was measured 5 minutes later. Data from the 4 patients, in whom Vrel was estimated at zero end-expiratory pressure (ZEEP), are identified as such throughout this report. The ventilator was then switched to a pressure control mode at a rate of three breaths per minute so that the lungs could be inflated to an airway pressure of 40 cm H2O for 5 seconds. Inflation and deflation volume, flow, and pressure were recorded using the NICO monitoring module. IC, defined as the amount of gas entering the lungs between the pressures of 0 or 5 and 40 cm H2O was recorded on the NICO system, so it could be subsequently compared with the volume estimates derived from the ventilator's digital display. IC measurements were made in triplicate, whereby maneuvers with phasic respiratory muscle activity as judged by pressure and flow patterns were rejected post hoc from further analysis. The inflation maneuver was to be aborted on the basis of predefined safety termination criteria but in no instance were these met . The experiment concluded with a repeat measurement of Vrel before the patients were returned to their original ventilator settings.Patients were mechanically ventilated with an Engstrom GE Carestation ventilator  at settings previously determined by the primary care providers Table . The GE P value of less than 0.05.Normal values for TLC, vital capacity (VC), and residual volume were derived from reference values provided by Goldman and Becklake . The ela2.Clinical diagnosis and baseline ventilator data were obtained from the patients' electronic medical records Table . Eleven r = -0.66), the relationship was dominated by two outliers . Consequently, neither Pplat nor ERS helped predict the reduction in effective lung size in patients with lung injury.As expected, TLC was substantially reduced in the majority of patients, averaging 59% ± 23% of the predicted value (Table r = -0.63) .For the group, the ratio of Vrel/TLC, which averaged 0.45 ± 0.11, was not statistically different than the 0.47 ± 0.04 predicted for these individuals during health in the supine posture [2O, consistent with previous observations on the time course of recruitment of atelectatic regions in anesthetized humans [r = 0.76) , so that TLC could have been estimated from BMI and IC within ± 0.4 L in all but two instances.With the exception of patients 3 and 13, who essentially had normal lung volumes, IC was reduced, averaging 61% ± 26% of the predicted normal value for the entire group. Inspiratory flow invariably fell to zero during the 5-second inflation to 40 cm Hd humans . The vol2O in each instance, indicating that a Pplat threshold of 30 cm H2O does not guard against hyperventilation of aerated, recruitable regions of the injured lung.Since providers had scaled VT to PBW, the variability in VT when expressed as a percentage of predicted TLC was relatively small Figure . For thepost hoc adjustments of ventilator-displayed volumes to account for temperature, humidity, and tubing compliance. As recently reported, precision, accuracy, and handling of volume information differ widely among commercially available mechanical ventilators [Because the GE Carestation ventilator, which was used in these experiments, does not provide a numeric display of delivered volume when set in a pressure control mode, we compared ventilator-recorded expired volumes following TLC inflations with those measured with NICO. On average, the expired volume displayed on the ventilator was 5% ± 10% smaller than that measured with NICO. In part, this discrepancy reflects tilators .The main conclusion from this small observational study is that measuring the IC of intubated patients helps predict effective lung size. Our premise entering this study was that sizing the recruitable lung is important for individualizing patient care. Our research did not test the imperative of this premise. Nevertheless, we find its underlying rationale compelling. It is generally accepted that lungs, particularly when injured, are vulnerable to additional damage by both cyclic recruitment/derecruitment and overinflation. The two injury mechanisms frequently coexist in the same lung. While prevention of the former calls for an increase in parenchymal stress , prevention of the latter mandates a stress reduction, which is usually accomplished by limiting Pplat. With increasing lung impairment, the upper and lower volumes and hence stress safety boundaries within which both imperatives may be accomplished approach one another. In other words, the 'safe' inflation pressure amplitude, defined as the difference between optimal PEEP (one that maximizes recruitment) and a 'safe' Plat (one that minimizes the risk of overdistension), approaches zero or may even assume a negative value. Whereas sizing the recruitable lung does not address the choice of best PEEP or mean airway pressure per se, it does provide information about the probability that a chosen VT will encroach on upper or lower lung volume (or both) or stress safety boundaries.2O provides a reasonable estimate of the injured lungs' total capacity. In normal humans, TLC is almost completely determined by the size and recoil properties of the lungs because the lungs' compliance near TLC approaches zero whereas that of the chest wall remains finite. As a result, in upright normal humans, the intrathoracic pressure near TLC approximates 10 cm H2O [2O as a surrogate of stress injury risk is implicitly based on these estimates. It is now apparent that the lungs of many recumbent patients, particularly those with increased BMI or distended abdomens or both, are not fully expanded at a PRS of 30 cm H2O [2O. It is nevertheless likely that, in patients with extensive alveolar flooding and collapse or with morbid obesity or with both, even a PRS of 40 cm H2O does not guarantee full lung inflation. The choice of 40 cm H2O thus represents a compromise between patient safety and biologic certainty.We assumed that the TGV at a transrespiratory system pressure (PRS) of 40 cm H0 cm H2O . The wid0 cm H2O . TherefoOur data are entirely in line with observations by Chiumello and colleagues , who emp2O yielded an acceptable IC estimate, even though we refrained from using neuromuscular blocking agents. Repeat IC estimates (available in 10 of 14 patients) varied by less than 12%, averaging ± 5% for the group. None of our attempts to inflate the thorax to 40 cm H2O pressure had to be aborted for cardiovascular reasons. Limiting the duration of inflation to 5 seconds undoubtedly enhanced the tolerance of the IC 'recruitment' maneuver. It is of note that, within the limits of our flow detection capabilities (>1 L/minute), a 5-second inflation appeared sufficient to fully expand all recruitable lung units. This observation is in keeping with computer tomography-based estimates of alveolar recruitment of atelectatic lung regions [We set out to measure Vrel and consequently IC at/from a volume at ZEEP. We abandoned this approach after four patients because reducing airway pressure to ZEEP frequently induced coughing, always runs the risk of oxygen desaturation, and was not essential for the objectives of our experiment. While the small sample size precludes a statistical evaluation of this change in experimental design, we are unable to detect the expected bias (lower Vrel/TLC and greater IC when Vrel is measured at ZEEP) in our data. Over 50% of inflations to 40 cm HWhile we expected that Vrel and, by inference, IC would serve as surrogates of lung impairment, namely of disease-related loss of lung units, we were surprised how strongly Vrel/TLC correlated with BMI. This observation underscores the importance of chest wall mechanics on lung function of recumbent patients with injured lungs. It is very much in line with recent esophageal manometry-based estimates of chest wall recoil in this population and undermines the rationale for limiting airway inflation pressure and, by inference, PEEP therapy to a singular Pplat value ,16. On aMeasuring the IC by means of the inherent hardware/software systems of commercially available mechanical ventilators can be challenging. Bench tests of mechanical ventilators used in our practice generally support the manufacturer's stated volume accuracy of ± 10% (data not shown). Compensation algorithms accounting for tubing compliance, gas temperature, and humidity vary greatly among vendors . Therefo2O, when adjusted for body weight/habitus, is sufficient to estimate the capacity of the injured lung at the bedside. We did not and cannot offer an opinion on the critical size of any IC- or TLC-based VT scaling factor nor do we know of specific data on its interactions with mean lung volume or PEEP. Consistent with hypotheses put forth by Chiumello and colleagues [We have provided evidence that measuring the volume of gas that enters the lungs during a brief inflation to 40 cm Hlleagues , we beli2O remains a reasonable initial care goal, we draw attention to the importance of BMI as a determinant of Vrel/TLC and will be less hesitant to exceed this threshold in patients with abdominal distension, but preserved TLC. Alternatively, we are likely to reduce VT to less than 6 mL/kg PBW long before Pplat reaches 30 cm H2O in nonobese patients with small effective lung capacities. Needless to say, validation of these approaches will require preclinical and clinical efficacy trials.The dependence of Vrel on BMI, which we have observed, indirectly supports the esophageal manometry-based conclusions of Talmor and colleagues  and thos2O, is reduced to varying degrees in mechanically ventilated patients with injured lungs.• Total lung capacity (TLC), defined as thoracic gas volume (TGV) at an airway pressure of 40 cm H2O.• TLC can be calculated by measuring the TGV at relaxed end-expiration (Vrel) and then adding the inspiratory capacity (IC), defined as the volume of gas which enters the lungs during a 5-second inflation to an airway pressure of 40 cm H• Because in recumbent patients body mass and habitus are important determinants of Vrel, TLC may be estimated with reasonable accuracy from IC and body mass index alone.• Future clinical trials in patients with injured lungs should consider data on chest wall mechanics and effective lung capacity.ALI: acute lung injury; ARDS: acute respiratory distress syndrome; BMI: body mass index; ERS: elastance of the respiratory system; IC: inspiratory capacity; PBW: predicted body weight; PEEP: positive end-expiratory pressure; Pplat: plateau airway pressure; PRS: transrespiratory system pressure; TGV: thoracic gas volume; TLC: total lung capacity; VC: vital capacity; Vrel: lung volume at relaxed end-expiration; VT: tidal volume; ZEEP: zero end-expiratory pressure.The authors declare that they have no competing interests.JSM and SRH screened and identified patients, obtained informed written consent, carried out all bedside measurements, and contributed to the data analysis. RAO contributed to study design and participated in study conduct and data analysis. RWS and CFB participated in study conduct and, together with SRH, were responsible for validating methods and approach at the bench. RDH conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.


Plasmodium and anopheline mosquitoes. Specifically targeting early sporogonic stages, we initially screened AMPs for toxicity against a mosquito cell line and P. berghei ookinetes. Promising candidate AMPs were fed to mosquitoes to monitor adverse fitness effects, and their efficacy in blocking rodent malaria infection in Anopheles stephensi was assessed. This was followed by tests to determine their activity against P. falciparum in An. gambiae, initially using laboratory cultures to infect mosquitoes, then culminating in preliminary assays in the field using gametocytes and mosquitoes collected from the same area in Mali, West Africa. From a range of 33 molecules, six AMPs able to block Plasmodium development were identified: Anoplin, Duramycin, Mastoparan X, Melittin, TP10 and Vida3. With the exception of Anoplin and Mastoparan X, these AMPs were also toxic to an An. gambiae cell line at a concentration of 25 µM. However, when tested in mosquito blood feeds, they did not reduce mosquito longevity or egg production at concentrations of 50 µM. Peptides effective against cultured ookinetes were less effective when tested in vivo and differences in efficacy against P. berghei and P. falciparum were seen. From the range of molecules tested, the majority of effective AMPs were derived from bee/wasp venoms.A new generation of strategies is evolving that aim to block malaria transmission by employing genetically modified vectors or mosquito pathogens or symbionts that express anti-parasite molecules. Whilst transgenic technologies have advanced rapidly, there is still a paucity of effector molecules with potent anti-malaria activity whose expression does not cause detrimental effects on mosquito fitness. Our objective was to examine a wide range of antimicrobial peptides (AMPs) for their toxic effects on  Breaking the complex life cycle of malaria by blocking its development in the mosquito is one area of research being pursued for malaria control. Currently, the mosquito itself, or microbes that live within it, are being genetically modified to provide toxic or lethal outcomes to the parasite. However, this usually comes with a cost to the lifespan and reproductive capabilities of the mosquito, resulting in a strong disadvantage if these modified organisms were to be released in the wild. This work aimed to identify a group of molecules suitable for inclusion in genetic modification strategies, which are toxic to malaria parasites, but have no costly side-effects to the mosquito. Within this group of molecules, toxins from bee and wasp venoms were prominent in their effects on mouse and human parasites. These particular molecules may prove effective in novel malaria control strategies and such venoms may also be a promising source of additional anti-malaria toxins. In the pursuit of malaria eradication, novel tools are in constant demand due to the lack of an effective vaccine and the emergence of pesticide-resistant insects and drug-resistant parasites An. stephensi, has been achieved on many occasions  and non-membrane active , as reviewed in Plasmodium spp. in vitro and several have been tested in vivo via infections of laboratory-reared anopheline mosquitoes  and P. falciparum from cultured sources (B) and field sources (C).Promising AMP candidates were assessed for their anti-P. berghei gametocytes and fed to An. stephensi mosquitoes (in vivo experiment as they were much less toxic to P. berghei ookinetes at a concentration of 50 µM than the other peptides. Mosquito wing sizes did not differ significantly between batches of mosquitoes used in these experiments (p = 0.477) and we therefore make the assumption that, on average, fully engorged females took similar sized bloodmeals and received similar parasite loads. The only consistently effective peptide at this concentration was Melittin, significantly reducing parasite prevalence by an average of 10% (p = 0.019) and intensity by 68%  over the three replicates  or intensity (p = 0.651).Melittin, Mastoparan X or TP10 dimer were tested We carried out a preliminary investigation using vectors and parasites from the same malaria endemic district to validate the efficacy of specific peptides in semi-natural conditions. Infecting recently colonised mosquitoes with parasites from gametocyte carriers was extremely challenging, and often resulted in no, or very low, infections. We tested 20 gametocyte carriers, fed to over 8500 mosquitoes; 75% of which took a full blood meal. Wing lengths were consistent throughout the experiments (p = 0.635) and egg numbers were not significantly different in mosquitoes fed with any peptide compared to controls (p = 0.397). Feeds from only nine gametocyte carriers resulted in mosquito infections, with gametocyte numbers ranging from 5–90 gametocytes/µl, producing infections with a prevalence range of 0–46% and intensity of 0–23 oocysts per midgut. Despite this low success rate, we were able to test two of our top candidate AMPs; TP10 dimer (n = 3 replicates) and Vida3 dimer (n = 2 replicates). Over the replicates, TP10 dimer did not provide significant anti-parasitic effects on either prevalence (p = 0.699) or intensity  these differences may reflect either stage-specific activity or a difference in the experimental environment. If the former, the choice of transgene promoters that direct AMP expression to maturing ookinetes would be critical to maximising the impact of any chosen effector molecule Our seven candidate peptides showed greater efficacy against enriched ookinete cultures than against parasites in the mosquito. As An. gambiae cell line, to assess potential impacts on mosquito fitness and help determine speed and mode of action. Of the seven peptides that displayed anti-malarial effects, five also caused a reduction in viability of insect cells. Despite this, feeding AMPs to mosquitoes did not have any significant negative impact on mosquito longevity or fecundity over a 10 day period that included two separate blood meals. These contrasting results may relate to physiological differences between the midgut epithelial cells and cultured cells. The latter lack a glycocalyx, which may act as an initial protective layer in midgut cells In parallel, we tested all AMPs for toxicity to an P. bergheiper se. However, it is possible that perceived fitness costs in transgenic strains may be associated with specific insertion sites, plasmid constructs or loss of genetic heterogeneity as a result of inbreeding One peptide (TP10 dimer) did reduce mosquito longevity in 2 of 4 replicate experiments during the latter stages of a 30 day assessment. However, since no effects were seen prior to 10 days (encompassing at least 2 egg batches) any fitness cost when expressed as a transgene would likely be minimal. This is because the majority of reproductive potential is likely to be realized before this time. To date, investigations of transgenic mosquito fitness indicate variable outcomes. For example, SM1 transgenics showed no fitness cost when fed on uninfected blood P. falciparum and An. gambiae. TP10 dimer and Melittin lowered both prevalence and intensity of P. falciparum infections produced from laboratory cultures. It has already been reported that the monomer of TP10, added to blood meals at 30 µM, reduced parasite prevalence by 35–45% Some candidate AMPs were additionally investigated using in vivo and in vitro is the midgut environment. Although we have no information on the stability of our AMPs when added to blood, it has been reported that very few peptides survive proteolytic degradation longer than a few minutes via transgenes under the control of a midgut promoter, or if expressed outside of the midgut to target later stages of the parasite.One possible reason for differences in vitro-produced P. falciparum gametocytes is the most feasible method for large-scale screening of novel AMPs. What may be best practice, however, is to focus on testing recently isolated parasites from malaria endemic areas, combined with vectors collected from the same area Studying AMPs in field conditions in Mali proved challenging, as very few membrane feeds produced infections. Whilst every blood sample drawn was gametocyte-positive, only one sample was able to provide an infection where AMPs were effectively tested. For practical reasons, we conclude that the use of Whilst we are largely unaware of how these AMPs work in isolation or in synergy, the data presented here provide specific information on our candidate AMPs under given conditions. The Sua 4.0 cell line data confirm the lytic mode of action of Melittin One benefit of the non-specific nature of AMP activity may be the slow emergence of resistance. Parasites would require profound changes in membrane structure to develop resistance to this class of molecules. Yet, resistance must always be considered as a possibility, and Peschel and Sahl present several mechanisms of how this could evolve Trypanosoma and Leishmania spp. P. falciparum and Trypanosoma brucei bruceiPlasmodiumWe have concentrated here specifically on the effects of AMPs on malaria parasites. In the natural environment, AMPs may come into contact with other microbes in both an advantageous or negative manner. On the positive side, AMPs active against malaria may also have activity against other human pathogens such as An. gambiae is still a challenging species for transgenesis and logistical difficulties in transgene dispersal and reproductive barriers must be overcome WolbachiaPlasmodium activity that deserves further exploration. If used in synergy and in multiple mosquito compartments, these should be able to eliminate parasites within the vector or be used as blueprints to rationally develop new synthetic AMPs. There is clearly a need to consider how chosen effector molecules might best be deployed through transgenic technologies. Both transgenic and paratransgenic strategies are considered by Wang and Jacobs-Lorena in a recent review An. gambiae. We further suggest that the concentration of the effector molecule, in relation to parasite load, is an important determinant of success. Thus, multiple peptides, acting in synergy, could perhaps achieve a complete transmission blockade within the mosquito. We highlight AMPs from the venom of bees and wasps as a future source of novel anti-Plasmodium effector molecules. Over the last few years, bee, wasp and hornet venoms have attracted attention as potential bioactive substances, with new AMPs regularly being described . At the study site in Nyaganabougou, ethical approval from community leaders and written informed consent from parents of minors was obtained for all blood samples. Every effort was made to minimise distress and all parasite-positive individuals identified by the study were subsequently treated by trained medical staff.For animal work at Keele: Animals were housed in the Central Animal Facility at Keele University, which is designated by the Home Office. All work was carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 as amended by EU Directive (2010/63/EU). Protocols were approved by veterinary staff and conducted by trained personnel under Project Licence PPL 40/2997. Every effort was made to minimize suffering.For animal work in Paris: Animals were housed in the Pasteur Institute Animal Facility, which is accredited by the French Ministry of Agriculture (Accreditation A75-15-31). All work was conducted in accordance with French and European regulations on care and protection of the Laboratory Animals . Protocols were approved by veterinary staff and performed in compliance with the NIH Animal Welfare Insurance #A5476-01 issued on 31/07/2012. Every effort was made to minimise suffering.All reagents were purchased from Sigma UK unless otherwise stated. Experiments were conducted at the University of Keele, UK, the Malaria Research and Training Centre (MRTC) at the University of Bamako, Mali and CEPIA at the Pasteur Institute, Paris, France.ad libitum on 10% glucose supplemented with 0.05% PABA (para-amino-benzoic acid) and females provided with defibrinated horse blood  for egg production in the UK, O+ human blood in Bamako. In Paris, larvae were fed dry cat food pellets, adults were fed 10% sucrose without PABA and the colony maintained by feeding on anaesthetized rabbits. For P. falciparum infections, gametocytes were mixed with AB+ human blood/serum and infected mosquitoes maintained on 10% sucrose supplemented with 0.05% PABA. Wing lengths of experimental mosquitoes were recorded throughout to check for any differences in mosquito size, as differences would affect bloodmeal volume, egg production and numbers of parasites ingested.Mosquitoes were maintained in standardized conditions An. gambiae, M form, according to the method of Fanello Plasmodium falciparum infection in Mali for a further 20–30 generations. An. stephensi mosquitoes  were established as laboratory colonies in Bamako and Keele and were verified as  SDA 500 , the besA total of 33 peptides were rehydrated in sterile distilled water to a stock concentration of 1 mM and stored at −20°C in aliquots suitable for individual experiments. All peptides were used in assays at a final concentration of 50 µM, unless otherwise stated. This choice of concentration was based on reported stimulated endogenous insect AMP concentrations ranging from 1–100 µM, An. gambiae Sua 4.0 cell line was used To establish potential effects of AMPs on mosquito cells, the 4/ml in 195 µl of supplemented Schneider's insect medium, with the addition of 5 µl  of the test peptide. Cells were maintained for 3, 6, 24 or 48 h, after which they were resuspended and a sub-sample from duplicate wells counted using a haemocytometer. To establish cell viability after 48 h (whether simply arrested in development or non-viable), resuspended cells were added to an equal volume of 5% erythrosin B vital dye and viable cells excluding the stain were counted. The viability of 100 cells in triplicate wells was recorded. Each AMP assay was performed in triplicate and included a negative control minus the peptide  and a further control of 5 µl of 10% SDS (sodium dodecyl sulphate) substituted for the AMP as this causes 100% cell lysis.Each AMP was tested separately to establish if it affected cell viability and growth. Sua 4.0 cells were seeded in 48 well microtitre plates  at a density of 30×10An. gambiae  were used to establish the effects of AMPs on mosquito longevity and fecundity in the absence of confounding factors introduced with parasite infection Female in vitro. As protocols for the conversion of P. falciparum gametocytes to ookinetes do not provide high yields, the rodent malaria parasite, P. berghei ANKA, was used to assess the effects of AMPs on early sporogonic stages. Ookinete cultures of P. berghei were established from gametocytaemic blood as previously described 5/ml in a volume of 47.5 µl Schneider's insect medium (Invitrogen) with 2.5 µl of the test AMP . Ookinetes were incubated for 30 min in conjunction with triplicate control wells containing medium with 2.5 µl of water instead of AMP. A sub-sample of parasites was then added to an equal volume of 5% erythrosin B to assess viability . Each of the 33 AMPs was screened in this way and assays were performed 3 times. AMPs displaying a high degree of toxicity (>90% of ookinetes displaying erythrosin B staining) underwent further ookinete assays using a concentration of 100 µM AMP. To assess synergistic effects, combinations of two peptides were also tested at a concentration of 25 µM per AMP, in the assay described above.Our primary aim was to target parasites in mosquitoes before they cross the midgut wall, therefore AMPs were first assessed for their ability to kill ookinetes P. berghei ookinetes in vitro were subsequently tested on P. berghei parasites in vivo  of AMP. This mixture was fed to 100 nulliparous 3–5 day old female An. stephensi. A further 475 µl of blood from the same infection was mixed with 25 µl of distilled water to act as a control. Mosquitoes were maintained at 19°C for optimum parasite development. Between 30 and 50 mosquitoes were dissected after 10 days. Mosquito wing length, numbers of retained eggs and oocysts were recorded in triplicate experiments carried out on different mosquito cohorts.Five of the seven most effective peptides against P. falciparum NF54 gametocytes were produced through large-scale automated culture at the Pasteur Institute, Paris 2, 1% O2, 94% N2). Fresh red blood cells (RBCs) were added to obtain a 7% haematocrit. Fourteen days after initiating the culture, gametocyte maturity was assessed on thin Giemsa-stained blood smears, and male gametocyte maturity verified by exflagellation tests. Gametocyte cultures were centrifuged for 5 min at 1500 rpm and the ∼500–600 µl pellet resuspended in fresh RBCs and AB human serum to give a final haematocrit of 40%.An. gambiae mosquitoes were left to feed in the dark for 15 min and only fully engorged females were transferred to small cages and provided with 10% sucrose containing 0.05% PABA. After 8 days, 30 midguts were dissected and stained with bromo fluorescein for the detection of oocysts.Blood containing mature gametocytes (475 µl) was added to 25 µl of the test AMP . Peptides were replaced by 25 µl of sterile deionized water for each control. The blood/parasite/peptide mixture was placed in a Hemotek membrane feeder, previously warmed to 37°C. For each feed, 70 to 100 nulliparous female An. gambiae Mali colony used for this study were originally collected in the village of Kenieroba (a distance of 15 km from Nyaganabougou). Potential male and female gametocyte carriers between the ages of 6–10 were screened by thick smear and parasite species and density determined by local staff, led by Saibou Doumbia.Nyaganabougou, a village approximately 60 km south-west of the Malian capital, Bamako, was used as a site for the source of donors of malaria-infected blood. Due to its proximity to the river Niger, which provides perennial mosquito breeding sites, Nyaganabougou has high numbers of infected individuals and an extended transmission season. Mosquitoes used to establish the Gametocyte carriers were taken to the Malaria Research and Training Centre at the University of Bamako for blood donation. Approximately 4 ml of blood was extracted from carriers and immediately divided into 3 Hemotek membrane feeders for testing 2 separate AMPs and one control for each carrier. Peptides were added to 950 µl of gametocytaemic blood for a final concentration of 50 µM. All parasite-positive individuals were subsequently treated appropriately for parasites and associated symptoms, as determined by, and carried out by, local health workers in Nyaganabougou.An. gambiae  were starved overnight, and allowed to feed on the gametocytaemic blood/AMP mix for 30 min. Engorged females were maintained on 10% glucose with 0.05% PABA for 8 days before dissection. No oviposition sites were provided so that fecundity could be measured by determining retained eggs. Mosquito wing length, numbers of retained eggs and oocysts were recorded, and triplicate peptide experiments were carried out on different mosquito cohorts, using blood from different donors. Where possible, a minimum of 50 mosquitoes were dissected for each condition.Approximately 120 adult female P. berghei ookinetes were assessed by the Wilcoxon signed rank test and oocyst burdens of both rodent and human malaria were compared via the Mann-Whitney U test. Differences in infection prevalence and mosquito sizes (wing length) used in all in vivo studies were assessed using a 2-sample t-test. Longevity studies were analyzed by Kaplan-Meier tests, where survival curves were compared using the Wilcoxon test to weight early failures (immediately after the first feed) more heavily. The number of eggs produced per mosquito after the first feed was analyzed by a one-way ANOVA.Analyses were performed using Minitab 16 statistical software. Data were checked for normality (Anderson-Darling) and replicates were checked for between-experiment variation using one-way ANOVA. Sua 4.0 cell growth and viability assays were also analyzed using one-way ANOVA. The effects of AMPs on Table S1Peptide sequence, activity and source.(DOC)Click here for additional data file.Table S2Effect of AMPs on mosquito longevity over 10 days.(DOC)Click here for additional data file.Table S3Effect of AMPs on mosquito oviposition over 10 days.(DOC)Click here for additional data file.


To examine the relationship between human papillomavirus (HPV) and giant cell arteritis (GCA) of the temporal artery.The study group consisted of 22 cases of histologically positive/biopsy confirmed GCA. The control groups consisted of 21 histologically negative temporal artery biopsies and fifteen cases of vascular margins of nephrectomies. For detection of the presence of HPV, two methods were used: 1) polymerase chain reaction (PCR) with INNO-LiPA HPV Genotyping Extra, 2) Cervista™ HPV HR. All cases were from the files of the Barnes-Jewish Hospital and Washington University in St. Louis.TM) confirmed the aforesaid results with 100% concordance with the exception of three cases which had low genomic DNA for which it was not possible to perform the test. The differences in HPV positivity between the histologically positive and negative temporal artery biopsies and between the histologically positive temporal artery biopsies and controls were both statistically significant .HPV DNA was detected by PCR and genotyping in 16 of 22 (73%) histologically positive cases of GCA and in only five of 21 (24%) histologically negative temporal artery biopsies. Among the vascular margin controls, only three of 15 (20%) were positive for HPV DNA. The second, independent method . Further studies are necessary to elucidate the pathophysiology underlying this association. Giant cell arteritis is one of the most common causes of vasculitis involving the temporal artery. Other potential causes are Wegener granulomatosis, polyarteritis nodosa (PAN), and Buerger disease . GCA genHistologically, GCA shows transmural inflammation with mixed inflammatory cell infiltrate mostly consisting of lymphocytes, histiocytes, plasma cells, occasional neutrophils and rarely eosinophils Figure . This caInfectious agents have long been considered as a possible etiology of GCA. The concept is that GCA represents a chronic inflammatory response, triggered by an infectious agent, with subsequent inappropriate tissue response to injury. However, studies looking for organisms have had conflicting results. Some have demonstrated organism DNA, such as Herpes Simplex Virus, while the vast majority have failed to demonstrate an association. It is speculated that the inflammatory response may be triggered by an infectious agent. If a connection between GCA and HPV infection were to be established, the potential clinical implications are great, as GCA, with its possible vision loss -5, polymHuman papillomavirus (HPV) is an increasingly common human pathogen in recent decades. It is a mucosotropic virus which is not thought of as spreading systemically. However, HPV genotype 16 has been found integrated into the genome of bacteria isolated from cervical cancer biopsies, and there is also published data showing HPV viral particles within peripheral nerves and small vascular endothelial cells adjacent to oral and cervical cancers as demonstrated by transmission electron microscopy ,10. In tWith approval by the Washington University Human Research Protection Office (HRPO), we searched the Copath database of Barnes Jewish Hospital for temporal artery biopsy specimens from 1995 to 2008 and retrieved all specimens for which material was available. We identified approximately 60 cases, of which 43,  had material available for review. There were five males and 17 females in the histologically positive GCA group with a mean age of 78.9 years. In the histologically negative group, there were four males and 17 females with a mean age of 67 years, and the control kidney vascular margin group had six males and nine females with a mean age of 38.2 years. There were no statistically significant difference in gender among the groups (p = 0.176). The clinical diagnosis of GCA was based on the criteria of the American College of Rheumatology . HoweverTM HPV HR  assay.Two methods were utilized to evaluate for HPV DNA. The first, INNO-LiPA HPV Genotyping Extra, was used to perform the testing in our research laboratory of the division of anatomic and molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis. All blocks were then submitted to an outside laboratory  for independent preparation of genomic DNA and for testing employing the CervistaIn both laboratories to avoid potential contamination, a maximum physical separation between the pre- and post-amplification steps was used. Separate pipettes and other lab materials were used as a part of good laboratory practice. The FFPE blocks were cut and processed under strict conditions to prevent DNA from being carried over from one case to the next during microtomy. Also a new blade was used for each case and the area was cleaned. Ice cubes used to cool blocks were discarded between cases.With regards to the technical limitations of this study, it is well known that formalin fixation randomly fragments DNA in a duration-dependent manner, resulting in a partial degradation. The degree of fragmentation depends on the type and age of the sample and the conditions used for fixation. Due to this degradation, FFPE tissue is not suitable for amplification of large DNA segments. Nevertheless, PCR amplification of segments ranging up to 1300 base pair has been reported. On the other hand, incubation at an elevated temperature after proteinase K digestion partially removes formalin cross-linking of the DNA, improving yield as well as DNA performance in assays. Furthermore, in one of the techniques used in this study (INNO-LiPA HPV Genotyping), short-PCR-fragment (SPF 10) primers are employed, which amplify a 65 base pair segment of target DNA and this testing is considered to be one of the most sensitive PCR assays for the detection of HPV DNA.http://www.Gentra.com) as per the manufacturer's instructions from 10 μm sections cut from the paraffin blocks. The concentration of the prepped DNA was measured spectrophotometrically using Nanodrop. Detailed procedure information is available at their web site, but briefly, we placed five 10 micron sections of tissue into a 1.5 mL microcentrifuge tube and added 1.0 mL of xylene, vortexed, and incubated for five minutes with constant gentle mixing. Then we centrifuged it for five minutes at 13,000-16,000 x g. In the fume hood, discarded by pipetting the xylene supernatant and left behind the visible pellet (tissue). We repeated this xylene wash twice. We then added 1.0 mL 100% ethanol, vortexed, and incubated five minutes with constant gentle mixing at room temperature, centrifuged at 13,000-16,000 x g for five minutes to pellet the tissue, and discarded the ethanol. We repeated these ethanol washes twice. Subsequently we added 1.0 mL 70% ethanol, gently mixed, and centrifuged at 13,000-16,000 x g for five minutes at 4°C, removed all residual ethanol and allowed tissue pellet to dry by centrifugation under vacuum for five minutes. For cell lysis, we added 300 μl Cell Lysis Solution (Gentra Puregene™ kit) and gently vortexed for 30 seconds. Then we added three μl Puregene Proteinase K (20 mg/ml), and mixed by inverting 25 times and incubated the lysate at 55°C for three hours to overnight. We inverted the tube periodically during the incubation. Then was added three μl RNase A Solution to the cell lysate, and mixed by inverting the tube 25 times and incubated at 37°C for 15 min to one hour. For Protein Precipitation we cooled quickly the sample to room temperature by placing on ice and added 100 μl Protein Precipitation Solution (Gentra Puregene™ kit) to the cell lysate. The volume should be 1/3 of the Cell Lysis Solution in the tube. Subsequently, it is vortexed vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution uniformly with the cell lysate. Then, we centrifuged it at 13,000-16,000 x g for five minutes. The precipitated proteins formed a tight pellet. If the protein pellet was not tight, we vortexed vigorously for 20 seconds at high speed, and then incubated on ice for 5 min. We then centrifuged at 13,000–16,000 x g for three minutes. Using a pipette, we removed the supernatant containing the DNA (leaving behind the precipitated protein pellet) into a clean 1.5 mL microcentrifuge tube. We added two μl (for 400 μl supernatant) of a DNA carrier  to aid recovery of small DNA quantities and then vortexed them. We then added 400 μl 100% of isopropanol. Subsequently, it was mixed by inverting gently ~50 times until the white threads of DNA formed a visible clump, and then it was centrifuged at 13,000-16,000 x g for 10 minutes and the supernatant was poured off. We added 500 μl of 70% ethanol and inverted the tube to wash the DNA pellet. We centrifuged at 13,000-16,000 x g for 5 minutes, and carefully poured off the ethanol and inverted and blotted the liquid from the tube on clean absorbent paper and allowed to air dry for 10-15 minutes. Finally, we added 50 μl DNA Hydration Solution and incubated at 65°C for one hour to dissolve the DNA. We incubated at room temperature overnight with gentle shaking. Samples could then be centrifuged briefly and transferred to a storage tube. The concentration of the DNA used for INNO-LiPA HPV Genotyping Extra testing in each case was 50 ng.DNA was extracted using PureGene Kit  genome is amplified using short-PCR-fragment assay (SPF10 primers), and the resulting biotinylated amplicons are then denatured and hybridized with specific oligonucleotide probes.The INNO-LiPA HPV Genotyping An additional primer pair for the amplification of the human HLA-DPB1 gene is added to monitor sample quality and extraction. The length of the HLA-DPB1 fragment is 280 base pairs. All probes are immobilized as parallel lines on membrane strips. After hybridization and stringent washing, streptavidin-conjugated alkaline phosphatase is added, which binds to any biotinylated hybrid previously formed.Extra Amp) is used for standardized preparation of biotinylated amplified material. This amplification kit is based on the polymerase chain reaction (PCR) using SPF10 primers.Incubation with BCIP /NBT (Nitro-Blue Tetrazolium Chloride) chromogen yields a purple precipitate, and the results are visually interpreted using the reference guide provided. An amplification kit  as described by the manufacturer  was used for purification of genomic DNA from formalin-fixed, paraffin-embedded tissues according to the manufacturer’s instructions with exception of incubation time. Overnight incubation in proteinase K for digestion of proteins/contaminants is not recommended by Qiagen but is something that CPA Laboratory has found useful to increase the nucleic acid elution.Slides were cut from the original blocks, and The QIAamp DNA FFPE Tissue kit  bind to the target DNA recognition site. When those sequence specific oligonucleotides overlap by at least one base pair on the target sequence, an invasive structure forms that acts as a substrate for the Cleavase® enzyme. The enzyme cleaves the 5’ portion (flap) of the probe at the position of the overlap.CervistaM HPV HR  is a qua6– 107 fold signal amplification per hour. The reagents for this test are provided as three oligonucleotide master mixtures, which identify the 14 types of HPV arranged according to phylogenetic relatedness. Master mixture 1 identifies the positivity of genotypes 51, 56 and 66. Master mixture 2 (MM2) shows the positivity of genotypes HPV 18, 39, 45, 59 and 68 and master mixture 3 (MM3) reveals the positivity of HPV genotypes 16, 31, 33, 35, 52 and 58. By design, the released 5'-flaps bind only to their respective FRET oligonucleotides to generate target-specific signal. A positive result indicates that at least one of the 14 high-risk types is present in the DNA sample. For each case 150 to 200 ng of DNA prepped from the FFPE tissues at CPA Labs were utilized for the Cervista HPV testing. The sensitivity of this test is set at 5000 copies of HPV DNA. The Cervista assay has an internal control to verify if the assay worked. If the internal control does not pass, it suggests either the sample is degraded or extraction failed or the PCR did not work.The probes are present in large molar excess and cycle rapidly on and off the target sequence generating many cleaved 5’ flaps per target sequence. The cleaved flaps then bind to a universal hairpin fluorescence resonance energy transfer (FRET) oligonucleotide creating another invasive structure that the Cleavase® enzyme recognizes as a substrate. The enzyme cleaves the FRET oligonucleotides between the fluorophore and quencher molecule and produces a fluorescence signal as the cleaved flaps cycle on and off. For each copy of target, the combined primary and secondary reactions result in 10T-test and Fisher's exact tests to evaluate the correlation between the presence of HPV DNA and other variables. P values < 0.05 were considered significant. All statistical analyses were performed using the SAS v.9.1 system software .Statistical analyses were performed using the Chi-Square, Extra, 16 of the 22  biopsy-confirmed GCA cases were positive for HPV. Twelve out of the 16 (75%) HPV-positive GCA-positive patients were female. Among these 16 positive cases, 5 had a HPV 6 alone (31%), 3 cases had HPV 16 alone (19%), 7 cases had both types 6 and 16 (44%), and only one case was extensively "multi-genotype" showing types 6, 16, 31, 33, and 40 (6%) . Two cases which were HPV type 6 by INNO-LiPA (cases # 4 and 6) had low genomic DNA when prepped for the CervistaTM HPV HR testing, so could not be evaluated by this method. All positive cases of the CervistaTM HPV HR were from the Master Mix 3 which includes the HPV genotypes 16, 31, 33, 35, 52 and 58. As expected, all cases for which genotype 6 was present in the INNO-LiPA method were negative with the Cervista method since low risk types are not evaluated in that assay. All negative cases with INNO-LiPA HPV Genotyping Extra were negative also with CervistaTM HPV HR testing. As such, there was 100% agreement between the two testing methods on the histologically positive temporal arteritis cases.Using the INNO-LiPA HPV Genotyping Extra, of which 3 (60%) were genotype 16, one (20%) was genotype 6 and one (20%) was genotype 52. CervistaTM HPV HR testing revealed positivity of Master Mix 3 for all 4 cases which were positive by INNO-LiPA HPV Genotyping Extra for high risk HPV. Case number 2 which was negative with INNO-LiPA was of low genomic DNA with Cervista method. As such, there was 100% agreement between the two testing methods on the histologically negative temporal artery biopsy cases.Among the 21 cases of GCA histologically negative biopsies, 5 were positive for HPV by INNO-LiPA HPV Genotyping Extra. Two cases showed genotype 16 (1 case of Wilms' tumor and 1 of acute and chronic pyelonephritis), and one genotype 6 . CervistaTM HPV HR testing revealed positivity of Master Mix 3 for both cases which were positive for high risk HPV by INNO-LiPA HPV Genotyping Extra. As such, there was 100% agreement between the two testing methods on the 15 vascular margin control cases.Among the 15 nephrectomy vascular resection margins (controls), 3 were positive for HPV by INNO-LiPA HPV Genotyping The association between HPV positivity and histologically confirmed GCA was statistically significant (p = 0.0013). There was also a statistically significant association between HPV positivity and Caucasian race (p = 0.0339). No other associations were statistically significant.In multivariate analysis Table , subjectGiant cell  arteritis is a chronic vasculitis involving medium and large size arteries that typically affects individuals older than 50 years of age.,3,4 AlthHistologically, the disease progresses from minimal involvement of vessels, with only collections of lymphocytes confined to the internal or external elastic lamina or adventitia to a panarteritis with segmental areas of necrosis of the arterial wall and extensive destruction of the elastic laminae, a feature that can be clearly demonstrated with special stains for elastic fibers Figures  and 2.The etiology of the inflammatory reaction in the GCA has not been identified. However, it has been demonstrated that there is a restricted clonal expansion of tissue-infiltrating T cells in these lesions, which suggests that they are reacting to a specific antigen located within the affected arterial wall which is thus eliciting the disease . The mosChlamydia pneumoniae, Mycoplasma pneumoniae , and Parvovirus B19 [C. pneumonia and herpes simplex virus (HSV). However, further studies were unable to detect the presence of these infectious agents. Cooper et al. [C. pneumonia, HSV, varicella zoster virus (VZV), Epstein–Barr virus (EBV), or human herpesvirus 7 (HHV7). In addition, Rodriguez-Pla et al. [C. pneumoniae in temporal artery GCA. Due to this conflicting and contradictory data, no conclusive link has, to date, been demonstrated between a GCA and a specific infectious agent.Infectious agents have been considered in the past as the etiology of GCA. Elling et al., by indirect serological evidence, found high incidence of GCA within a population associated with irus B19 . Publishirus B19  and Poweirus B19  reportedr et al.  and Cankr et al. , could na et al.  and othea et al. -23 couldThe role of human papillomavirus (HPV) in the development of cervical, head and neck, anal and skin cancers is well known. Molecular and epidemiological studies have shown that persistent HPV infection is the most important risk factor for cervical cancer ,25. HighEnterococcus , Staphylococcus , Bacillus and Corynebacterium) and demonstrated the HPV viral particles by transmission electron microscopy in those bacteria. In addition, HPV type 16 has been detected in peripheral nerves, and vascular endothelial cells [Interestingly, Ma et al.  reportedal cells . Anotheral cells . The rolal cells -32.In this study, we found that HPV DNA, of both high and low risk types, is present in formalin-fixed, paraffin-embedded temporal artery biopsy specimens, and in statistically significantly higher numbers in the arteritis specimens compared to histologically-negative temporal artery biopsies and nephrectomy vascular margin controls. The validity of these results was confirmed with exactly matching results by an outside laboratory that independently prepped DNA from the paraffin blocks and utilized a completely different detection method. Our results are admittedly surprising and bring many questions about what HPV's role, if any, would be in temporal arteritis. HPV has been associated with tumorigenesis, and many studies have investigated HPV-related modification of the immune system to establish infection. However, we are not aware of any literature citing HPV as an inciting agent for inflammatory diseases. Our findings do support the notion that HPV can disseminate systemically, but do not, by themselves, tell us anything specific about the pathophysiological relationship between the virus and temporal arteritis, if there even is one. Further studies are necessary to address this.It is important to mention that a negative temporal artery biopsy does not rule out the diagnosis of temporal artery GCA, since the changes can be patchy with skip areas of uninvolved artery. Although serial sectioning of the temporal artery biopsy is recommended, not all cases will be diagnosed histologically . ActuallIt is also worth noting that of the 16 histologically-proven GCA cases that had HPV, 11 were high risk genotype 16. Only five cases were positive for low risk HPV genotype 6. Although, the importance of distinguishing low risk from high risk HPV genotypes has been established in the pathogenesis of neoplasms, what its significance might be in inflammatory disorders such as GCA is currently unknown.In summary, we have identified HPV DNA in the majority of histologically-proven giant cell  arteritis specimens and validated these results by a completely independent outside laboratory assay. The association raises questions regarding the biology of HPV infections, when dissemination occurs, and what this dissemination means clinically. Such studies are required to understand the significance of the association in our series.The authors declare that they have no competing interests.AM conceived the study, carried out the molecular testing, analyzed the data, and drafted the manuscript. JDP and JSL, designed the study, collected data, helped interpret findings and critically revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/13/132/prepub


P > 0,05). The mean lumbar lordosis angle was 54,71 preoperatively and 53,18 postoperatively. Regarding the preoperative and postoperative measurements of lumbar lordosis angles, no statistically significant difference was found between the groups . Although breast size may be an important factor that affects body posture, reduction mammaplasty operations have little or no radiologic effect on the vertebral column.Some studies emphasized that anatomic mechanisms of vertebral aberrations could be associated with large breasts. The effect of mammaplasty operation on the vertebral column and body posture seems to be beneficial; in this trial, it was planned to investigate the objective radiologic effect of reduction mammaplasty on the posture of the vertebral column in a group of patients operated due to the large breasts. Thirty-four white women with large breasts were enrolled in this study. The patients were divided into three groups according to their breast cup sizes. Anteroposterior and lateral radiographs of the lumbosacral and thoracic spine were taken at baseline preoperatively, and the same radiographic images were taken in an average of 12 months later than the reduction mammaplasty operation. All were evaluated and compared for thoracic kyphosis angle and lumbar lordosis angle both preoperatively and postoperatively. The mean thoracic kyphosis angle was 40,53 preoperatively and 39,38 postoperatively. However, there was no statistically significant difference between the preoperative and postoperative measurements in all groups ( Hypertrophic large breasts are common and may be causative factor for impairment of spine function. The most common symptoms of women suffering from hypertrophic breasts are malposture, painful shoulder grooves, persistent submammary intertrigo, and back pain , 2. ReduIn a study, it was documented that breast size seems to be an important factor that affects body posture, especially the thoracic kyphosis and lumbar lordosis angles . The resThirty-four patients with breast hypertrophy were referred to our clinic for evaluation and appropriate therapy in the period of September 2010 and March 2011. We received approval by the Local Hospital Ethical Committee and also obtained the informed consent from the patients before study. The patients having at least one of the following criteria were excluded from the study: (1) systemic or vertebral disease who had undergone spinal surgery, (2) using any medication (3) patients <18 years of age or postmenopausal patients  , 10 and n = 10), Group II: D cup size (n = 11), and Group III: DD cup size (n = 13). There was no patient with A or B breast cup size. For radiologic evaluation, preoperatively anteroposterior and lateral radiographs of the thoracic and lumbosacral regions were obtained with the patients in a relaxed upright standing position without shoes. Body mass index (BMI) was calculated for each patient with using the following formula: weight/height2. The cup sizes of the breasts for every patient by calculating the difference between the overbust and underbund measurements are as follows: <6,5 cm (A cup), 6,5–13 cm (B cup), 13–19 cm (C cup), 19,5–26 cm (D cup), and >26 cm (DD cup).Age, weight, height, and preoperative cup sizes of the breasts and weight of resected tissues were recorded for every patient. Thirty-four patients were divided into three groups according to the preoperative breast cup sizes—Group I: C cup size  and the lumbar lordosis angle (the angle between the superior end plate of the first lumbar vertebra and the superior line of the sacrum) were measured by using Cobb's method [U test, and Pearson correlation analysis by using computerized statistical software  were used. All results are presented as means, ranges, and standard deviations. Differences between the groups in terms of these angles and clinical parameters such as age, BMI, and resected tissue volumes were analyzed and compared.For the statistical analyses of differences between three groups, Kruskal Wallis, Mann-Whitney P > 0,05).We performed a study of 34 women with various breast sizes. The mean age was 33,7 years . The age distribution was homogenous between the groups and there was not statistically significant difference (The breast cup size was C in 10 patients (Group I), D in 11 patients (Group II), and DD in 13 patients (Group III). For Groups I, II, and III, a balanced representation of women with respect to breast cup size  was observed.2 (range 26–34). There was no statistically significant difference between the three groups in terms of BMI   .P < 0,05); however, there was no significant difference between Groups II and III   of tissue was resected per breast. There was a statistically significant difference between the groups in terms of resected tissue volumes, as it was significantly lower in Group I  .P < 0,05). However, there was no significant difference between the preoperative and postoperative measurements in all groups   preoperatively and 39,38° ± 5,35° (range 25°–49°) postoperatively. Regarding the preoperative and postoperative thoracic kyphosis angles, the lowest score was found in Group I, and it was statistically significant  .P > 0,05)  preoperatively and 53,18° ± 7,95° (range 40°–70°) postoperatively. Regarding the preoperative and postoperative measurements of lumbar lordosis angles, there was no significant difference between the groups  .Overall, the complication rate was low. Delayed wound healing was observed in 2 patients, fat necrosis was observed in one, and postoperative infection developed in one patient.Malposture, painful shoulder grooves, submammary intertrigo, and back pain are the most common symptoms of women suffering from breast hypertrophy . Some auIn women presenting with reduction surgery, nonsurgical approaches including weight loss, physical therapy, special brassieres, and medications were not effective in providing permanent relief of macromastia-related symptoms . The maiIt was clearly documented that breast reduction mammaplasty is a good treatment for reducing symptoms of macromastia . DeformiIn a cohort study, Findikcioglu et al. emphasized that breast size seems to be an important factor that affects body posture, especially the thoracic kyphosis and lumbar lordosis angles . Large bTo the best of our knowledge, this is the second study quantitatively documenting the effect of breast reductions on the spinal posture. In a recently published paper, Findikcioglu et al. studied the same subject and showed that breast reduction surgery causes significant decrease in angles including thoracic kyphosis, lumbar lordosis, and sacral inclination . AlthougReduction mammaplasty is still one of the operations with the highest patient satisfaction. Although this radiologic study shows that reduction mammaplasty operations seem to have little or no radiologic effect, we still believe that this surgery is a medical necessity for patients with large breasts.


Barbus neumayeri from two river drainages in Uganda: the Rwembaita Swamp-Njuguta River System and the Dura River. We then examined two indices of population structure, GST and Jost's D, to detect links between oxygen availability and genetic variation and to determine if population genetic structure was associated with (i) dissolved oxygen regime (hypoxia or normoxia), (ii) geographical distance, or (iii) a combination of dissolved oxygen regime and geographical distance. Our results indicate that over a large scale (between drainages), geographical distance significantly affects the genetic structure of populations. However, within a single drainage, dissolved oxygen regime plays a key role in determining the genetic structure of populations. Within the Rwembaita-Njuguta system, gene flow was high between locations of similar oxygen regimes, but low between areas characterized by divergent oxygen regimes. Interestingly, GST analyses appear to yield less realistic measures of population structure than Jost's D, suggesting that caution must be taken when interpreting and comparing the results from different studies. These results support the idea that aquatic dissolved oxygen can act as a selective force limiting gene flow among populations of aquatic species and therefore should be considered when implementing conservation plans and assessing environmental impact of human activities.Population level response to hypoxia has become an issue of global significance because of increased frequency and intensity of hypoxic events worldwide, and the potential for global warming to exacerbate hypoxic stress. In this study, we sequenced two nuclear intronic regions and a single mitochondrial region across seven populations of the African cyprinid,  Nephelopsis obscura) residing in lakes with different dissolved oxygen (DO) regimes, were more consistent with oxygen acting as a selective agent than geographical distance. Similarly, at least two populations of the common skate (Dipturus batis) have been found to be reproductively isolated around the British Isles  dissolved oxygen regime (hypoxia or normoxia), (ii) physical barriers such geographical distance, and/or (iii) both oxygen regime and geography. Nuclear introns accumulate change at a relatively fast rate  and can often be easily amplified from a species of interest using primers designed from the relatively conserved flanking coding regions.Several recent studies have demonstrated strong patterns of interdemic variation in fish populations between low- and high-oxygen environments, particularly in traits related to oxygen uptake such as gill size  and geographical distance. The two main rivers Dura River and the Mpanga River, both eventually feed into the Lake George system . Basic pFish were sampled during the rainy season in January 2010 using baited minnow traps, and body mass and total length were measured in a small plastic bag with water. Pectoral fin clips were taken and stored in 95% ethanol for DNA analyses. Once the fin clips were sampled, fish were allowed to recover in a bucket of well-oxygenated water from their habitat, and they were released at the location of capture. Genomic DNA was extracted using the DNeasy Tissue Kit , and DNA concentration was estimated using an ND-8000 NanoDrop spectrophotometer .We used Polymerase chain reaction (PCR) to amplify two nuclear and one mitochondrial gene regions. The nuclear and mitochondrial loci have demonstrated utility in resolving question of population structure in other studies  nuclear locus was amplified using an oligonucleotide primer set previously optimized for various fish species, including cyprinids (Quattro and Jones The fourth intron of the Triosephosphate Isomeras A (TPI-A) nuclear locus was amplified using locus specific primers designed in a similar fashion to the CK primers (J. M. Quattro pers. comm.). Amplification of sufficient product to allow direct sequencing required semi-nested primary and secondary PCR reactions. The primer sequences are as follows: TPIDF1: 5′-YTG ATY GGS CAG AAG GTG GC-3′; 302 – TPIB4 R1: 5′-AGA ACC ACY TTR CTC CAG TC-3′; TPI4F2 – P301: 5′-GCA TYG GGG AGA AGC TRG AT-3′. The primary amplification was from TPIDF1 to 302 – TPIB4 R1. From this reaction, 2 μL of product was diluted in 100 μL water and 2 μL of this solution was used as template in a second PCR reaction from TPI4F2 – P301 to 302 – TPIB4 R1. In both primary and secondary reactions, the PCR parameters were as follows: an initial denaturation of 2 min at 90°C followed by 44 cycles of 45 sec at 90°C, 60 sec at 48°C, 60 sec at 72°C and a final extension time of 7 min at 72°C.A fragment of the 16S mitochondrial locus was amplified using “universal” primers: 16S AR: 5′-CGC CTG TTT AAC AAA AAC AT-3′; 16S BR: 5′-CCG GTC TGA ACT CAG ATC ACG T-3′ , nucleotide diversity (π), GST and FST-values were all calculated using DnaSP version 4.10  with a final visual optimization of the alignment. The number of haplotypes (within and between location), haplotype diversity , while the Dura River had the lowest diversity (Hd = 0.189). The maximum number of mutational steps between any of the haplotypes was four, although this maximum was only observed between a single individual from Mikana Stream and the haplotype B. The most common haplotype for this intron was haplotype A, found in 42 of 135 individuals . This pattern of FST showing similar, but not significant, patterns of differentiation to those in the GST analysis is consistent across all three loci and we have therefore presented only the GST values. Values for Jost's D dissimilarity ranged from −0.112 to 1.000, indicating variability between the Dura River population and all other populations . Due to the large distance between the Dura River population and all the other populations, additional analyses were conducted without the Dura River population. In the absence of the Dura River population, both GST and Jost's D were associated with the geographical distance in pairwise comparisons . By contrast, we did not find a significant relationship between either genetic metric and dissolved oxygen at this locus, even when the Dura River was not included in the analysis.Overall, we found a significant positive relationship between geographical distance and both Hd = 0.763), while the Dura River was completely fixed at this locus with a haplotype diversity of 0.000. The maximum number of mutational steps between any of the nine haplotypes obtained was two. The most common haplotype for the TPI intron was haplotype B found in 66 of 134 individuals .Similar to CK, ulations . Values pulation . AdditioR2 = 0.38, P < 0.0001 and R2 = 0.46, P < 0.0001 for GST and Jost's D, respectively). Nevertheless, when the Dura River was removed from the analysis, these relationships disappeared . By contrast, the relationships between the genetic metrics and dissolved oxygen between locations were significant when the Dura river was included , but stronger when this population was excluded from the analysis . The Mikana Stream, Njuguta River, and the Rwembaita Swamp all had only a single 16S haplotype and an Hd of 0.000. The most common 16S haplotype was haplotype A found in 112 of 134 individuals (83.6%). Haplotype B was observed in 17 of 134 individuals (12.7%) and only in individuals from the Dura River. The remaining haplotypes were found only in single individuals.We sequenced a 555 bp fragment of the 16S locus and identified seven haplotypes  with an GST and Jost's D analyses suggest significant genetic differences at this locus between some of the populations . However, no correlation was observed between geographical distance and either GST (P = 0.363) or Jost's D (P = 0.834) when the Dura river was excluded from the analysis. Additionally, there was no correlation between the genetic metrics and dissolved oxygen even when the Dura River was removed from the analysis.Similar to the results from the nuclear loci, there was a positive and strong correlation between geographical and genetic distance in the 16S data set . This isolation suggests that habitat-specific selection pressures may contribute to reduced gene flow , as has been suggested in other studies . Such discrepancies may cause misleading conclusions with respect to population structure leading to inappropriate management implications and we therefore advocate for a better investigation of the properties associated with different metrics used to estimate genetic differentiation. Furthermore, it is clear that GST may yield less realistic measure of population differentiation than Jost's D and caution must be taken when interpreting and comparing the results from different studies. Recently, Raeymaekers et al. (D and GST in distinguishing population genetic structure and conclude that no single measure can quantify all aspects of genetic structure. The differences in resolution observed here between D and GST are consistent with this conclusion. Interestingly, both their empirical and theoretical work suggest that D is more useful in distinguishing longer term processes, and GST in recent demographic events, while our empirical results appear to point in the opposite direction. This difference in conclusion suggests that either the separation of the B. neumayeri populations that we have studied is more complicated than we appreciate, or, that more work is necessary to completely understand the difference in performance of these two metrics.This study also allowed us to compare and contrast the results from distinct metrics of genetic structure. Some limitations that have been pointed out for s et al.  empiricaB. neumayeri across dissolved oxygen regimes. We also observe strong genetic differentiation of the Dura River location from all other sites, likely a function of the large swimming distance and the presence of geographical barriers between the Dura River location and all of the other samples sites. The remaining six sampling locations, although geographically close to each other, present levels of genetic divergence associated with dissolved oxygen regime. Earlier studies on the biochemistry of this system found significant differences between different oxygen regimes (Martínez et al. Our study detected significant population differentiation in the African cyprinid