Mycobacterium tuberculosis culture isolation and drug sensitivity testing (DST) using solid (LJ) and liquid (BACTEC-MGIT-960) media in Nigeria.This study compares This was a cross sectional survey of adults attending reference centres in Abuja, Ibadan and Nnewi with a new diagnosis of pulmonary tuberculosis (TB) or having failed the first-line TB treatment. Patients were requested to provide three sputum specimens for smear-microscopy and culture on LJ and BACTEC-MGIT-960. Positive cultures underwent DST for streptomycin, isoniazid, rifampicin and ethambutol.527 specimens were cultured. 428 (81%) were positive with BACTEC-MGIT-960, 59 (11%) negative, 36 (7%) contaminated and 4 (1%) had non-tuberculosis mycobacteria (NTM). 411 (78%) LJ cultures were positive, 89 (17%) negative, 22 (4%) contaminated and 5 (1%) had NTM. The mean (SD) detection time was 11 (6) and 30 (11) days for BACTEC-MGIT-960 and LJ. DST patterns were compared in the 389 concordant positive BACTEC-MGIT-960 and LJ cultures. Rifampicin and isoniazid DST patterns were similar. Streptomycin resistance was detected more frequently with LJ than BACTEC-MGIT-960 and ethambutol resistance was detected more frequently with BACTEC-MGIT-960 than LJ, but differences were not statistically significant. MDR-TB was detected in 27 cases by LJ and 25 by BACTEC-MGIT-960 and using both methods detected 29 cases.There was a substantial degree of agreement between the two methods. However using the two in tandem increased the number of culture-positive patients and those with MDR-TB. The choice of culture method should depend on local availability, cost and test performance characteristics. Mycobacterium tuberculosis complex (MtbC) is the accepted reference standard for confirmation of TB infection and is necessary for drug susceptibility testing (DST). There are several methods for culturing MtbC using solid and liquid media. Although solid media has been used for over 100 years, liquid culture media is increasingly being introduced in low and middle income countries (LMIC). Solid culture media is cheaper and more widely available, but is labour-intensive, less sensitive and slower than liquid culture [Culture of  culture . Liquid th among the high MDR-TB burden countries. The Nigerian TB and Leprosy Control Programme (NTBLCP) has given high priority to improve the identification and treatment of cases with MDR-TB and has recently commissioned the development of diagnostic and treatment facilities in referral centers across the country. DST capacity however remains limited because most of these facilities are concentrated in large urban centers.Nigeria, the most populous country in Africa, has one of the largest burdens of TB. Clinical response to treatment is often less than optimal and WHO estimates that 2.2% and 9.4% of new and re-treatment TB cases have Multi drug resistant TB (MDR-TB), ranking 15Despite the increasing availability of DST facilities in West Africa, there are very few head-to-head comparisons of the MtbC DST patterns obtained by liquid and solid systems, to inform laboratories considering whether to replace one method for the other or to run the two in parallel.Here we compared the DST results obtained when specimens were cultured using solid  and an automated liquid culture media (BACTEC Mycobacterium Growth Indicator Tube (MGIT) 960).This cross sectional study was conducted in Abuja, Ibadan and Nnewi, in Nigeria. The aim was to compare the isolation rate of MtbC from sputum using LJ and the BACTEC-MGIT-960 and then compare the level of agreement of DST to first-line anti-TB drugs using each type of media. Adults (>18 years old) with sputum smear-positive microscopy were enrolled between August 2009 and July 2010. Both newly diagnosed patients and those who had failed first-line treatment (patients continuing to be smear-positive after 5 months of treatment) were prospectively enrolled until 500 new and 100 re-treatment patients were enrolled. Patients enrolled were not receiving treatment at the time of enrolment. Exclusion criteria included patients unable to produce sputum or to provide informed consent to participate and smear-negative cases. The sample sizes were calculated to assess the prevalence of drug resistance in new and retreatment patients, as described in Lawson et al. . SpecimeThree sputum samples were submitted from each patient for direct Ziehl Neelsen smear microscopy. If the patient was smear-positive, the best quality specimen (defined as the most muco-purulent) was sent for culture and DST at Zankli Medical Research Laboratory (ZMRL) in Abuja within seven days of collection. Samples were refrigerated prior to shipment and maintained in cold-chain during shipment with ice packs. At ZMRL, specimens were decontaminated using Petroff’s method (4% NAOH) and cultured on LJ and on an automated BACTEC-MGIT-960 system  ,6. Two hPure AFB cultures were tested for their sensitivity to streptomycin (STR), isoniazid (INH), rifampicin (RIF) and ethambutol (EMB) using the proportion method in LJ ,8 and inZankli Medical Research Laboratory undergoes external quality assurance of DST for first-line anti-TB drugs through the WHO Supranational Reference Laboratory Network (SRLN) for BACTEC-MGIT-960 and LJ. The most recent EQA of the laboratory results was 100% for STR, INH, RIF and EMB when using BACTEC-MGIT-960; 100% for STR, RIF and EMB and 80% for INH when using LJ.All data were analyzed using Epi-info . Proportions were compared using Chi squared tests and the degree of agreement was compared using the Kappa statistic. Ethical approval was obtained from the Federal Capital Territory Health and Research Ethics Committee and Zankli Ethical Research Review Board. Participants provided their written informed consent to participate, following the procedures approved by the ethics committees.A total of 527 sputum specimens were cultured on LJ and the BACTEC-MGIT-960. The mean  age of the patients was 34 12, 7–82) years, with 315 (60%) being male. The mean (range) time to detection was 11 (1–33) and 30 (7–56) days for BACTEC-MGIT-960 and LJ, respectively. The results stratified by the participating centres are shown in Table 2, 7–82 yFor the purpose of the head to head comparison, only patients examined with results for both culture systems were included in Table DST was conducted on both BACTEC-MGIT-960 and LJ media in the 389 concordant culture-positive isolates. The DST results obtained by LJ and BACTEC-MGIT-960 are shown in Figure Twenty nine patients were classified as having MDR by at least one of the two methods. Of these 27 were identified by LJ and 25 by BACTEC-MGIT-960 (Kappa 0.876).Tuberculosis drug resistance is increasing in many LMICs and Nigeria is not an exception ,9,10. MoNTPs often need to decide on the culture method that should be implemented in the country. Among the aspects that need to be considered are the sensitivity of the method and its susceptibility to contamination. The higher sensitivity of liquid over solid culture is widely accepted  and accoServices should also be aware of the delay of both culture methods which affect clinical management. Although this delay is more prolonged with LJ, it is still significant with liquid culture and clinical decisions are often made before culture and DST results are available for both methods .http://www.who.int/tb/challenges/xdr/xdrconsultation/en/index.html). If the results of both methods were considered valid, these discrepancies would result in the identification of a higher number of drug resistant strains with important cost and patient care implications. This is thus an area that requires further research.Liquid culture has been reported to detect more cases of first-line drug resistance than LJ by some  but not Another consideration for choosing solid or liquid culture is the cost of the systems and the availability of consumables. Liquid culture is several times more expensive than solid media and although consumables for LJ media are universally available in Nigeria, there is only one supplier for BACTEC-MGIT-960 and we have experienced difficulties maintaining stocks. The test performance characteristics, its overall cost and availability therefore have important implications for deciding the DST method of choice in a country. In our context, and in other settings with limited resources, laboratories should be encouraged to develop solid culture capability and to add liquid culture only when the resources required become available.The study reported here has several limitations. We only included patients with smear-positive disease and excluded smear-negative cases. This was based on the algorithm for testing in Nigeria, in which patients with smear-negative results are not routinely cultured. Smear-negative but culture-positive patients however would benefit from DST and it is possible that their exclusion could lead to some bias. This bias could be due to the yield of culture (as liquid culture may be more sensitive in smear-negative patients) and the performance of DST, as patients with paucibacillary TB or milder forms of the disease could have different DST patterns than those with high bacillary loads.In conclusion, liquid culture is more sensitive than solid culture, but there was a high degree of agreement between the two systems. Their simultaneous use resulted in a higher recovery rate and detection of more drug resistance than using only one of the two methods. The mean time to detection was shorter with liquid culture and its monitoring can be automated to facilitate dealing with large number of specimens. However this method is more costly and prone to contamination . Solid aThe authors have no conflict of interest to declare. Beckton Dickinson did not participate in the analysis, interpretation or reporting of this study.LL conceived the study, conducted the statistical analysis and prepared the initial protocol. NE carried out the laboratory culture and DST at ZMC and participated in the drafting of the manuscript. STA and JOL participated in the design of the study and facilitated the study implementation in Abuja. GNU, OMS, JNE identified and recruited patients in the participating hospitals, CMP provided expert mycobacteriology expertise and interpretation of the data; MAY participated in the protocol development, data analysis and drafting the manuscript and LEC supported protocol development, data analysis and interpretation and prepared the final manuscript. All authors read and approved the final manuscript.


Since rupture-prone plaques commonly contain high amounts of activated macrophages, imaging of the macrophage content may provide a useful tool for the evaluation of plaque vulnerability. The purpose of this study was to explore the uptake of 68Ga was investigated in atherosclerotic LDLR-/-ApoB100/100 and C57BL/6N control mice at 3 h after injection. The ex vivo biodistribution of the 68Ga was assessed and autoradiography of aortic cryosections was defined. In vivo imaging of 68Ga was performed using a small animal positron emission tomography PET/CT scanner.Uptake of ionic 68Ga-radioactivity was higher in atherosclerotic plaques than in healthy vessel wall  and adventitia . The autoradiography signal co-localized with macrophages prominently as demonstrated by Mac-3 staining. In both mice strains, the highest level of radioactivity was found in the blood.Our results revealed that the uptake of 68Ga-radioactivity uptake in the aortic plaques of atherosclerotic mice, especially at the sites rich in macrophages. While the uptake of 68Ga was promising in this animal model, the slow blood clearance may limit the usability of 68Ga as a PET tracer for clinical imaging of atherosclerotic plaques.We observed a moderate but significantly elevated  Atherosclerosis is a chronic inflammatory disease of artery wall with infiltration of monocytes into subendothelial space and their differentiation into macrophages. Macrophages express pro-inflammatory and chemotactic molecules thus playing an essential role in the formation, progression, and pathogenicity of atherosclerotic plaques. The population of macrophages in atherosclerotic lesions is heterogeneous. The majority of macrophages transform into foam cells by accumulating lipids in their cytoplasm, while other macrophages may have vascular protective and repair properties. Furthermore, some macrophages may secrete enzymes that degrade the extracellular matrix and fibrous cap, possibly leading to a rupture of the atherosclerotic plaque. Significantly higher quantities of macrophage-rich plaque areas have been reported in atherosclerotic patients with acute coronary syndromes than in patients with stable angina. Since the rupture-prone plaques commonly contain high amounts of activated macrophages, imaging of the macrophages may provide a useful tool for determining plaque vulnerability -5.68Gallium  is a generator-produced, positron-emitting radionuclide with a high positron yield (89%) possessing suitable physical properties for in vivo PET imaging. 67Gallium (67Ga) has traditionally been used to image inflammation with gamma cameras. Since 68Ga owns the same chemical characteristics as 67Ga, it is assumed that ionic 68Ga could accumulate at the sites of macrophage-rich areas in inflammatory lesions. Furthermore, 68Ga-based PET may have some added advantages compared to 67Ga-based SPECT, such as a better resolution for the detection of inflammatory plaques fluoro-D-glucose (18F-FDG), we have measured the plaque/wall ratio of 2.7 ± 1.1 approximately in the same aged LDLR-/-ApoB100/100 mice as used in the present study [68Ga-tracers  and 18F-tracers  are not necessarily very much different from each other [68Ga has a higher kinetic energy compared to 18F, which could decrease the imaging spatial resolution, particularly when a small plaque lesion is imaged with a high resolution small PET/CT. Based on these earlier observations, 18F-FDG seems to be more promising PET tracer for imaging of atherosclerotic plaques than 68Ga.In the present study, the plaque/wall ratio of -deoxy-2-Ffluoro-Dch other . However68Ga between the atherosclerotic mice and controls was found in bone and liver. The uptake of 68Ga-radioactivity in the whole aorta of atherosclerotic mice was higher than that in the control mice, but the difference was not significant. The uptake in the inflammatory plaques could partly explain the lower uptake of 68Ga in the liver of atherosclerotic mice. More likely this may be due to liver failure caused by high fat content, which can occur in older LDLR-/-ApoB100/100 mice fed with a Western-type diet. Concerning the uptake of 68Ga in the bone, we assume that in the young control mice (aged 2.5 months), active bone remodeling may have contributed to the higher bone uptake as compared to the older LDLR-/-ApoB100/100 mice (aged 10 to 11 months).Clinical observations indicate that in normal circumstances, the major proportion of the gallium retained in the body is localized in the bone and liver, and that inflammatory lesions alter this biodistribution, frequently causing decreased hepatic uptake . In the 68Ga-radioactivity in inflammatory lesions is complex, and the majority of 68Ga is probably delivered into the inflammatory lesions through the increased permeability at the site of atherosclerotic arteries. In the present study, accumulated 68Ga-radioactivity was detected in the macrophages-rich plaques suggesting that 68Ga accumulates at the site of inflammation. In addition to that, calcified areas of atherosclerotic plaques were also detected by 68Ga, which may be explained by the competitive binding of gallium ions to the Ca2+ and Mg2+ binding sites [68Ga to distinguish macrophages-rich plaques and calcified plaques.The accumulation of ng sites . Since t68Ga in inflamed atherosclerotic plaques was promising as measured by autoradiography, the slow blood clearance limits the use of ionic 68Ga for in vivo imaging of atherosclerotic plaques.To some extent, gallium may also be taken up by atherosclerotic plaques due to its binding to the circulating transferrin and avidly to the transferrin receptors at the site of atherosclerotic arteries. Transferrin receptor expression has been shown to correlate positively with macrophage infiltration in human carotid atherosclerotic lesions . Moreove68Ga-radioactivity uptake in the aortic plaques of atherosclerotic mice, especially at the sites rich in macrophages, as compared to areas of healthy vessel wall. While the uptake of 68Ga-radioactivity was prominent in the atherosclerotic plaques of LDLR-/-ApoB100/100 mice, the slow blood clearance limits the feasibility of ionic 68Ga for clinical imaging of atherosclerotic plaques especially in coronary arteries.Autoradiography analysis showed a moderate, but significantly higher The authors declare that they have no competing interests.68gallium and participated in writing of the manuscript. VJOL involved in designing the analysis and interpretation of data, and in drafting and revising the manuscript. PL involved in designing the experiments and revising the manuscript. SY-H, JK, and AR contributed the conception and design, critically contributed and revised the manuscript and enhanced its intellectual content. All authors have approved the final content of the manuscript.JMUS contributed the conception and design, performed the experiments, analyzed and interpreted data and drafted the manuscript. IL performed the experiments, acquired, analyzed and interpreted data and participated in writing of the manuscript. HJS prepared the


Aim of this study is to summarize and re-evaluate our eight year experience in the application of a water safety plan (WSP) in a 1500-bed teaching hospital.WSP is based on a strategy integrating surveillance, maintenance, continuous chlorine-based disinfection and end-point filtration in critical areas. The strategy includes also molecular typing of the isolates and virulence genes expression analysis to detect the occurrence of adaptative changes in strains colonizing the water system.Recently, monochloramine treatment was applied in selected areas.3 CFU/L. After eight-years of integrated strategy, Legionella was still present but the positive supply points were reduced to 22% (54/241) in the last year, and the samples exceeding 103 CFU/L were cut down to 18%. All isolates were identified as L. pneumophila sg1, two predominant and persisting clones, one of which showing increased chlorine tolerance. Long-time exposure to chlorine enhanced the ability to express more promptly some virulence genes involved in intracellular protozoa infection. The application of end-point filtration in high-risk areas is therefore required until a new effective disinfectant is introduced. Following the substitutions of chlorine dioxide by monochloramine, eradication of planktonic Legionella was observed although long-time effects have to be evaluated.Before the disinfection-filtration strategy, Legionella was isolated in 67% of samples (54/81), 79% of these samples exceeding 10Standard environmental surveillance methods may not be sufficient to determine the most effective disinfection method and should be accompanied by evaluation of the susceptibility to sanitising agents.None declared.


The default mode network (DMN) is a set of brain regions that consistently shows higher activity at rest compared to tasks requiring sustained focused attention toward externally presented stimuli. The cognitive processes that the DMN possibly underlies remain a matter of debate. It has alternately been proposed that DMN activity reflects unfocused attention toward external stimuli or the occurrence of internally generated thoughts. The present study aimed at clarifying this issue by investigating the neural correlates of the various kinds of conscious experiences that can occur during task performance. Four classes of conscious experiences  that varied along two dimensions (“task-relatedness” and “stimulus-dependency”) were sampled using thought-probes while the participants performed a go/no-go task. Analyses performed on the intervals preceding each probe according to the reported subjective experience revealed that both dimensions are relevant to explain activity in several regions of the DMN, namely the medial prefrontal cortex, posterior cingulate cortex/precuneus, and posterior inferior parietal lobe. Notably, an additive effect of the two dimensions was demonstrated for midline DMN regions. On the other hand, lateral temporal regions  were specifically related to stimulus-independent reports. These results suggest that midline DMN regions underlie cognitive processes that are active during both internal thoughts and external unfocused attention. They also strengthen the view that the DMN can be fractionated into different subcomponents and reveal the necessity to consider both the stimulus-dependent and the task-related dimensions of conscious experiences when studying the possible functional roles of the DMN. During the last decade, the default mode network (DMN) of the brain—a network of brain regions that includes the medial prefrontal cortex (MPFC), the posterior cingulate cortex (PCC)/restrosplenial cortex (Rsp), the medial and lateral temporal lobes, and the posterior inferior parietal lobes (pIPL)—has become the object of intensive focus and research in neuroscience Some authors have argued that higher DMN activity corresponds to a shift of perspective from current external information to internally generated cognitions The proposal that DMN activity corresponds to the occurrence of mind-wandering and internal cognitions is not consensual, however. Some authors have argued that DMN activity might support the general, unfocused monitoring of the external environment rather than internal thoughts These two views on the possible function of the DMN  are not necessarily mutually exclusive. It is conceivable that activity in the DMN reflects a general state of unfocused attention in which both internal representations  and task-unrelated external stimuli  are gathered and monitored A number of previous fMRI and PET studies have begun to investigate the possible function of the DMN by examining correlations between brain activity and different types of conscious experiences. Some studies have inferred mind-wandering indirectly, by varying task demands to influence the probability of task-unrelated thoughts and estimating the frequency of mind-wandering episodes by collecting data outside the scanner Although these neuroimaging findings suggest that mind-wandering is associated with the recruitment of both DMN and executive network regions, it should be noted that participants were simply asked to report whether they were totally focused on the proposed task (on-task reports) or were distracted by task-unrelated thoughts (off-task reports). This dual-choice format does not permit to clearly distinguish between mind-wandering, external distractions, and task-related interferences, such that these three types of conscious experiences might have been mixed into the same response category In order to clarify the role of the DMN in mind-wandering versus unfocused external attention, the present study used a factorial design in which participants reported their conscious experiences in terms of both task-relatedness and stimulus-dependency while they performed the SART. Thus, four possibilities of responses to thought-probes were included, corresponding to the four classes of conscious experiences defined above. Our first aim was to investigate the brain regions that were specifically associated with the two defining dimensions of these conscious experiences. Furthermore, we examined the brain regions that were specifically related to (1) task-related interferences, (2) external distractions, and (3) mind-wandering, in comparison to being fully focused on the SART. A conjunction analysis was also performed to identify the brain regions that were more active during mind-wandering compared to both task-related interferences and external distractions. Finally, region of interest (ROI) analyses were performed in order to investigate whether distinct subregions of the MPFC that have been identified in previous studies (see above) are differentially sensitive to task-relatedness and stimulus-dependency. Results of these analyses demonstrate the relevance of our finer-grained conceptualization of ongoing conscious experiences for explaining variations in DMN activity. Both the “task-relatedness” and “stimulus-dependency” dimensions were indeed related to activity in several regions of the DMN, suggesting that these regions underlie cognitive processes that are active during both internal thoughts and unfocused external attention.All participants gave their written informed consent to take part in the study, in line with the Declaration of Helsinki, and the study was approved by the Ethics Committee of the Medical School of the University of Liège.Twenty-two right-handed adults (17 women) aged between 18 and 30 years (mean age = 22 years) participated in the experiment. None of the participants had any history of neurological or psychiatric disorder.The version of the SART used in the study was adapted from Christoff et al. Each block was immediately followed by a thought-probe which interrupted the task. For each probe, participants were asked to characterize the conscious experience they had in the few trials prior to the probe, according to the two dimensions “task-relatedness” and “stimulus-dependency” described above . Four poIn order to familiarize participants with the thought-sampling method, they first performed a version of the SART with thought-probes outside the scanner in an interval ranging from five to one day(s) before the fMRI session. In this first session, after being presented with the instructions and before performing the SART, participants were trained in classifying ten sentences representing different conscious experiences in order to familiarize them with the four response categories, and were presented with a short example of the SART . Then, they performed 40 blocks of the SART. They were given a paper sheet with the instructions for responding to the probes and were told that they could keep it for the whole SART and check it in case they felt uncertain about the definition of the four response possibilities. A final training of approximately six minutes was also performed in the scanner immediately before the fMRI session, during the structural MR acquisition.3). Thirty-two 3-mm thick transverse slices (FOV 22×22 cm2) were acquired, with a distance factor of 30%, covering the whole brain. Between 1360 and 1472 functional volumes were acquired. The first three volumes were discarded to account for T1 saturation. A structural MR scan was obtained at the beginning of the session . Head movement was minimized by restraining the subject's head using a vacuum cushion. Stimuli were displayed on a screen positioned at the rear of the scanner, which the subject could comfortably see through a mirror mounted on the standard head coil.Data were acquired on a 3 Tesla scanner  using a T2* sensitive gradient echo EPI sequence  implemented in MATLAB . Functional scans were realigned using iterative rigid body transformations that minimize the residual sum of squares between the first and subsequent images. They were normalized to the MNI EPI template (voxel size: 2×2×2 mm) and spatially smoothed with a Gaussian kernel with full-width at half maximum (FWHM) of 8 mm.fMRI data were preprocessed and analyzed using SPM5 . For completeness, the supplementary tables also list other regions that survived a threshold of p<0.001, uncorrected for multiple comparisons with a minimum cluster size of 15 voxels, but these regions are not discussed further.The individual summary statistics images of these four linear contrasts were then entered in second-level analyses, corresponding to random-effects models. A 2 (task-relatedness)×2 (stimulus-dependency) whole-brain voxel-wise repeated-measures analysis of variance (ANOVA) was first performed, which allowed us to examine the brain regions that are specifically related to the two dimensions of interest in this study. Then, we investigated the brain activations specific to task-related interferences relative to being fully focused on the task (task-related interferences>on-task), external distractions relative to being fully focused on the task , and mind-wandering relative to being fully focused on the task (mind-wandering>on-task), using Finally, ROI analyses were performed in order to investigate the possible distinction between a rostral portion versus a more caudal portion of the MPFC. Gilbert et al. SD = 112). Response withholding accuracy for targets was 62.28% (SD = 16.94). Regarding thought-probes, participants reported being on-task for 31.59% (SD = 14.27) of probes, task-related interferences for 22.13% (SD = 7.62) of probes, external distractions for 25.44% (SD = 10.04) of probes, and mind-wandering for 20.83% (SD = 11.17) of probes.Mean response time for non-targets was 405 msec  = 0.66; p = 0.42; partial 2η = 0.03] or of stimulus-dependency , and the interaction was not significant either . A second ANOVA was conducted on the variability of RTs : we observed a main effect of task-relatedness , no main effect of stimulus-dependency , and a significant interaction , indicating that reports of being completely focused on the task were preceded by more stable RTs than the three other classes of reports . There was no significant difference between the three other kinds of reports.Next, we examined the impact of the four types of reports on response times (RTs) for the five non-target stimuli preceding each probe. A 2 (task-relatedness)×2 (stimulus-dependency) repeated measures ANOVA on mean RTs was performed. Mean values and standard deviations are detailed in F = 7.74; p<0.05; partial 2η = 0.27], no main effect of stimulus-dependency , and a significant interaction , indicating that reports of being completely focused on the task were preceded by fewer errors to the targets than the three other kinds of reports . There was no significant difference between the three other types of reports.We also examined the effect of the four types of reports on response accuracy for the whole block of trials. We performed a 2 (task-relatedness)×2 (stimulus-dependency) repeated measures ANOVA on the number of errors to the target stimuli. Mean values and standard deviations are detailed in SD = 4.03) for reports of being fully focused on task, −0.09 (SD = 3.07) for task-related interferences reports, 1.73 (SD = 4.46) for external distractions reports, and 0.23 (SD = 3.05) for mind-wandering reports. Examination of the 95% confidence intervals revealed that the frequency of reports of being fully focused on task decreased with time on the task . None of the other kinds of reports significantly increased/decreased in frequency with time on the SART: task-related interferences , external distractions , and mind-wandering . Combining these indexes revealed that the time spent on the SART had no effect on task-unrelated reports  and stimulus-independent reports . These results indicate that the time spent on the SART had minor impact on the distribution of responses to the thought-probes in this study and thus cannot fully account for the fMRI results.In order to examine whether the time spent on the SART had an impact on the distribution of responses to thought-probes, an index of the effect of time on probe responses was calculated for each of the four kinds of reports according to the following formula: number of reports for the 32 last blocks of the SART – number of reports for the 32 first blocks of the SART. Mean values of this index were −1.86 ×2 (stimulus-dependency) whole-brain voxel-wise ANOVA was performed. The main effect of task-relatedness revealed clusters of activation in the medial prefrontal cortex (MPFC), the posterior cingulate cortex (PCC)/precuneus, the left posterior inferior parietal lobule (pIPL)/occipital cortex, the left anterior inferior parietal lobule (aIPL), the right middle frontal gyrus and the left parahippocampal cortex (PHC)/restrosplenial cortex (Rsp). Examination of the parameter estimates for each condition revealed that all of these brain regions were more active during intervals preceding task-unrelated reports  than during intervals preceding task-related reports , except for the left aIPL and the right middle frontal gyrus for which the opposite effect was observed . Other clusters of activations included the bilateral inferior/middle temporal gyrus (ITG/MTG), the left MTG and the left inferior frontal gyrus. Examination of the parameter estimates revealed that all of these regions were more active in intervals preceding stimulus-independent reports  than in intervals preceding stimulus-dependent reports  and in the two regions previously associated with task-related conscious experiences in the whole brain ANOVA: the left aIPL  and the right middle frontal gyrus ; see also Considering that task-related interferences and external distractions were associated with activations in distinct subregions of MPFC  compared to being on-task see , we alsoThe common involvement of DMN regions in both task-unrelated reports and stimulus-independent report, as well as the absence of interaction effect for these regions and the results of the direct contrasts suggest an additive effect of the “task-relatedness” and “stimulus-dependency” dimensions of conscious experiences on DMN activity. Mind-wandering would be associated with the highest level of DMN activity, task-related interferences and external distractions with intermediate levels of DMN activity, and being fully focused on the SART with the lowest level of activity in this network. We then conducted a conjunction analysis testing the conjunction null hypothesis: [(mind-wandering>task-related interferences) ∩ ] to confirm that DMN regions are indeed more active during mind-wandering compared to both task-related interferences and external distractions. This conjunction analysis revealed clusters of activations in the MPFC, the PCC/precuneus, and left MTG  = 5.39; p<0.05; partial 2η = 0.20], revealing that the two regions showed higher activity for task-unrelated reports, and a main effect of stimulus-dependency , revealing that the two regions showed higher activity for stimulus-independent reports. The interaction between task-relatedness and stimulus-dependency was not significant , indicating an additive effect of these two dimensions. Furthermore, the main effect of regions was not significant , and none of the interaction effects involving regions were significant: task-relatedness×regions , stimulus-dependency×regions , and task-relatedness×stimulus-dependency×regions , indicating that the two ROIs showed similar patterns of activations as a function of the two dimensions of conscious experience under investigation here. Overall, these results are thus in line with the results of the whole brain analyses, showing an additive effect of “task-relatedness” and “stimulus-dependency” on MPFC activity.Finally, because (1) our clusters of activation in the MPFC were rather extended and (2) it has been suggested that closely juxtaposed portions of the MPFC may perform different cognitive functions, a 2 (task-relatedness)×2 (stimulus-dependency)×2 (regions) repeated measures ANOVA was performed on the parameter estimates for two subregions of the MPFC that have been differentially linked to external attention and mind-wandering/mentalizing in previous studies Previous neuroimaging studies suggest that mind-wandering is associated with increased DMN activity. However, in these studies, participants were simply asked to report whether they were totally focused on the proposed task (on-task reports) or were distracted by task-unrelated thoughts (off-task reports), which does not permit to clearly distinguish between mind-wandering, external distractions, and task-related interferences. The present study aimed at clarifying this issue by investigating the neural correlates of the various kinds of conscious experiences that can occur while participants perform a task requiring sustained focused attention and the cognitive processing of external stimuli. Four classes of conscious experiences  that varied along two dimensions (“task-relatedness” and “stimulus-dependency”) were sampled using newly designed thought-probes while participants performed the Sustained Attention to Response Task SART .We found that reports of task-unrelated conscious experiences were associated with increased activity in the MPFC, the PCC/precuneus, and the pIPL, all regions of the DMN These results are important because they help reconcile different proposals that have been made regarding the functional role of the DMN. For instance, Gusnard and Raichle The MPFC, PCC/precuneus, and pIPL were engaged during both task-unrelated and stimulus-independent conscious experiences. This result suggests that these regions underlie cognitive processes active during both unfocused attention toward external stimuli and internal thoughts. A possibility is that increased activity in these DMN regions  reflects the engagement of cognitive processes involved in monitoring the self-relevance of the ongoing content of consciousness The moderate level of DMN activity related to external distractions and task-related interferences might suggest that both are intermediate states preceding the occurrence of mind-wandering. There is some behavioral evidence that the occurrence of mind-wandering is not an all or nothing phenomenon but follows a gradual temporal sequence Another interpretation of our findings would be that the increased midline DMN activity, and especially MPFC activity Among the DMN regions, the lateral temporal lobes were linked to stimulus-independent reports but not to task-unrelated reports. This finding indicates that distinct subregions within the DMN are related to specific dimensions of conscious experiences, which is in agreement with recent studies demonstrating the heterogeneity of the DMN and its fractionation in subcomponents that might support different cognitive processes Stimulus-independent reports were also characterized by activity in the inferior frontal gyrus, a region that has been linked to executive functions and goal-directed activities Finally, in contrast to the posterior lateral prefrontal activation related to stimulus-independent reports, a more anterior region of the middle frontal gyrus and the aIPL were specifically activated in relation to task-related reports. The middle frontal gyrus is usually considered as being part of the dorsal attention network, a set of dorsal brain regions supporting top-down focused attention toward external stimuli To conclude, this study demonstrates that task-relatedness and stimulus-dependency are both related to DMN activity. Notably, an additive effect of these two dimensions of conscious experiences was demonstrated for the MPFC and PCC/precuneus. On the other hand, other DMN regions were specifically related to only one dimension, with activity in lateral temporal regions being solely related to stimulus-independent reports. These results have two broad implications. First, they reveal the necessity to take both dimensions into account when attempting to determine the functional role of the DMN. This could help resolve controversies as to the role of the DMN in internal versus external attention Table S1Brain regions (outside a priori areas of interest) associated with the main effects of task-relatedness and stimulus-dependency, as well as the cross-over interaction effect in the whole-brain ANOVA.(DOC)Click here for additional data file.Table S2Brain regions (outside a priori areas of interest) associated with task-related interferences and mind-wandering compared to being fully focused on task.(DOC)Click here for additional data file.Table S3Brain regions (outside a priori areas of interest) more active during task-related interferences compared to external distractions.(DOC)Click here for additional data file.Table S4Brain regions (outside a priori areas of interest) more active during mind-wandering compared to both task-related interferences and external distractions .(DOC)Click here for additional data file.


Xenopus laevis indicate that T3 actions in both the epithelium and the rest of the intestine, most likely the underlying connective tissue, are required for the formation of adult stem cells. Molecular analyses suggest that cell-cell interactions involving hedgehog and BMP pathways are critical for the establishment of the stem cell niche that is essential for the formation of the adult intestinal stem cells.The mammalian intestine has long been used as a model to study organ-specific adult stem cells, which are essential for organ repair and tissue regeneration throughout adult life. The establishment of the intestinal epithelial cell self-renewing system takes place during perinatal development when the villus-crypt axis is established with the adult stem cells localized in the crypt. This developmental period is characterized by high levels of plasma thyroid hormone (T3) and T3 deficiency is known to impair intestinal development. Determining how T3 regulates adult stem cell development in the mammalian intestine can be difficult due to maternal influences. Intestinal remodeling during amphibian metamorphosis resembles perinatal intestinal maturation in mammals and its dependence on T3 is well established. A major advantage of the amphibian model is that it can easily be controlled by altering the availability of T3. The ability to manipulate and examine this relatively rapid and localized formation of adult stem cells has greatly assisted in the elucidation of molecular mechanisms regulating their formation and further revealed evidence that supports conservation in the underlying mechanisms of adult stem cell development in vertebrates. Furthermore, genetic studies in  The intestinal epithelium is responsible for the principle physiological function of this organ: digestion and absorption of nutrients. Throughout adult life, the vertebrate intestinal epithelium undergoes self-renewal through the proliferation of the adult stem cells. In mammals, the stem cells are localized in the crypt of intestine while the absorptive epithelial cells, the most abundant epithelial cell type, and secretory cells are present along the villus of the crypt-villus axis -4. As thWhile intestinal development occurs quite early during mammalian embryogenesis, the maturation of this adult epithelial self-renewing system takes place during the so-called postembryonic development -9, the pin vivo when compared to mammalian postembryonic development, where maternal influences complicate the studies on the embryos/neonates.Amphibian metamorphosis resembles postembryonic development in mammals ,20. ThisXenopus laevis[Xenopus laevis and tropicalis, the premetamorphic tadpole intestine is a simple tubular structure made of mostly a single layer of larval epithelial cells with little connective tissue or muscles except in the single fold, the typhlosole, where the connective tissue is abundant , their mouse homologs also have their peak levels of expression in the intestine within the first 2 weeks after birth [Many of the genes regulated by T3 during he years -91. Somehe years ,90, supponserved -97, consorphosis . Furtherer birth , when thXenopus laevis line that expresses a dominant positive TR under the control of a heat shock-inducible promoter for organ culture studies. The dominant positive TR could not bind to T3 but functioned as a constitutively liganded TR to induce metamorphosis when expressed after heat shock treatment of premetamorphic tadpoles [Organ culture studies as reviewed above have suggested that the non-epithelium, most likely the connective tissue, is required for T3-induced formation of adult progenitor/stem cells during intestinal metamorphosis. Gene expression analyses have shown that many genes are regulated by T3 in the epithelium or connective tissue or both. To investigate whether T3 actions in the epithelium and non-epithelium have specific roles in adult intestinal stem cell development, we made use of a transgenic tadpoles . Thus, ttadpoles . Expresstadpoles .The above findings as well as other studies suggest that during metamorphosis, T3 signals in the larval epithelium and the rest of the intestine (the non-epithelium), mostly the connective tissue, have distinct effect on epithelial transformations Figure . T3 actiAs discussed above, the ECM remodeling is likely important for intestinal metamorphosis. EMC remodeling is largely mediated by matrix metalloproteinases (MMPs), a superfamily of Zn-dependent membrane-bound or secreted endopeptidases -108. MMPXenopus laevis ST3 was added to the T3-treated organ cultures of premetamorphic intestine, it inhibited T3-induced ECM remodeling and larval epithelial cell death [MMPs have long been implicated in amphibian metamorphosis. In fact, the first MMP, collagenase, was isolated as a collagen-degradation enzyme from the resorbing tadpole tail . Gene exll death . In addill death , a procell death .Complementary to the organ culture studies, transgenic overexpression of ST3 under the control of a heat shock-inducible promoter  in premein vitro[in vitro and during metamorphosis [The exact mechanism by which ST3 functions remains to be investigated. Compared to other MMPs, ST3 has much weaker activities toward known ECM proteins but much higher activities toward a few non-ECM proteins such as α1-protease inhibitor, at least in vitro-125. Intorphosis -128 and orphosis ,128. TraXenopus laevis intestine [A number of signal transduction pathways have been shown to be important for intestinal development in mammals. Among them are the Shh, WNT, BMP, and Notch pathways ,129,130.ntestine ,131. It ntestine . We haventestine . These intestine . More imntestine .Xenopus laevis intestine. Furthermore, BMPR-IA, a type I receptor of BMP-4, is expressed in both the developing connective tissue and progenitor/stem cells of the adult epithelium during metamorphosis, suggesting that BMP-4 affects both the connective tissue and adult epithelium during intestinal metamorphosis. Consistently, in intestinal organ culture, the addition of BMP-4 protein not only repressed cell proliferation in the connective tissue but also promoted differentiation of the adult epithelial cells [In addition to Shh, bone morphogenetic protein-4 (BMP-4), a member of the TGFβ superfamily of signaling molecules, is also a T3 response gene . BMP-4 ial cells . MoreoveXenopus metamorphosis [BMP-4 is a known target of Shh during mammalian gut development -138. Likorphosis . Thus, torphosis .Xenopus intestinal metamorphosis have led to the identification of many T3 response genes that are likely involved in the development of the adult stem cells and many such genes, such as those of the Shh and BMP-4 signaling pathways, likely have similar functions during the maturation of the adult intestine in mammals. A major future challenge will be to investigate the functions of such genes in vivo. The recent advancements in gene knockout studies in Xenopus by using gene-specific nucleases [The mammalian adult intestinal stem cells have long been used as a model to study tissue renewal and stem cell maintenance. The establishment of the self-renewing system of the intestinal epithelium is largely conserved in vertebrates. It takes place during the postembryonic period when plasma T3 levels are high, and more importantly, depends on T3. In amphibians, this postembryonic period is the T3-dependent metamorphosis. Increasing evidence indicates that the formation of the self-renewing epithelium of the adult intestine involves the formation of the adult intestinal stem cells that are distinct from the larval/neonatal intestinal epithelial stem cells across vertebrate species. Analyses of ucleases ,140 undoThe authors declare that they have no competing interest.All authors participated in the writing of the review. All authors read and approved the final manuscript.


Understanding the dynamics of the microbial communities that, along with their secreted enzymes, are involved in the natural process of biomass composting may hold the key to breaking the major bottleneck in biomass-to-biofuels conversion technology, which is the still-costly deconstruction of polymeric biomass carbohydrates to fermentable sugars.However, the complexity of both the structure of plant biomass and its counterpart microbial degradation communities makes it difficult to investigate the composting process.Liriodendron tulipifera) wood-chips and mown lawn grass clippings (85:15 in dry-weight) and used as a model system. The microbial rDNA abundance data obtained from analyzing weekly-withdrawn composted samples suggested population-shifts from bacteria-dominated to fungus-dominated communities. Further analyses by an array of optical microscopic, transcriptional and enzyme-activity techniques yielded correlated results, suggesting that such population shifts occurred along with early removal of hemicellulose followed by attack on the consequently uncovered cellulose as the composting progressed.In this study, a composter was set up with a mix of yellow poplar  and solid-state fermentation for the production of cellulolytic enzymes and biofuels. The intertwining matrix of biopolymers  of which plant cell walls are composed poses a major obstacle to the deconstruction of these walls to simple sugars and chemicals that can serve as raw materials for the fermentative production of alternative liquid fuels and other bioproducts. This major bottleneck in biomass conversion technology can be mitigated by 1) reducing plant biomass recalcitrance through genetic engineering of energy crops, thereby 2) minimizing the requirement for thermo-chemical feedstock pretreatment, 3) improving performance of the enzymes used for saccharification, and 4) introducing the one step conversion concept, or consolidated bioprocessing (CBP), in which enzyme production, enzymatic hydrolysis, and fermentation are combined for microbial production of biofuels using biomass as substrates , and xyterature ], the prTrichoderma spp. ITS , endoglucanase  and beta-glucosidase (BGL). The gene expression profiling of the cellulases is shown in Figure Meanwhile, to assess the expression of cellulase genes of genus Interestingly, as shown in Figure Phanerochaete chrysosporium is a model fungus that can degrade lignin without "touching" the cellulose of the wood. Like other white rot fungi, P. chrysosporium secretes an array of peroxidases and oxidases that attack lignin . We pree review .P. chrysosporium's ligninase genes into CBP strains (so far mainly bacteria and yeasts) in order to further enhance their lignocellulolytic functions.Composting in the current study can be viewed as a natural variant of CBP in rotary composters that involves the interactions of microbes and their secreted enzymes with plant cell walls. Here, we propose to incorporate into the strategy for the development of CBP one observed attribute of woody biomass compost, which is the observed fungal dominance in the late phase of composting when more cellulose is exposed. Such observation is in line with the natural degradation of biomass in wood litter with a higher fungal contribution (67-99%) to the total microbial mass (see review ). BecausAs the examined representative fungal xylanases, cellulases and ligninases were differentially expressed at different composting stages in this study, the timing of adding or expressing such enzymes in a CBP should also be more precisely regulated according to the progress of lignocellulosic substrate degradation.T. reesei [Penicillium janthinellum [B. subtilis ASH [B.licheniformis A99 [Aspergillus tamarii [A. terreus M11, using various lignocellulosic materials as carbon source [Mortierella isabellina on the substrates of crusted freshly harvested sweet sorghum have been used to produce biodiesel [M. isabellina cannot utilize cellulose and xylan, it is reasonable to speculate that a co-culture with lignocellulolytic fungi such as T. reesei and P. chrysosporium may reduce the substrate cost by increasing the portion of useable materials in the substrate mixture.Solid-state fermentation (SSF) is a process wherein the growth of microorganisms takes place on a solid substrate in absence or near absence of free water, but with enough moisture to support microbial growth and metabolism . It has . reesei -51 and Phinellum , to prodilis ASH ,53, B.lirmis A99  and Aspe tamarii , and to n source . Compostiodiesel . As M. iTo the best of our knowledge, this project constitutes the first comprehensive study of the natural composting of yellow poplar biomass. The following observations and implications highlight the value of using biomass compost for demystifying the enigma of "natural biomass conversion" in terms of lignocellulolytic gene expressions, enzymatic activities and their effects on deconstruction of plant biomass materials.CtCBM3-GFP fluorescence labeling experiments, enzymatic activity analyses and functional gene expression profiling suggest that more hemicelluloses were degraded in the early stages of composting and that the celluloses in the biomass were thereby progressively more "unwrapped" and exposed at the later stages.First, the Second, a significant microbial population shift observed in this study suggests that studies of the conditions at the transition points between bacteria- and fungus-dominated stages are critical for identifying new microbial systems that are potentially applicable to biomass conversion. Future metatranscriptomic analysis via mass-scale sequencing during these stages and at their transitions can provide great potential for discovering novel cellulolytic microbes and enzymes.Poa pratensis L.) and the lawn was not fertilized in 2007 or during the preceding year. To inoculate the sample with microbes, composted material taken from a mixed, already-established natural compost pile of trimmed tree branches and twigs was added. Water was added to moisten the sample, and after the entire contents of the composter were mixed thoroughly, the initial moisture content of the mixture was determined by oven-drying of representative aliquots, to be approximately 58%. The composter was rotated once a week, beginning on the 7th day after the composter setup, in order to aerate the samples. At each of the designated sampling dates , the composted material was thoroughly mixed by rotating the composter bin and by stirring the materials inside with bars, both horizontally and vertically. After mixing, a total of about 200 g of compost was sampled from various depths below the compost mass surface and mixed. An aliquot was air-dried and used to determine the water content, while the bulk of the withdrawn aliquot was stored at -80°C for later use in bio-imaging, microbial rDNA and gene transcriptional profiling , and enzyme-activity analyses (for the samples collected at week 3 and 18) as well as chemical compositional analysis (for the samples collected at week 1 and 27). The presented data are based on the studies of the compost samples from October 2007 compost samples. To test the reproducibility of the major measurements of this study, a second independent composting experiment was conducted in August 2009 following the same procedure as described above. The same sampling patterns and analyses were carried out for the second composting to track its microbial community composition (rDNA profiling) and cellulolytic gene expression, and the results obtained were consistent with the observation we made by using the samples from October 2007 compost (data not shown).A drum-shaped rotary composter Figure  was set Temperature and oxygen concentration were monitored constantly and recorded every other day during the composting process. Temperature at the center of the compost was measured using a 1522 Digital Indoor/Outdoor Thermometer  and a Windrow Thermometer ; the oxygen concentration was measured using an oxygen analyzer  at the center of the compost. To minimize the impact of the above-mentioned weekly (pre-sampling) rotation on the measurement of the temperature and oxygen concentration, these measurements were made before the scheduled rotation was conducted.CtCBM3-GFP that was prepared in our previous work [CtCBM3-GFP was carried out in a blotting buffer  at room temperature for 30 min. The sample was washed three times in washing buffer  followed by centrifugation (1000× g for 1 min). The final pellet was transferred to a slightly different buffer  and subjected to microscopic analysis. Samples including original, untreated material as control, and material sampled at 6, 15, and 24 weeks composting, were imaged using an Olympus inverted fluorescence microscope. Representative images were selected for display based on analysis of 10-14 microscopic fields from each of the examined compost samples.The structural changes in plant biomass produced by the microbial decay community was assessed by both white-light and fluorescence microscopy. For sample preparation, small amounts of the frozen composted samples were immersed in water to thaw and soften the material. Single pieces of the composted biomass were then chosen and hand-cut in an orientation that would result in a transverse cross-sectioning. The thin sections were kept in water until being deposited onto a glass coverslips for microscopic analysis. Chemically-specific labeling of the composted material utilized a family-3 carbohydrate-binding module (CBM) fused with a green-fluorescent-protein tag, ous work . LabelinCompositional analysis of the composted materials was performed by using method described in the literature .Total microbial genomic DNA was extracted from 5-g compost samples using the standard procedure provided by the Ultra Clean Mega Soil DNA Kit . To increase the purity of extracted genomic DNA, a further purification of DNA samples was conducted using the Qiagen DNA Purification Kit . The prepared DNA samples were quantified using a Nanodrop 1000 Micro-Volume UV-vis Spectrophotometer  and stored at -80°C until used for the real-time PCR analysis using microbial rDNA primers, as described later.The total fungal RNA of the compost was extracted from 1 g of each compost sample, which was ground to fine powder in liquid nitrogen using a mortar and pestle, followed by the extraction and purification protocol for filamentous fungi using Qiagen RNeasy Plant Mini Kit . The above extracted total fungal RNAs were treated with DNase I  to eliminate the genomic DNA contamination. One microgram of purified total RNA was reverse-transcribed using SuperScript III Reverse Transcriptase with random primers  according to the manufacturer's kit manual. The prepared fungal cDNA samples were stored at -20°C until used for the real-time RT (reverse transcription) PCR amplification using functional gene primers, as described later.Real-time PCR, using the universal primer sets for archaeal, bacterial and fungal rDNA Table  and the Phanerochaete chrysosporium, all other primers for cellulase- and hemicellulase-encoding genes were based on sequences from 2-4 different species of the same genus, and can therefore be viewed as group primers at the sub-genus level  and specifying a Tm value between 58°C and 62°C and an amplicon size between 100 and 250 bp.In parallel, real-time RT (reverse transcription) PCR was employed to profile the selected genes encoding cellulolytic enzymes over the time course of composting, using the above prepared fungal cDNAs as templates and the primers designed as follows. The designing of the primers Table  for thesel Table . These sPCR analyses were performed using the iCycler iQ real-time detection system . The iCycler iQ optical system software  was used to compile PCR protocols and set up the plate. Each microtiter plate well contained a 20-μL mixture of the following: 10 μL 1X iQ SYBR Green Supermix, which contained all the nucleotides, polymerase, reaction buffer, and SYBR green dye , 5 μL forward and reverse primers, giving final optimal concentrations of 300 nM for each, and 5 μL of DNA as template (2.5 ng per well). The PCR conditions for microbial rDNA amplification were described in the literature for the source of individual primers. For the primer pairs targeting functional genes listed in Table The PCR specificity for functional genes was confirmed at three levels. First, at the primer designing step, the designed primers were run through a Basic Local Alignment Search Tool (BLAST) search against the nucleotide collection (nr/nt) of NCBI. The designed primer should only hit only the target gene of the target species; any primer that matched sequences for organisms other than target species was abandoned. Second, at the end of real-time RT-PCR, the dissociation curve  analysis of PCR end products was performed by ramping the temperature from 60°C to 95°C at a rate of 1°C per 30 s; a PCR reaction with high specificity should have a single melting peak. Third, PCR products were analyzed by agarose gel electrophoresis to check for the presence of single band, with some PCR product bands being gel-purified using QIAquick spin columns  and sequenced.Approximately 5 g of frozen composted material was withdrawn from each of the representative samples at 3 weeks and 24 weeks into the composting process. Approximately 2.0 g of each sample was wet-ground with a mortar and pestle in 20 mM sodium acetate buffer, pH 5.0 at room temperature, then suspended in 20 mL of the same buffer to produce a uniform, readily-pipettable slurry. The remainder of each frozen compost sample was weighed, oven-dried , and reweighed to obtain a rough estimate of the biomass solids content of the slurry produced from that sample.2CO3 in one well of a 96-well, flat-bottomed polystyrene plate . Product fluorescence was then read in a Tecan GENios plate reader  with excitation/emission wavelengths at 340/465 nm. Relative enzyme activities were reported as raw fluorescence readings, minus the fluorescence of an identically-treated blank with acetate buffer substituted for the biomass slurry.For the assay procedure, 0.8 mL of each well-mixed compost slurry was pipetted into each of three triplicate 2.0-mL HPLC vials. To each assay vial was added 0.2 mL of a 0.5-mM solution of a given 4-methyl-umbelliferyl-β-D-glycoside substrate  in 20 mM sodium acetate buffer, pH 5.0. Each vial was crimp-sealed, and the contents were mixed well by manual inversion before being incubated for 3 hours in a 30°C air incubator, with constant mixing by inversion at a rate of 10/min. At the end of the 3-h incubation, solids in the reaction mixtures were removed by centrifugation, and fluorescence of the cleavage product was developed by mixing 0.1 mL of each supernatant with 0.1 mL of 2 M NaCtCBM3: Clostridium thermocellum carbohydrate-binding module 3; EGL: Endoglucanase; ITS: Internal transcribed spacer; LiP: Lignin peroxidase; MnP: Manganese peroxidase; PCR: Polymerase chain reaction; rDNA or rRNA: ribosomal DNA or RNA; XYN: Xylanase.BGL: Beta-glucosidase; CBH: Cellobiohydrolase; CBM: Carbohydrate-binding module; CBP: Consolidated bioprocessing; The authors declare that they have no competing interests.SYD, MPT and HW designed and coordinated the study, and revised the manuscript. HW, MPT and JOB conducted the compost setup and maintenance, sample harvesting, data analysis, and manuscript preparation. HW, MH and YL conducted DNA and RNA extraction, primer selection and PCR reactions. HW and QX carried out fluorescence microscopy study of composted samples. Enzymatic activity assay was carried out by JOB. MEH was consulted. All authors read and approved the final manuscript.


The innate response to infection by an Old World arenavirus is initiated and mediated by extracellular and intracellular receptors, and effector molecules. In response, the invading virus has evolved to inhibit these responses and create the best environment possible for replication and spread. Here, we will discuss both the host’s response to infection with data from human infection and lessons learned from animal models, as well as the multitude of ways the virus combats the resulting immune response. Finally, we will highlight recent work identifying TLR2 as an innate sensor for arenaviruses and how the TLR2-dependent response differs depending on the pathogenicity of the strain. Old World arenaviruses cause a wide spectrum of disease in humans, determined by the strain of virus and the immune status of the patient. Lassa virus (LASV) affects an estimated 2 million people each year, causing mild, flu-like illness to severe multisystem disease. Lymphocytic choriomeningitis virus (LCMV) generally causes a mild, self-limiting illness in the immunocompetent person with rare progression to severe or fatal encephalitis, but can cause a hemorrhagic fever-like disease in patients in which the virus was acquired through solid organ transplantation, and are thus immunocompromised.Because of strict regulations, studies involving LASV can be quite cumbersome. Since the earliest symptoms of LASV infection are non-specific, patient data is unreliable and incomplete. In order to understand the immune responses that contribute to LASV pathogenesis, or lack thereof, the use of surrogate arenaviruses, both Old and New World, in cell culture and animal model experiments helps to build a better picture of how Old World arenaviruses can cause severe disease.in vitro or in vivo . No. No20]. TLR2-dependent proinflammatory responses are induced at the plasma membrane. However, we have demonstrated that UV or heat inactivated-LCMV is no longer able to elicit such a response. Additionally, multiplicity of infection, as well as replication, was strongly linked to the level of cytokine induction. Taken together, this data indicates that live replicating virus is necessary for efficient induction of proinflammatory immune responses. Further, because inactive virus was unable to induce TLR2-dependent responses, the possibility exists that arenaviruses stimulate secretion of a danger-associated molecules, capable of stimulating TLR2. Since live replicating virus is necessary for TLR2-dependent cytokine induction, it is probable that TLR2 stimulation occurs after membrane fusion and release of genomic material into the host cytoplasm. Co-infection experiments demonstrated the ability of pathogenic LCMV-WE to actively inhibit the cytokine response elicited by LCMV-ARM C 20]. L. L20]. LIn agreement with previous studies, LCMV-WE could not inhibit exogenous stimulation of TLR2-induced cytokine responses. The addition of LTA, a TLR2/6 ligand, in the presence of LCMV-ARM or LCMV-WE infection elicited cytokine production equivalent or greater than LTA treatment alone. This implies that the manner in which LCMV-WE inhibits the TLR2-dependent cytokine response is arenavirus-specific, or possibly specific to the type of ligand. It is possible that LCMV-WE may be capable of inhibiting TLR2-dependent cytokine production by other viruses. Further investigation with other viruses known to stimulate TLR2-dependent cytokine production in co-infection experiments with LCMV-WE will answer the specificity of LCMV-WE inhibition as ligand type plays an important role in TLR2 driven responses.Two important questions remain: how does TLR2 signaling contribute to resistance/recovery from arenavirus infection and how could the virus inhibit TLR2 signaling? To determine the importance of TLR2 signaling in protection against arenavirus infection, the correct animal model must be used. Mouse models of arenavirus infection rely on IFN-I rather than inflammatory cytokines and cytotoxic T cell responses. These mouse models provide more information for chronic infections rather than severe acute infections, such as those caused by LASV in humans. The non-human primate model most closely resembles the disease progression seen in humans. However, methods to decipher the contribution of TLR2 are more difficult to pursue in such models. The use of function-blocking antibody treatment may provide some insight. Studies could then be directed toward the activation of APC, T cells, or viral load, in the absence or reduction of TLR2 signaling. These types of studies could help elucidate the contribution of TLR2 signaling in the model presented below , in whicAlong with the global effects of TLR2 signaling in arenavirus infection, it is still necessary to determine how arenaviruses stimulate the TLR2-dependent response and how this is inhibited during pathogenic arenavirus infection . We haveIdeally, one would like to singly express protein components of arenaviruses to determine if they stimulate TLR2. However, the requirement for live replicating virus confounds these efforts. The use of virus replicon particles (VRP) in which a replication defective virus particle encodes a gene under a separate promoter may help shed some light on this issue. VRPs could be used to express a particular gene of interest from either pathogenic or non-pathogenic arenaviruses. Additionally, using reverse genetics, proteins of pathogenic arenaviruses could be interchanged with the non-pathogenic protein, providing the replication necessary while singly expressing the pathogenic component.in vitro, we have provided a possible explanation for the development of severe disease in LASV infection. Further work in this area could entail looking for differences in TLR2 stimulation, TLR2 mutations, and TLR2-related SNPs to determine why some, but not all infected patients, succumb to LASV infection. Additionally, having identified the differences in TLR2-dependent cytokine responses between pathogenic and non-pathogenic virus infection, this work provides an avenue for development of therapeutics. With limited treatments available to use against LASV infection, use of TLR2 agonists may provide activation of the type of immune response necessary to clear virus more effectively and increase chances for survival. It is not clear whether activation of TLR2 signaling should be taken into account in vaccine design for arenaviruses. The ideal vaccine candidate for Old World arenaviruses would stimulate an efficient cytotoxic response and generate long lasting memory. Knowing the role of TLR2/Mal/MyD88 signaling in generating specific CD4+ and CD8+ T cell responses, and taking into consideration the inability of LASV to induce memory markers in T cells, it remains paradoxical that ML29, containing a small genomic segment from Lassa virus, has impaired induction of IL6 and IL8, yet is an excellent attenuated vaccine in non-human primates. Further reverse genetic and systems studies will be necessary to assess the role of the proinflammatory response in pathogenesis and protection.By comparing the differences in TLR2-dependent responses during infection by arenaviruses of differing pathogenic potential for humans and non-human primates


Physical activity is known to have health benefits across population groups. However, less is known about changes over time in socioeconomic differences in leisure-time physical activity and the reasons for the changes. We hypothesised that class differences in leisure-time physical activity would widen over time due to declining physical activity among the lower occupational classes. We examined whether occupational class differences in leisure-time physical activity change over time in a cohort of Finnish middle-aged women and men. We also examined whether a set of selected covariates could account for the observed changes.The data were derived from the Helsinki Health Study cohort mail surveys; the respondents were 40-60-year-old employees of the City of Helsinki at baseline in 2000-2002 . Follow-up questionnaires were sent to the baseline respondents in 2007 . The outcome measure was leisure-time physical activity, including commuting, converted to metabolic equivalent tasks (MET). Socioeconomic position was measured by occupational class . The covariates included baseline age, marital status, limiting long-lasting illness, common mental disorders, job strain, physical and mental health functioning, smoking, body mass index, and employment status at follow-up. Firstly the analyses focused on changes over time in age adjusted prevalence of leisure-time physical activity. Secondly, logistic regression analysis was used to adjust for covariates of changes in occupational class differences in leisure-time physical activity.At baseline there were no occupational class differences in leisure-time physical activity. Over the follow-up leisure-time physical activity increased among those in the higher classes and decreased among manual workers, suggesting the emergence of occupational class differences at follow-up. Women in routine non-manual and manual classes and men in the manual class tended to be more often physically inactive in their leisure-time (<14 MET hours/week) and to be less often active (>30 MET hours/week) than those in the top two classes. Adjustment for the covariates did not substantially affect the observed occupational class differences in leisure-time physical activity at follow-up.Occupational class differences in leisure-time physical activity emerged over the follow-up period among both women and men. Leisure-time physical activity needs to be promoted among ageing employees, especially among manual workers. Health behaviours, such as leisure-time physical activity tend to be socioeconomically patterned. Such patterning is complex as socioeconomic position covers a range of social, economic and material circumstances from childhood to adulthood . The maiCross-sectional studies suggest that people in higher education ,4 and ocIn the last few decades, increases in leisure-time physical activity have been reported in Finland , Canada According to a study on trends in Finland, education, occupational class and household income differences in leisure-time and commuting physical activity generally remained relatively small between 1978-2002 . Follow-It is known that low leisure-time physical activity in general is associated with higher weight ,11,13, sWe hypothesised that class differences in leisure-time physical activity would widen over time due to declining physical activity among the lower occupational classes. Our first aim was to examine occupational class differences in leisure-time physical activity, and subsequent changes, over a follow-up of 5 to 7 years. Our second aim was to examine the effect of covariates on changes in the occupational class differences in leisure-time physical activity.The data were derived from the Helsinki Health Study cohort mail questionnaire surveys administered to employees of the City of Helsinki, Finland. At baseline in 2000-2002, the data covered 8 960 employees aged 40-60-years  . The folThe analyses for this study were carried out among those with data on occupational class (missing data for 118), leisure-time physical activity (missing data for 135) and all the covariates (missing data for 123). After exclusions for missing data, the final data consisted of 5 652 women and 1 279 men.The Helsinki Health Study protocol has been approved by ethics committees of the Department of Public Health, University of Helsinki, and the City of Helsinki health authorities, Finland.The respondents were asked to estimate the average weekly hours of physical activity during their leisure time (including commuting) within the previous year . There wThe amount of leisure-time physical activity was assessed by approximate metabolic equivalent tasks (MET) taking into account the intensity, duration and frequency  and calcModerate-intensity leisure-time physical activity for about 30 minutes at least five times a week is recommended by the national guidelines in Finland as well as other countries -27. ApprOccupational class was used as an indicator of socioeconomic position. Information on occupational class was derived from the City of Helsinki personnel registers for those who gave written permission for data linkage (77%) . For the2) . Physical and mental health functioning were measured on the physical component summary (PCS) and the mental component summary (MCS) of the Short Form 36 (SF-36) questionnaire [All the covariates were self-reported and taken from the baseline survey, except employment status which was taken from the follow-up survey. Thirteen per cent of women were single, 10% cohabiting, 58% married, 16% divorced and 3% were widowed. The corresponding figures for men were 11%, 15%, 64%, 10% and 1%. Twenty-two per cent of women and 25 per cent of men were smokers at baseline. Body mass index (BMI) was calculated as the weight in kilograms divided by the height in metres squared  was used for the analyses.At baseline 24% of women were physically inactive and there were no differences between the occupational classes Table . The preThirty-seven percent of women were physically active in their leisure-time at baseline and there were no differences between the occupational classes Table . At follTwenty-five per cent of men were physically inactive at baseline and as with women there were no differences between the occupational classes Table . At follAmong men 43% were physically active during their leisure-time and there were no differences between the occupational classes at baseline Table . Among mNext logistic regression analysis was used to examine the effects of the covariates on the occupational class differences in leisure-time physical activity at follow-up. Firstly, we examined physical inactivity. Among women, after adjusting for age, manual workers (OR 1.60 95% CI 1.31-1.97) and routine non-manual employees (OR 1.34 95% CI 1.14-1.57) were more likely to be physically inactive at follow-up than professionals Table . We furtSecondly, we examined the physically active. Among women, after adjusting for age, manual workers (OR 0.69 95% CI 0.57-0.83) and routine non-manual employees (OR 0.79 95% CI 0.69-0.91) were less likely to be physically active at follow-up than professionals  were more likely to be physically inactive at follow-up than those in the upper classes  were less likely to be physically active at follow-up than those in the upper classes . However none of these additional covariates had a substantial effect on the emergent occupational class differences. We also checked the effects of covariates measured at follow-up but the results were similar to those using covariates measured at baseline.Occupational class differences in health and functioning tend to widen towards late middle-age, with manual workers' health being the worst . Health Therefore we conducted control analyses and adjusted for the physical functioning subscale of the SF-36 which more precisely measures health problems that restrict physical activities such as running and brisk walking. The physical functioning subscale is by definition a purer measure of physical functioning than the physical component summary which is a composite measure . The difFurther control analyses showed that the follow-up physical functioning score attenuated the association more than the other examined covariates (data not shown), however, the baseline adjustment had no effects. This suggests that there were differences in physical functioning between the occupational classes at follow-up that affect leisure-time physical activity. In other words leisure-time physical activity decreased among the lower occupational classes partly due to poorer health as suggested by previous research . Our sinOne also might ask whether similar developments could be observed in other health behaviours. With regard to the consumption of healthy food, for example, the socioeconomic gap has remained stable, and food habits increasingly tend to follow national guidelines . FurtherGiven that our study was based on an occupational cohort we used occupational class for the analyses, which reflects material resources and working conditions, in particular. We used education as a parallel socioeconomic indicator in our control analyses. Education is an indicator reflecting knowledge and affecting unhealthy behaviours. However, the results of the control analyses were similar to those reported above (data not shown).The baseline survey was conducted in spring and the follow-up in autumn, but because physical activity was measured over the previous year, there should be no seasonal effects on the results. In this study leisure-time physical activity was self-reported, which is common practice in epidemiological studies ,34,35. PWe examined leisure-time physical activity while some other studies have also examined occupational physical activity. Working life has changed over the decades, and is generally less physically demanding . This isThe response rate was acceptable both at baseline (67%) and follow-up (83%), but non-participation was still a concern and may have biased the findings. Our analysis of the baseline non-response indicated that the associations in question were unlikely to be severely affected . AccordiAll the respondents were from the Helsinki metropolitan area and employed at baseline by the City of Helsinki. The results therefore cannot be generalised to the whole population of Finland, and not even to the employed population at large. Nevertheless, the cohort is large and diverse, and the study was planned to enable studies of the socioeconomic differences in lifestyles and health. A further strength was the use of identical questions at baseline and follow-up in assessing occupational class differences in leisure-time physical activity.Occupational class differences in leisure-time physical activity emerged during the follow-up of 5 to 7 years: there was an increase in activity among the upper classes and a decrease among the lower classes. In the interests of health promotion and disease prevention it is important for ageing people of all occupational classes, but especially for manual workers to maintain and increase physical activity. Efforts should also be made to reduce the socioeconomic differences. In the future mechanisms behind the socioeconomic differences in leisure-time physical activity should be further examined.BMI: body mass index; MCS: the mental component summary; MET: an activity metabolic equivalent task; PCS: the physical component summary; SF-36: the Short Form 36;The authors declare that they have no competing interests.TS carried out the statistical analysis, interpreted the results and drafted the manuscript. TS, JL, TL, OR and EL contributed to designing the study, interpreting the results and drafting the manuscript. All the authors critically reviewed the manuscript and approved the final version.


A qualitative comparison of the PET scans was performed blinded to the PSA and ALP results. A semi-quantitative comparison was made by measuring the maximum standardised uptake values (SUVmax) in five bone metastases in each patient. The means of the five SUVmax measurements in each subject were used as a quantitative measure of global metastatic activity at each time point.Five patients with castrate-resistant prostate cancer and bone metastases with no known soft tissue disease received 100 kBq/kg of radium-223  and minimal change  in two patients with increased PSA at 12 weeks. Four patients showed similar reductions in mean SUVmax and ALP at 12 weeks.Three patients showed a PSA decline at 12 weeks  whilst two patients showed PSA increases . All five patients showed a reduction in ALP of greater than 25%. The qualitative assessment of the 18F-fluoride PET is more accurate than the qualitative comparison of scans in assessing response in bone metastases, correlating with the PSA response and ALP activity and offering a potential imaging biomarker for monitoring treatment response in bone metastases following treatment with 223Ra-chloride.The semi-quantitative  Prostate cancer is the commonest cancer in men in the UK and is the second most common male cancer worldwide . Bone me99mTc-methylene diphosphonate (MDP) scintigraphy. However, it has been shown that the bone scan is limited in its sensitivity to measure treatment effect, with only 52% of responders showing scintigraphic improvement and 62% of non-responders showing scintigraphic deterioration at 6 to 8 months in one early study of patients with breast cancer [However, bone metastases are notoriously difficult to monitor during treatment, and in practice a combination of clinical, biochemical (e.g. prostate-specific antigen (PSA), serum alkaline phopshatase (ALP)) and imaging assessments are used . Howevert cancer -10, but t cancer  and quan18F-fluoride was first described as a bone imaging agent nearly 40 years ago [18F-fluoride PET and PET/CT has shown increased diagnostic accuracy compared to 99mTc-MDP planar and/or single-photon emission computed tomography (SPECT) imaging in prostate and other cancers [18F-fluoride PET might allow an accurate and timely measurement of the treatment response in bone metastases from prostate cancer. A previous report describes changes in 18F-fluoride uptake measured with PET following bisphosphonate treatment of glucocorticoid-induced osteoporosis [223Ra)-chloride , a bone-seeking alpha emitter with a half-life of 11.4 days, using semi-quantitative 18F-fluoride PET and to compare with changes in biochemical markers including PSA as a tumour marker and ALP as a bone formation marker.ears ago , but it  cancers -18. PET oporosis , but to 18F-fluoride PET imaging, PSA and ALP assessments were performed to assess treatment response. Biochemical results were retrieved from an institutional electronic patient record corresponding to the timing of the PET scans. Approval for this study was obtained from a research ethics committee and the national Administration of Radioactive Substances Advisory Committee, and the patients provided written informed consent.This imaging study was performed as a pilot substudy of an open-label phase 1 trial of Alpharadin in patients with bone metastases and castration-resistant prostate cancer. Repeated 99mTc-MDP bone scintigraphy and with no evidence of nodal or visceral disease on CT scan, were included in this pilot study. The mean age was 63.2 years .Five male subjects with osteoblastic bone metastases from castration-resistant prostate cancer, as determined by 223Ra-chloride intravenously at baseline and after 6 weeks. PSA, ALP and 18F-fluoride PET measurements were performed at baseline before the first administration, at 6 weeks just before the second administration and at 12 weeks to assess treatment response.The subjects received 100 kBq/kg of 18F-fluoride PET imaging was performed 1 h following injection of 250 MBq 18F-fluoride on a Gemini PET/CT scanner .A whole body Data were acquired for 3.5 min per bed position following a low-current (50 mAs) CT scan performed for attenuation correction and lesion localisation. Scans were assessed qualitatively by visual inspection for evidence of response at 6- and 12-week time points compared to the baseline scan and without knowledge of the PSA or ALP results. The images were analysed using the Hermes Gold 3 software . For the semi-quantitative assessment, five representative bone metastases were chosen that were greater than 2 cm in diameter in each subject. Lesions were selected to represent both axial and appendicular sites when present and the maximum standardised uptake value (SUVmax) calculated from an ROI of each lesion. The average SUVmax of the five lesions was then calculated for each patient at each scanning time point.The qualitative interpretation resulted in a classification of stable diseases in all five cases with no perceptible difference in uptake or number of lesions Figure . In the 18F-fluoride with PET in patients with bone metastases from prostate cancer receiving systemic therapy with 223Ra-chloride. Additionally, it has shown that the semi-quantitative analysis using SUVs is able to measure changes not detected by qualitative visual inspection.This pilot study has shown the feasibility of measuring changes in the uptake of 18F-fluoride uptake in bone metastases and in the biochemical parameters, PSA and ALP at 12 weeks. A more consistent correlation was present at 12 weeks than at 6 weeks. In two other subjects (D and E), small changes in 18F-fluoride uptake of less than 20% were seen, accompanied by small rises in PSA of less than 20% in both subjects but with larger changes in ALP  at 12 weeks.In this study, three subjects  showed a comparable reduction in 18F-fluoride is a bone-specific tracer and rather than indicating tumour metabolic activity directly, it is a marker of the local osteoblastic reaction of bone that accompanies most bone metastases [18F-fluoride uptake and ALP at 12 weeks in four out of five of the subjects  is a temporal discordance in changes with intrinsic tumour activity. Although not observed in this pilot study, the osteoblastic reaction to a metastasis by bone may subside at a different rate than the metabolic activity of the malignant tumour tissue, or an initial increase in osteoblastic activity may occur as part of a healing response in the surrounding bone of responding metastases, the so-called flare phenomenon [18F-fluorodeoxyglucose or radiolabelled choline, changes in 18F-fluoride uptake were similar to changes in PSA at 12 weeks.A potential weakness of an indirect assessment of tumour activity by measuring parameters that primarily reflect osteoblastic activity (i.e. ALP and enomenon . However18F-fluoride uptake is a closer predictor of the biochemical PSA response than at 6 weeks. Previous reports of the flare phenomenon measured biochemically with ALP [18F-fluoride PET scan is a more reliable measure of tumour response than the 6-week time point. An assessment at 12 weeks after commencing systemic therapy for bone metastases remains at an early enough time point to be clinically relevant in informing clinical management decisions and for measuring early response in clinical trials.Our study suggests that a 12-week semi-quantitative assessment of with ALP  or with 18F-fluoride PET was insensitive to changes at 6 or 12 weeks in this study, and it is possible that for the same reason, a qualitative interpretation of the 99mTc-MDP bone scintigraphy has not found universal favour and is relatively insensitive as a response assessment method, unless there are unequivocal new lesions beyond the expected flare period. In the absence of better imaging biomarkers, PET also has the inherent advantage over the conventional 99mTc-MDP bone scintigraphy of more accurate and absolute quantification of radioactive tracer concentrations, and therefore lends itself to a quantitative approach. A potential weakness of the methodology employed in this study was that the semi-quantitative assessment was only performed in five lesions in each subject. However, the lesions were chosen to represent different areas of the skeleton and whilst a global measure of skeletal metastases would be of utility, this has practical limitations, particularly in patients with extensive disease. A full kinetic analysis of the dynamic PET and blood data would also be of interest, but this approach, whilst potentially more sensitive to small changes in bone clearance of tracer, is also limited in practicality.A qualitative visual assessment of response using 18F-fluoride PET to measure early response in bone metastases are limited. In addition, it is not possible to extrapolate these results to different forms of systemic therapy, including hormones or chemotherapy, or in different tumour types where the mechanism of action of individual drugs and the differences in biological behaviour of tumour types and effects on the skeleton may differ. Nevertheless, we believe that the results are of sufficient interest to encourage further research into using 18F-fluoride PET as a biomarker of response in bone metastases in a variety of cancers and treatment types.These results are from a small pilot study, and the conclusions on the ability of 18F-fluoride PET as a method to monitor the treatment response in bone metastases following systemic therapy with 223Ra-chloride at clinically relevant intervals.The results of this pilot study suggest there may be a role for semi-quantitative ALP: alkaline phosphatase; MDP: methylene diphosphonate; PET: positron emission tomography; PSA: prostate-specific antigen; SUVmax: maximum standardised uptake value.CP is a consultant to Algeta ASA.GC study design, data acquisition, analysis, manuscript drafting and approval. CP study design, coordination of study, manuscript drafting and approval. SC data acquisition, manuscript drafting and approval. BJ coordination of study, patient recruitment, manuscript drafting and approval. AKA study design, coordination of study, manuscript drafting and approval. VL study design, data acquisition, analysis, manuscript drafting and approval.