PDB ID: 1JIE) was used as the template to model the protein followed by loop generation and development of troponin complex for both the troponin I wild type and four mutants (NCBI ID: PRJNA194382). The structural study was carried out to determine the effect of mutation on the structural stability and protein-protein interactions between three subunits in the complex. These mutations, especially the arginine to glutamine substitutions were found to result in local perturbations within the troponin complex by creating/removing inter/intra molecular hydrogen bonds with troponin T and troponin C. This has led to a decrease in the protein stability and loss of important interactions between the three subunits. It could have a significant impact on the disease progression when coupled with allelic heterogeneity which was observed in the cases carrying these mutations. However, this can be further confirmed by functional studies on protein levels in the identified cases.Hypertrophic Cardiomyopathy (HCM) is an autosomal dominant disorder of the myocardium which is hypertrophied resulting in arrhythmias and heart failure leading to sudden cardiac death (SCD). Several sarcomeric proteins and modifier genes have been implicated in this disease. Troponin I, being a part of the Troponin complex , is an important gene for sarcomeric function. Four mutations (1 novel) were identified in Indian HCM cases, namely, Pro82Ser, Arg98Gln, Arg141Gln and Arg162Gln in Troponin I protein, which are in functionally significant domains. In order to analyse the effect of the mutations on protein stability and protein-protein interactions within the Troponin complex, an in silico study was carried out. The freely available X-ray crystal structure ( Cardiac hypertrophy in HCM is a “compensatory” phenotype due to increased cardiac myocyte stress or altered CaThe cardiac myocyte is made of myofibrils and myofilaments. Myofibrils are made up of repeating units called sarcomere which are the basic contractile units of the myocyte. It contains the contractile proteins which are of 2 types- a) thick filament proteins -Myosin heavy chain (MYH7) and cardiac Myosin-binding protein-C (MYBPC3) which is reported to be implicated mostly in HCM b) thin filament regulatory proteins – Tropomyosin (TPM1), cardiac Troponin (cTn) and Actin (ACTC). The myosin has 2 heads with ATPase function which interacts with binding sites on actin forming cross bridges that leads to contraction –relaxation coupling. Tropomyosin is a rod shaped protein that is interdigitated between the alpha helical strands of actin.2+ concentration surrounding the contractile apparatus 2+ sensitivity is conferred on troponin by the troponin C subunit, which is an EF-hand Ca2+-binding protein. At micro molar Ca2+ concentrations troponin C binds strongly to troponin I, which is central to thin filament regulation, since it is an actin-binding inhibitory protein which on its own inactivates thin filaments and thus prevents it from binding and inhibiting actin. Hence, tropomyosin rolls out of the way of the actin filament active sites, so that myosin can attach to the thin filament and produce force and/or movement. In the absence of calcium, tropomyosin interferes with this action of myosin, and therefore muscles remain relaxed Troponin is attached to tropomyosin at regular intervals, and lies within the groove between actin filaments. In cardiac muscle, troponin and tropomyosin form the principal mechanism by which contractility is regulated in response to the CaThe integrated nature of the troponin-tropomyosin switch explains how mutations in any component of it can lead to a common outcome 2+-independent interactions between the carboxy terminal domain of Troponin C, the amino terminal domain of Troponin I comprising the first 98 residues and the last 50 residues of Troponin T The three subunits of the troponin complex are held together by Ca2+, respectively Multiple Troponin C and Actin binding domains within Troponin I have been localized to the car boxy region of the protein and this carboxy portion of Troponin I is designated the inhibitory peptide (IP) region which toggles between actin and Troponin C in the absence and presence of Ca2+-independent interactions with Troponin C The amino terminal domain of Troponin I interacts with Troponin T and is involved in the CaIn a recent study on the Indian population, screening of all the exons including the exon-intron boundaries of the Troponin I gene in 101 HCM patients along with 160 healthy controls revealed a total of 16 mutations, including 15 SNPs, and a 4 bp deletion polymorphism The high prevalence of Arginine → Glutamine mutations (4%) at three positions  and the absence of remaining reported mutations observed in other populations (2–7%), clearly elucidates the unique origin of Indian populations Hence, it was found fit to evaluate the influence of the mutations  in the disease progression by predicting the molecular changes in the protein through in silico models generated with the help of standard bioinformatics tools. This study is the first of its kind, considering that there is no available literature yet on the in silico modelling of the cardiac protein Troponin I and its mutant analogues, with reference to Indian population.These mutations could be predicted to significantly affect the disease progression due to protein instability in combination with allelic heterogeneity.th position is adjacent to the site of mutation, Arg98Glu but does not influence the side conformation of the neighbouring residues. The main chain conformations of all the non-glycine residues in the model structure were found to be in the allowed regions of Ramachandran plot. The model structure consists of three long helices connected by loops similar to the template structure. The model structure generated was continuous for the region comprising residues 40 to 191 wherein the helix 2 and helix 3 are connected by a loop (residues 137 to 146). This loop was not observed in the template structure  at positions 80 and 97 which is cysteine in the present protein . Though tructure .de novo modelled loop of 10 residues and model structure of mutants was submitted in BioProject at NCBI with accession ID of PRJNA194382.The co-ordinates for the residues 137–146 were generated by loop modelling program and resulted in 3-D structure of continuous region of Troponin I protein from residues 40–191. The region of residues from 137 to 146 was modelled as loop . Hence, The change at residue 98 from Arg to Gln due to point mutation does not affect the overall conformation of the protein. The point mutation occurs in Helix 2 but does not disturb the stability of the α-helical secondary structure. However, the two residues, Arg and Gln, differ in both size and charge as Arg is larger and more positively charged compared to the smaller Gln. The Arg98 in wild type Troponin I forms a intramolecular hydrogen bond with side chain of Gln94 of Troponin I and two intermolecular hydrogen bonds with OD1 and OD2 of Asp221 of Troponin C protein . This faThe residue Arg141 in Troponin I occurs in the loop region. This basic Arg residue in the mutant is replaced by the shorter Gln residue. The Arg141Gln point mutation produces a minor local conformational change due to the difference in the observed contacts, resulting in an increase in the potential energy  . The guaThe 162 residue present in Helix 3 of Troponin I is positioned away from the region of contact between the three subunits of troponin complex. Thus the substitution of this residue may not affect the protein-protein interaction, but the residue Arg162 forms intra chain hydrogen bonds with carboxylate oxygen of Glu165 and hydroxyl group of Ser166 . Loss ofIn the native structure, the Proline residue, located in the loop region, is not involved in any intra or intermolecular interactions. Proline possesses an imino group and the change to Serine introduces a polar hydroxyl group which unlike Proline has hydrogen bonding capabilities. But hydroxyl group of Ser82 side chain in mutated protein does not form any inter molecular hydrogen bond and hence potential energy does not change much  . HoweverThis study mainly highlights on arginine to glutamine substitution mutations in Troponin I which are found to be highly prevalent in the Indian population leading to sudden cardiac death (SCD) These mutations mainly show the effect of hydrogen bonds on the stability of protein as well as stability of intermolecular assembly. In the unfolded state, all potential hydrogen bonding partners in the extended polypeptide chain are satisfied by hydrogen bonds to water. When the protein folds, these protein-to-water hydrogen bonds are broken and only some are replaced by sub-optimal intra-protein hydrogen bonds. Despite the small contribution made to protein stability by hydrogen bonds, if an intramolecular hydrogen bond in a protein is disrupted, without the possibility of forming a compensating hydrogen bond to solvent, that protein will be destabilized The novel mutation Arg98Gln in exon 6 of Troponin I gene, was observed in a 28 years old severe hypertrophic obstructive cardiomyopathy patient (HOCM) with interventricular septum (IVS) thickness of 25 mm and his 5 years old asymptomatic son, who has a family history of sudden cardiac death The dominant Arg141Gln mutation in exon 7 of Troponin I gene was observed in two individuals with severe familial asymmetric septal hypertrophy (ASH+), with interventricular septum (IVS) thickness of 25 and 28 mm, respectively The dominant Arg162Gln mutation in exon 7 of Troponin I gene was observed in an individual with severe asymmetric septal hypertrophy (ASH) with mean thickness of 29 mm had abnormal echocardiogram/ECG It is known that the non-random use of synonymous codons creates codon usage bias The mutant does not introduce any conformational variation as it is housed in the region of the stable helical secondary structure but results in a reduction in the protein stability as indicated by lesser negative potential energy  compared to wild type. This region of the protein does not make direct contact with other subunits of the complex and hence the allelic heterogeneity may be involved in the disease progression which is supported by the fact that four out of 7 Individuals with the allelic heterogeneity had presented with severe septal hypertrophy (ASH++) with the mean thickness of 27, 28, 29, 32 mm and the ECGs were abnormal in all the four individuals C → T transition resulting in the replacement of proline with serine (Pro82Ser) in exon 5, lying within the troponin C binding domain, was observed in 2 HCM patients and a control individual. This mutation improves the protein-protein interactions between the TnI and TnC subunit due to an additional hydrogen bond being formed.Destabilisation leads to rapid degradation of the mutant protein by cellular proteases, which reduces the steady-state concentration of the protein within the cell. The susceptibility of a protein to such proteolytic breakdown depends upon its kinetics of monomer folding and oligomer assembly and upon the intrinsic (thermodynamic) stability of its functional native-state conformation Hence three of the four mutations destabilise the protein while the Pro82Ser mutation stabilises the protein complex.in silico protein modelling for the first time. It was hypothesized that these mutations  may be responsible for gross protein instability which in turn might affect its interactions with other sarcomeric proteins, hence leading to the pathogenesis of cardiac hypertrophy in the HCM patients. The hypothesis proved correct since it was found that these mutations lead to local perturbations in the protein making the complex less stable and it was observed that allelic heterogeneity may also be involved in the disease progression.The effect of 4 exonic mutations (1 novel) of troponin I which were identified in the Indian population, were analysed by in silico in this study. Further functional studies need to be done to establish protein turnover in wild type and mutant cell lines in order to validate the hypothesis that the protein instability induced due to the mutations and allelic heterogeneity are responsible for the disease progression and severity.The conformational changes induced in the protein structure due to the various point mutations were analysed PDB id: 1J1E) was available The effect of the mutations  on the structure and function of the Troponin I protein was analysed. The X-ray crystal structures of human cardiac Troponin I protein in complex with Troponin C and Troponin T  of Troponin I was developed individually using structure developed for native protein along with newly generated loop with the help of ‘Build Mutant’ protocol of DS 2.0. This protocol changes the selected residue into the desired residue and optimizes the conformation of mutated residue along with neighbouring residues using MODELER. The modelled region of the mutant structure was refined individually by subjecting to similar energy minimization protocol that was used for the native structure. The potential energy of native troponin I and four mutants was calculated using ‘calculate energy’ protocol available in DS 2.0 in the presence of distance dependent dielectric constant and spherical cut-off model of electrostatics with the help of CHARMm force field (c33b1).The crystal structure of Troponin I protein complexed with Troponin C and Troponin T was used as framework to develop the complex structure of Troponin that includes the modelled region of Troponin I (137–146). The structure of native as well as mutant Troponin I along with modelled region (137–146) was superimposed on the chain corresponding to Troponin I of crystal complex. This newly developed complex was refined by three steps of energy minimization to relax the constraints on the geometry in the newly built structure of the complex. The energy minimization was performed using the force field parameters of CHARMm (c33b1)


Cerebrospinal fluid (CSF) α-synuclein is reduced in synucleinopathies, including dementia with Lewy bodies, and some studies have found increased CSF α-synuclein in Alzheimer’s disease (AD). No study has explored effects of CSF α-synuclein on brain atrophy. Here we tested if baseline CSF α-synuclein affects brain atrophy rates and if these effects vary across brain regions, and across the cognitive spectrum from healthy elders (NL), to patients with mild cognitive impairment (MCI) and AD.Baseline CSF α-synuclein measurements and longitudinal structural brain magnetic resonance imaging was performed in 74 NL, 118 MCI patients and 55 AD patients. Effects of baseline CSF α-synuclein on regional atrophy rates were tested in 1) four pre-hoc defined regions possibly associated with Lewy body and/or AD pathology , and 2) all available regions of interest. Differences across diagnoses were tested by assessing the interaction of CSF α-synuclein and diagnosis .The effects of CSF α-synuclein on longitudinal atrophy rates were not significant after correction for multiple comparisons. There were tendencies for effects in AD in caudate  and brainstem . CSF α-synuclein had significantly different effects on atrophy rates in NL and AD in brainstem (P=0.037) and caudate (P=0.006).Discussion: With the possible exception of caudate and brainstem, the overall weak effects of CSF α-synuclein on atrophy rates in NL, MCI and AD argues against CSF α-synuclein as a biomarker related to longitudinal brain atrophy in these diagnostic groups. Any effects of CSF α-synuclein may be attenuated by possible simultaneous occurrence of AD-related neuronal injury and concomitant Lewy body pathology, which may elevate and reduce CSF α-synuclein levels, respectively.  Cerebrospinal fluid (CSF) α-synuclein is reduced in most patients with synucleinopathies, including Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) –4. This AD patients with Lewy body pathology have accelerated cognitive decline , suggestWe tested the a priori hypothesis that baseline levels of CSF α-synuclein have effects on regional atrophy rates. We tested this primarily in amygdala (which is a region where AD patients are likely to have both Lewy body pathology  and earlData used in the preparation of this article were obtained from the ADNI database (adni.loni.ucla.edu). Details about the ADNI are given in the Acknowledgments section. Written informed consent was obtained from all participants and the study was conducted with prior institutional ethics approval.We examined volumetric changes in regions of interest (ROI) in NL, MCI and AD. Structural magnetic resonance imaging brain scans at multiple time points  because we made use of data from as many subjects as possible with both imaging and CSF.This was an ADNI study. The ADNI was launched in 2003 by the National Institute on Aging (NIA), the National Institute of Biomedical Imaging and Bioengineering (NIBIB), the Food and Drug Administration (FDA), private pharmaceutical companies and non-profit organizations, as a $60 million, 5-year public-private partnership. The primary goal of ADNI has been to test whether serial MRI, PET, other biological markers, and clinical and neuropsychological assessment can be combined to measure the progression of MCI and early AD. Determination of sensitive and specific markers of very early AD progression is intended to aid researchers and clinicians to develop new treatments and monitor their effectiveness, as well as lessen the time and cost of clinical trials. The Principal Investigator of this initiative is Michael W. Weiner, MD, VA Medical Center and University of California – San Francisco. ADNI is the result of efforts of many co-investigators from a broad range of academic institutions and private corporations, and subjects have been recruited from over 50 sites across the U.S. and Canada. The initial goal of ADNI was to recruit 800 subjects but ADNI has been followed by ADNI-GO and ADNI-2. To date these three protocols have recruited over 1500 adults, ages 55 to 90, to participate in the research, consisting of cognitively normal older individuals (NL), people with MCI, and people with early AD. Inclusion criteria for NL subjects were mini-mental state examination (MMSE) scores 24-30, Clinical Dementia Rating (CDR) 0, no depression, no MCI, and no dementia. MCI subjects had MMSE 24-30, memory complaint, and objective memory loss (adjusted for education) measured by Wechsler Memory Scale Logical Memory II, CDR 0.5, absence of significant levels of impairment in other cognitive domains, essentially preserved activities of daily living, and no dementia. AD subjects had MMSE 20-26, CDR 0.5-1, and met the NINCDS-ADRDA criteria for probable AD. For up-to-date information on ADNI, see http://www.loni.ucs.edu/ADNI/Research/Cores/index.shtml), which included two T1-weighted MRI scans using a sagittal volumetric magnetization prepared rapid gradient echo (MP-RAGE) sequence. Only one of the MP-RAGE sets was used for analysis. The ADNI MRI quality control center at the Mayo Clinic selected the MP-RAGE image with higher quality and corrected for system-specific image artifacts, as previously described  or k=48 [all available ROI]). All statistics were done using R . The software “Medical Image Processing, Analysis and Visualization”  was used to plot statistics on brain images.All tests were two-sided and considered significant at P<0.05. When indicated in the manuscript, P-values were corrected for multiple comparisons, using a false discovery rate (fdr) correction. Corrections were applied to all tested regions in each diagnostic group , P=0.49 for age, and P=0.65 for sex. In the results presented in this paper on the effects of CSF α-synuclein on atrophy rates, we adjusted all models for APOE genotype, age and sex. However, the main results were only marginally different when not adjusting for these co-variates (data not shown).Age, sex, and CSF α-synuclein:time interaction effect was not significantly associated with missing visits (P=0.13), suggesting that study drop-out was not associated with baseline CSF α-synuclein.Several subjects lacked MRI data for one or more visit see , but thea priori defined primary ROI for this study were brainstem, caudate, amygdala and hippocampus. See APOE and sex. The patterns of atrophy rates differed across the subgroups. In brainstem, all groups had significant and similar atrophy rates . In caudate, the atrophy rate was significantly different from zero only in AD . In amygdala and hippocampus, all groups had significant atrophy rates, with steeper slopes in AD  and MCI  than in NL . We tested the interactions between diagnoses and time to assess if the differences in slopes were significant, and found significantly different slopes for AD versus NL in caudate (P=0.021), MCI versus NL in amygdala (P=0.022), AD versus NL in amygdala (P=0.023), MCI versus NL in hippocampus (P<0.001), and AD versus NL in hippocampus (P<0.001). The p-values in this paragraph are not adjusted for multiple comparisons, since they are not part of the primary aim of the study.The a priori defined ROI, within the groups of NL, MCI and AD patients. Effects were adjusted for age, APOE and sex. There were no significant effects of CSF α-synuclein after correction for multiple comparisons. See We next tested if baseline CSF α-synuclein had effects on atrophy rates in the We next compared effects of baseline CSF α-synuclein on atrophy rates across diagnostic groups, testing NL versus MCI, and NL versus AD. For NL versus AD, significant CSF α-synuclein:diagnosis:time interactions were found in 1) the brainstem, where AD patients with low baseline CSF α-synuclein had higher atrophy rates while the opposite was seen in NL , and 2) the caudate, where AD patients with high baseline CSF α-synuclein had higher atrophy rates while the opposite was seen in NL . The interaction in caudate was not present after removal of two AD subjects with high CSF α-synuclein and large atrophy rates.We finally tested effects of CSF α-synuclein on longitudinal volume changes in all available ROI (N=48) in NL, MCI and AD tested separately. No longitudinal change was significantly affected by baseline CSF α-synuclein after fdr correction for multiple comparisons. Before correcting for multiple comparisons, there were effects in NL in the lateral ventricle ; and in AD in the transverse temporal region , in the banks of the superior temporal sulcus , in the caudate , and in the inferior temporal region . To visualize this exploratory analysis, we generated whole brain maps showing t-values for the effects of CSF α-synuclein on regional volume change , panel BThe key findings of this study, which to our knowledge is the first study on effects of CSF α-synuclein on brain atrophy, were that (1) baseline CSF α-synuclein had no significant effects on atrophy rates in amygdala, caudate, brainstem and hippocampus in the individual groups of NL, MCI and AD; (2) when comparing groups, there were some significant differences across the diagnostic groups, with stronger effects of CSF α-synuclein on atrophy rates in AD than in NL in caudate and brainstem; (3) in an exploratory analysis of all available brain regions, the effects of CSF α-synuclein seemed to vary across brain regions and diagnosis, although no effects reached statistical significance after correcting for multiple comparisons.a priori defined brain regions amygdala, caudate, brainstem and hippocampus, within the diagnostic groups. This finding argues against the use of CSF α-synuclein as a stand-alone indicator of future risk of brain atrophy within the cognitive spectra from NL to MCI and AD. It is possible that the combination of CSF α-synuclein with other biomarkers have stronger effects on brain atrophy than individual biomarkers. A recent study found that the combination of CSF α-synuclein with the core CSF AD biomarkers Aβ42, total tau (T-tau) and phosphorylated tau (P-tau) enhances the diagnostic accuracy for MCI to AD conversion compared to the individual biomarkers [The first major finding of this study was that baseline CSF α-synuclein lacked significant effects on atrophy rates in the omarkers . Future The second major finding of this study was that although the effects of CSF α-synuclein on atrophy rates were non-significant within the individual diagnostic groups, there were significant differences between NL and AD in brainstem and caudate, when comparing these diagnostic groups with each other. In the brainstem, AD patients with low baseline CSF α-synuclein had high atrophy rates, while the opposite was seen in NL. A possible interpretation of this is that concurrent Lewy body pathology, as reflected by low CSF α-synuclein levels, are more strongly associated with brainstem atrophy in AD than in NL. The brainstem is interesting, since it is a typical region for early Lewy body deposits . HoweverThe final part of this study was an exploratory analysis of all available regions. After correction for multiple comparisons, there were no significant effects of CSF α-synuclein. However, although the effects were non-significant, the patterns seemed to differ between NL and MCI/AD. In NL and MCI, the effects of CSF α-synuclein varied, and atrophy rates were related to either high or low CSF α-synuclein . In AD, high atrophy rates tended to be related to high CSF α-synuclein, with some exceptions, for example the brainstem, where high atrophy rates tended to be related to low CSF α-synuclein. This difference between groups may be related to random variations, since atrophy rates were larger in AD. We observed that longitudinally increased ventricle volumes generally were related to low CSF α-synuclein. Further studies of white matter structure and lesions may be useful to clarify if atrophy of periventricular regions is related to Lewy body pathology, as reflected by low CSF α-synuclein.It should be emphasized that this was not a study of subjects afflicted with disorders known to be primarily associated with α-synuclein pathology. Both PD and DLB are characterized by the presence of Lewy Bodies and α-synuclein in the brain. Patients with PD are being studied in the Parkinsons Progressive Biomarkers Initiative and CSF α-synuclein is being collected from these patients. Therefore, the results of the PPMI project  will be interesting to compare with the ADNI data. We know of no study measuring longitudinal atrophy in DLB subjects with CSF α-synuclein measurements.Limitations of this study include the small sample size, and the lack of autopsy follow-up. Also, since only a subset of AD and MCI patients are likely to have concomitant Lewy body pathology, it is possible that a larger study cohort is needed to detect any effects of CSF α-synuclein on Lewy body related atrophy. This is especially true if CSF α-synuclein is affected by opposite forces in AD, with Lewy body pathology reducing levels and neuronal injury increasing levels, attenuating any effects of the biomarker, as has recently been suggested . AnotherThis is the first study of CSF α-synuclein and brain atrophy rates in NL, MCI and AD. The findings were generally negative, with small or no effects of baseline CSF α-synuclein and atrophy rates. Future studies may incorporate other biomarkers to identify AD patients with likely concomitant Lewy body pathology, and test if these patients have unique relationships between CSF α-synuclein and patterns of atrophy.Figure S1Y-axes show slopes for individual subjects from linear mixed effects models, adjusted for age, and x-axes show baseline CSF α-synuclein levels. The panels show data for the lateral ventricles in NL, MCI and AD. Regression lines were generated by regressing CSF α-synuclein levels on the slope. B-coefficients and p-values are presented for the interaction CSF α-synuclein:time.(TIFF)Click here for additional data file.


To investigate the effects of G-CSF or GM-CSF therapy in non-neutropenic patients with sepsis.A systematic literature search of Medline, Embase and Cochrane Central Register of Controlled Trials was conducted using specific search terms. A manual review of references was also performed. Eligible studies were randomized control trials (RCTs) that compared granulocyte-colony stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF) therapy with placebo for the treatment of sepsis in adults. Main outcome measures were all-cause mortality at 14 days and 28 days after initiation of G-CSF or GM-CSF therapy, in-hospital mortality, reversal rate from infection, and adverse events.P = 0.44; P for heterogeneity = 0.31, I2 = 15%). Compared with placebo, G-CSF or GM-CSF therapy did not significantly reduce in-hospital mortality . However, G-CSF or GM-CSF therapy significantly increased the reversal rate from infection . No significant difference was observed in adverse events between groups . Sensitivity analysis by excluding one trial did not significantly change the results of adverse events .Twelve RCTs with 2,380 patients were identified. In regard to 14-day mortality, a total of 9 death events occurred among 71 patients (12.7%) in the treatment group compared with 13 events among 67 patients (19.4%) in the placebo groups. Meta-analysis showed there was no significant difference in 28-day mortality when G-CSF or GM-CSF were compared with placebo (relative risks (RR) = 0.93, 95% confidence interval (CI): 0.79 to 1.11, There is no current evidence supporting the routine use of G-CSF or GM-CSF in patients with sepsis. Large prospective multicenter clinical trials investigating monocytic HLA-DR (mHLA-DR)-guided G-CSF or GM-CSF therapy in patients with sepsis-associated immunosuppression are warranted. Despite improvements in antimicrobial therapy and supportive care, the incidence of sepsis continues to rise and sepsis is now the third leading cause of infectious deaths in the United States , with a ex vivo whole blood cultures of patients with severe sepsis [To date, one approach is the use of granulocyte colony stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF), to augment myeloid cell functions in patients with sepsis. G-CSF, widely used in reducing the duration of febrile neutropenia following cytotoxic chemotherapy, has been shown to stimulate the production of neutrophils and modulate the function and activity of developing and mature neutrophils . Comparee sepsis ,26.So far, G-CSF and GM-CSF have shown promise in the treatment of infection in non-neutropenic hosts in many animal models -30. AddiWe followed the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses for reporting our meta-analysis and results .We searched Pubmed, Embase, and the Cochrane Central Register of Controlled Trials (to 25 October 2010) to identify potentially relevant trials. We restricted the search to trials on adults and used the search terms "granulocyte colony-stimulating factor", "granulocyte colony stimulating factor, recombinant", "GCSF", "filgrastim", "lenograstim", "sargramostim", "pegfilgrastim", "granulocyte-macrophage colony-stimulating factor", "granulocyte-macrophage colony stimulating factor, recombinant", "GMCSF", "molgramostim", AND "sepsis", "septicemia", "septicaemia", "septic shock". We restricted the findings of this search with a highly sensitive search strategy recommended by the Cochrane Collaboration for identifying all randomized controlled trials . In addiWe considered trials that investigated the therapeutic effects of G-CSF or GM-CSF administered intravenously or subcutaneously in adults with sepsis. Sepsis was defined according to the American College of Chest Physicians/Society of Critical Care Medicine consensus criteria  or was eFull text versions of all eligible trials were obtained for quality assessment and data extraction independently by two reviewers. Extracted data were entered into Microsoft Excel 2007 and were checked by a third reviewer. Disagreement or doubt was resolved by discussion. Abstracted data included study design , patient characteristics, study methodology , intervention  and main outcomes. The quality of trials was assessed with the methods recommended by the Cochrane Collaboration for assessing risk of bias . The criThe primary outcomes of this meta-analysis were all-cause mortality at 14 days and 28 days after initiation of G-CSF or GM-CSF therapy. Secondary outcomes included in-hospital mortality, reversal rate from infection, and adverse events. The definition of reversal of infection referred to resolution of all signs, symptoms and laboratory assessment of infection or recovery from sepsis, which varied among trials due to different origins of sepsis. Adverse events were defined as organ dysfunction that was life threatening, required treatment and prolongation hospitalization, or was associated with death of the patient.P-value of χ2 test less than 0.10 and I2 greater than 50%). Potential sources of heterogeneity were identified by subgroup analysis on the basis of G-CSF and GM-CSF or/and by sensitivity analyses performed by omitting one study in each turn and investigating the influence of a single study on the overall meta-analysis estimate. Publication bias was assessed using funnel plots. A P-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using Review Manager, version 5.0 .Analyses were on an intention-to-treat basis. We calculated a weighted treatment effect across trials using fixed-effect model. The results were expressed as relative risks (RRs) with 95% confidence intervals (CIs) for dichotomous outcomes. We considered using random-effects model only in case of heterogeneity  [P = 0.44; P for heterogeneity = 0.31, I2 = 15%; Figure n = 2,044) [P = 0.60; P for heterogeneity = 0.13, I2 = 41%). Meanwhile, subgroup analysis of the other three trials of GM-CSF (n = 89) [P = 0.28; P for heterogeneity = 0.91, I2 = 0%).Data for 28-day mortality were extracted from nine trials = 2,133) -44,46,483 -44,46,= 2,044) ,42,43,46P = 0.86; P for heterogeneity = 0.80, I2 = 0%; Figure n = 945) [P = 0.95; P for heterogeneity = 0.67, I2 = 0%). Meanwhile, subgroup analysis of the other two trials of GM-CSF (n = 51) [P = 0.76; P for heterogeneity = 0.38, I2 = 0%).In-hospital mortality for patients who were treated with G-CSF or GM-CSF was 54/501 (10.8%), and that for patients treated with placebo was 54/495 (10.9%), according to five trials ,41,44,47n = 945) ,39,47 shP = 0.002; P for heterogeneity = 0.47, I2 = 0%; Figure n = 1,261) [P = 0.007; P for heterogeneity = 0.84, I2 = 0%). The other one trial of GM-CSF (n = 33) [P = 0.03).Data for reversal rate from infection were available from four studies -39,44. T= 1,261) -39 showeP = 0.62) [P for heterogeneity = 0.03, I2 = 58%). On the basis of the results of the sensitivity analysis, one study was excluded [P = 0.44; P for heterogeneity = 0.17, I2 = 36%; Figure Overall, there were no significant differences in adverse events between treatment group and placebo group according to the data from seven trials  ,43,45,47excluded . ExclusiDuring the past few decades, there were a variety of trials investigating the effect of G-CSF or GM-CSF therapy in patients with sepsis. However, consistent results have not been reported and no individual study has definitively established whether G-CSF or GM-CSF bring clinically important benefits to septic patients. In the present meta-analysis, we found no significant differences in all-cause mortality at 14 days or 28 days, in-hospital mortality, or adverse events between the G-CSF or GM-CSF group and placebo group in adult patients with sepsis. However, our result indicated that G-CSF or GM-CSF therapy was associated with a significant increase in reversal rate from infection.P = 0.05) in severe septic patients due to melioidosis [P <0.001) [et al. [With respect to mortality, a previous meta-analysis suggested that addition of G-CSF or GM-CSF to antibiotic therapy in preterm infants with suspected systemic infection did not significantly reduce all cause mortality at 14 days or in-hospital mortality . Recentlioidosis . Meanwhi <0.001) . However [et al. , none of [et al. . As oppo [et al. ,51. Rece [et al.  exploredex vivo tumor necrosis factor (TNF)-α production after LPS challenge [et al. observed that G-CSF-induced restoration of mHLA-DR was not only accompanied by an increased lymphocyte proliferation and Th1 cytokine production (interleukine (IL)-2 and interferon (IFN)-γ) in response to PHA but also by a better capacity to release inflammatory cytokines in a whole blood model after LPS challenge [et al. demonstrated that addition of GM-CSF to the standard treatment of patients with nontraumatic abdominal sepsis reduced the rate of infectious complications, shorten the duration of antibiotic therapy and the length of hospital stay [P = 0.002). However, it did not bring a significant benefit in 28-day mortality . Due to the complexity of sepsis and associated complications, the severity of sepsis varied largely. Therefore, G-CSF or GM-CSF therapy might not be able to bring a significant mortality benefit if administrated regardless of the specific situation in individual patient, especially the immunological state of enrolled patients.Treatment with G-CSF or GM-CSF enhanced cellular functions that were critical in infectious diseases, such as neutrophil, monocyte, and macrophage activation and increased circulating white blood cells -54. In ahallenge . Moreovehallenge . Theorettal stay . Rosen atal stay  observedet al demonstrated that G-CSF caused leukocyte stiffness but attenuated inflammatory response without inducing lung injury in septic patients [et al. demonstrated that low-dose GM-CSF was associated with improved gas exchange without pulmonary neutrophil infiltration and was not associated with worsened acute respiratory distress syndrome or the multiple organ dysfunction syndromes in patients with sepsis-associated respiratory dysfunction [P = 0.62; P for heterogeneity = 0.03, I2 = 58%). Definitions of adverse events in included studies were heterogeneous and sometimes absent, which might bring about heterogeneity. Sensitivity analysis did not significantly change the results of adverse events .Administration of G-CSF or GM-CSF theoretically may increase the prevalence of adverse events which include immunologically mediated organ dysfunction, such as acute respiratory distress syndrome (ARDS), to which patients with severe sepsis are particularly prone . Howeverpatients . Anotherpatients ,42. Recefunction . ConsistThere are several limitations that need to be considered in the present study. First, the geographic regions covered in this meta-analysis included North America , Europe , Asia (Japan and Thailand) and Oceania . Therefore, our results limited generalizability to other regions . Second, considerable heterogeneity existed in the type and dosage of G-CSF and GM-CSF. Different baseline characteristics such as age and APACHE II score might have affected the outcome of patients' response to medical management and might have produced possible clinical heterogeneity. Finally, the sample sizes were highly variable across trials and the trial enrolling the maximum number of patients  containeWhile this meta-analysis demonstrated that G-CSF or GM-CSF therapy significantly increased the reversal rate from infection, it was not associated with a significant reduction in all cause mortality at 14 days or 28 days, in-hospital mortality or adverse events in patients with sepsis. Therefore, our present meta-analysis did not suggest routine use of G-CSF or GM-CSF in patients with sepsis. Large prospective multicenter clinical trials investigating mHLA-DR-guided G-CSF or GM-CSF therapy in patients with sepsis-associated immunosuppression are warranted.• The literature shows that G-CSF or GM-CSF result in no differential effectiveness in treating sepsis, in terms of all-cause mortality at 14 days or 28 days, in-hospital mortality or adverse events. However, G-CSF or GM-CSF therapy significantly increased the reversal rate from infection in patients with sepsis.• There was a lack of consensus in the literature in regard to the effect of G-CSF or GM-CSF versus placebo in treating sepsis in adults.• Larger prospective randomized controlled trials investigating monocytic HLA-DR (mHLA-DR)-guided G-CSF or GM-CSF therapy in sepsis are warranted.CIs: confidence intervals; G-CSF: granulocyte-colony stimulating factor; GM-CSF: granulocyte-macrophage colony stimulating factor; LPS: lipopolysaccharide; mHLA-DR: monocytic HLA-DR; PHA: phytohemagglutinin; RCTs: randomized control trials; RRs: relative risks.The authors declare that they have no competing interests.LB and FW conceived the study and wrote the manuscript. LB, FW and JZ designed and performed searches and participated in the extraction and analysis of the data. Both XD and JL designed the study, supervised all of the study work and statistical analysis, and helped with manuscript revisions. All authors read and approved the final manuscript.


Background. Abdominoscrotal sonogram is often used in boys with a nonpalpable testis to determine the presence of the testis. We describe our experience with the use of ultrasonography in boys with a nonpalpable testis. Methods. We retrospectively reviewed the medical records of boys aged less than 14 years, who underwent preoperative ultrasonography for an impalpable undescended testis (IUDT) between 2006 and 2010 in our institution. The results of sonography and laparoscopy were compared. Results. A total of 26 impalpable testes in 22 patients, including 4 with bilateral impalpable testes, were preoperatively assessed by ultrasonography for their localization. Sonography localized only 6 of the 26 (23%) testes, which were laparoscopically explored and followed by orchiopexy. Conclusion. Ultrasound is not reliable in the preoperative assessment of patients with impalpable testes. About 1-2% of boys at age 1 year have an undescended testis (UDT); this disorder is unilateral in about 90% of cases and bilateral in about 10% . Almost Among the radiologic methods used to try to “localize” the nonpalpable testis preoperatively are retrograde venography, computerized tomography (CT), magnetic resonance imaging (MRI), and sonography –12. AbdoWe retrospectively reviewed the medical records of all patients less than 14 years who underwent abdominoscrotal ultrasonography for impalpable undescended testes (IUDT) between 2006 and 2010 at our institution. Data reviewed included patient age at surgery, ultrasound results, and intraoperative laparoscopic findings. The results of sonography were compared with those of laparoscopy.We identified a total of 22 patients with 26 impalpable testes, including 4 patients with bilateral impalpable testes, who underwent ultrasonography during the study period. Average patient age at the time of laparoscopy was 31 months. Ultrasonography was successful in localizing 8 of 26 (31%) testes .Only 6 of 26 (23%) testes explored by laparoscopy were identified by sonography, with five of the detected testes localized at the inguinal ring on laparoscopy. In contrast, 7 of the 18 (38.9%) testes not seen on ultrasonography were at the internal ring, whereas two testes identified by ultrasonography were absent during surgical exploration.Cryptorchidism is one of the most common genitourinary disorders in young boys. Although the management of boys with palpable testes has been standardized, there are no formal guidelines for the management of boys with nonpalpable testes .Orchiopexy is necessary, whether or not ultrasonography localizes a testis. If ultrasonography fails to demonstrate a viable testis, surgical exploration is still necessary, because neither CT nor MRI has been shown to be reliable in diagnosing a vanishing testis. Consequently, routine preoperative imaging is generally unnecessary, although obese boys may require imaging, since physical examination for an undescended testis can be difficult . NeverthLaparoscopy is currently the most reliable diagnostic modality used in the management of impalpable testes. This method clearly shows the anatomy and provides visual information upon which a definitive decision can be based .It was found that sonography is unnecessary in boys with a nonpalpable testis, because it rarely, if ever, localizes a true nonpalpable testis, and it does not alter the surgical approach in these patients . UltrasoBrody  proposed


Voxel-based linear regression analysis revealed significant associations between CSF BACE1 activity and [11C]PIB tracer uptake in the bilateral parahippocampal region, the thalamus, and the pons. Our study provides evidence for a brain region-specific correlation between CSF BACE1 activity and in-vivo fibrillar amyloid pathology in AD. Associations were found in areas close to the brain ventricles, which may have important implications for the use of BACE1 in CSF as a marker for AD pathology and for antiamyloid treatment monitoring.The secretase BACE1 is fundamentally involved in the development of cerebral amyloid pathology in Alzheimer's disease (AD). It has not been studied so far to what extent BACE1 activity in cerebrospinal fluid (CSF) mirrors in vivo amyloid load in AD. We explored associations between CSF BACE1 activity and fibrillar amyloid pathology as measured by carbon-11-labelled Pittsburgh Compound B positron emission tomography ([ Although [11C]PIB binds to several types of cerebral Aβ, including plaque, nonplaque, and vascular deposits, it has only minimal affinity to other protein aggregates such as Lewy bodies or neurofibrillary tangles [11C]PIB tracer uptake and CSF markers related to amyloid pathology such as Aβ42 further underscore the relevance of [11C]PIB PET imaging as an AD biomarker PIB tracer uptake and BACE1 activity in CSF in order to provide evidence in support of BACE1 activity as an in-vivo biomarker for AD pathology.In the present study, we quantitatively analyzed CSF BACE1 activity and [18F]FDG as well as [11C]PIB PET imaging of the brain. The psychometric workup was based on the Consortium to Establish a Registry for AD (CERAD) neuropsychological battery, which includes the Mini-Mental-State Examination (MMSE). Patients with mild to moderate dementia, who had a typical metabolic pattern for AD on [18F]FDG PET scans [11C]PIB PET scans and BACE1 activity values were not used for diagnostic purposes. Written informed consent was obtained from all participants according to the 1975's Helsinki Declaration, and the study protocol was approved by the ethics committee of the Medical Faculty at the Technische Universität München.Thirty-one patients with probable AD were recruited from the outpatient memory clinic of the Department of Psychiatry and Psychotherapy at the Technische Universität München. The clinical diagnosis was established by consensus of two experienced clinicians according to the NINCDS-ADRDA criteria. The clinical assessment included patient and proxy interviews, cognitive testing, physical examination, routine blood and CSF sampling, structural MRI, and FDG PET, and [11C]PIB PET scans of the brain were acquired according to published standard procedures [11C]PIB PET data. For spatial transformation of [11C]PIB data, the individual standardized uptake value images (40–70 min p.i.) were coregistered to the corresponding high-resolution MRI scans and then normalized to the Montreal Neurological Institute space  with the warping parameters of the individual's MRI to obtain images comparable between patients. In order to adjust for interindividual differences in tracer uptake, a relative measure of [11C]PIB was obtained by calculating a cerebral to cerebellar vermis ratio as demonstrated previously [11C]PIB scans was divided by the individual mean value in the cerebellum. The individual normalized images were then smoothed with a Gaussian kernel of 10 × 10 × 10 mm.Structural MRI, PIB tracer uptake, a voxel-based linear regression analysis was performed within SPM8 with tracer uptake as the dependent variable and CSF BACE1 activity as the independent variable, adjusting for factors known to impact on [11C]PIB uptake and amyloid pathology including APOE genotype PIB PET scan and lumbar puncture was 29 ± 41 days. Men and carriers of an APOEε4 allele were slightly overrepresented in the sample. Visual inspection of the [18F]FDG PET scans demonstrated a typical pattern for AD in all patients, that is, a bilateral affection of temporal and/or parietal and/or posterior cingulate cortex [11C]PIB scans showed an AD-typical tracer uptake as previously described in all patients, including frontal and temporoparietal cortex, precuneus, posterior cingulate gyrus, and striatum, and less retention in sensorimotor, occipital, and cerebellar cortex [11C]PIB-negative scan. The CSF concentrations of tau and Aβ42 were in the expected ranges for this type of sample [Patient characteristics are shown in e cortex . The visr cortex . No patif sample .11C]PIB tracer uptake as the dependent variable and CSF BACE1 activity as the independent variable, adjusted for covariates as described above, revealed significant positive associations in the bilateral parahippocampal region, the right thalamus, and the left pons . No other brain region showed a significant correlation between BACE1 activity in CSF and [11C]PIB tracer uptake (The regression analysis with 1CPIB traβ and the activity of BACE1 in CSF can readily be measured [11C]PIB tracer uptake in the parahippocampal region, the thalamus, and the pons.APP cleavage by BACE1 is the rate-limiting step in the production of Ameasured ; thus, Cmeasured , 9 but aβ production, in the hippocampal region has been reported in APP/BACE1 transgenic mice [11C]PIB PET scans were not found in some of the previously reported brain regions including the prefrontal cortex PIB tracer uptake is strongest in brain regions close to the ventricles [11C]PIB uptake increase in this disease stage might further restrict the association to certain brain areas.In line with our recent finding that the correlation between CSF Antricles , our prentricles  as well ntricles . Therefo18F]FDG PET scans were included. Finally, although our cross-sectional study demonstrated a correlation between BACE1 activity and in vivo amyloid load, longitudinal studies are necessary to determine the stability or variability of this association over time.Some limitations of our study have to be mentioned. The patients were recruited from a specialized university center and may, therefore, not truly represent the whole population with AD. Furthermore, a modest number of patients were included and replication in larger samples would be helpful. An additional concern is the lack of pathologic confirmation of AD. However, the validity of present clinical diagnostic criteria compared with autopsy diagnoses has been reported to be very good in study cohorts recruited at specialized centers ; moreove11C]PIB tracer uptake may map the entire cerebral amyloid load. This observation suggests that BACE1 activity in CSF can only be considered a peripheral measure for AD pathology in relation to certain brain regions. Our results support the notion that CSF analysis may represent an important tool for the detection of ongoing intracerebral pathology, but complementation with imaging procedures may allow the assessment of actual presence, quantity, and regional distribution of pathology in the brain. It is also important to note that the decision for or against CSF and imaging biomarkers in clinical routine and treatment trials will also depend on the willingness among clinicians to perform and the willingness of patients to undergo lumbar puncture on the one hand and access to cyclotrons and PET scanners as well as financial considerations on the other hand. Our findings encourage further studies in order to explore the value of BACE1 activity in CSF as a reliable, affordable, and easily accessible AD biomarker. However, our results also narrow the possible applications of CSF BACE1 activity due to its selective correlation with brain regional amyloid pathology.To conclude, our results point to an association between BACE1 activity in lumbar CSF and in-vivo fibrillar amyloid pathology in regions adjacent to the brain ventricular system. Thus, our data implicate that CSF BACE1 activity reflects AD pathology in particular brain regions, whereas [


Progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease caused by JC virus (JCV), occurs mainly in immunocompromised patients. While JCV DNA is detected in the cerebrospinal fluid (CSF) from a certain proportion of patients suspected of having PML, JCV-negative patients may also develop brain lesions due to other infectious agents. This study assessed the prevalence of six herpesviruses in the CSF from patients diagnosed with or suspected of PML.Two hundred and ninety-nine CSF specimens and clinical data were collected from 255 patients, including 31 confirmed PML cases. Quantitative PCR assays were carried out to detect the genomic DNA of JCV, herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6).Herpesvirus DNAs were detected in the CSF specimens from 29 of 255 patients (11.4%). HSV-1 and CMV were detected in JCV-negative patients, whereas VZV and EBV were detected in both CSF JCV-positive and -negative individuals. The herpesvirus-positive patients had underlying disorders that caused immunosuppression, such as HIV infection, congenital immunodeficiencies, and hematologic malignancies, and presented with neurologic symptoms and MRI lesions, mainly in the cerebral white matter. The median values of CSF cell counts and protein levels in the herpesvirus-positive patients were slightly higher than those in the PML patients.The results demonstrate that herpesviruses are occasionally detected in the CSF from PML patients and immunocompromised individuals suspected of having PML. Thus, this study provides a significant basis for the diagnosis and treatment of neurological disorders in immunocompromised patients. Polyomaviridae, genus Polyomavirus[Progressive multifocal leukoencephalopathy (PML) is a rare but fatal demyelinating disease of the central nervous system (CNS) caused by JC virus (JCV), a small DNA virus belonging to the family yomavirus-3. Humanyomavirus-4. In soyomavirus-4. Althoyomavirus,3,5. In yomavirus-3,6.The detection of JCV DNA in the cerebrospinal fluid (CSF) by PCR is a reliable and less-invasive diagnostic marker of PML, particularly when combined with typical magnetic resonance imaging (MRI) patterns ,7. CSF tHerpesviruses, in particular, herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), or human herpesvirus 6 (HHV-6), are major etiological agents of encephalitis and other CNS infections in immunocompromised persons -13. ThisThe study was conducted under the approval from the Ethical Committee for Biomedical Science in the NIID . Informed consent from patients or their family members was also obtained. Patients suspected of having PML on the basis of neurological symptoms and/or MRI patterns were enrolled in this study. Upon request to the patients’ physicians, CSF testing for JCVDNA was routinely performed regardless of patient age, gender, underlying disease, or medical history. Two hundred and ninety-nine CSF specimens were collected by lumbar puncture from 255 patients from April 2007 to the end of January 2010, immediately frozen, and then transferred to the NIID for PCR testing. For 44 of the patients, CSF testing was repeated during their follow-up period. Clinical data were collected from the patients’ physicians through standardized questionnaires. The following data were analyzed: age, gender, underlying diseases, manifestations of neurologic symptoms, pattern of brain MRI lesions, and CSF leukocyte counts and total protein levels.Total DNAs were extracted from the CSF specimens using a QIAamp DNA Blood Mini Kit  and then used as PCR templates as described previously ,14,15. SP-value was corrected using the Benjamini-Hochberg method. Differences in the ages and CSF cell counts and protein contents between patient groups were compared by nonparametric analyses using the Mann–Whitney U test. All P-values less than 0.05 were judged to be statistically significant.The detection rates of herpesvirus DNAs in the CSF specimens and the sex ratios of patients in each group were statistically compared by means of a two-tailed Fisher’s exact test. For multiple testing, the resulting n = 52, 50 males, 2 females), hematologic disorders , autoimmune disorders , other diseases , and unknown . A total of 299 CSF specimens from 255 patients were subjected to the real-time PCR assay for JCV DNA, and 42 samples (14%) were found to be positive for JCV DNA. The median JCV load in these specimens was 3.2 × 104 copies/mL . The prevalence of JCV DNA and underlying diseases in the patient population are shown in Table The study population comprised 166 males and 89 females. The mean age of the subjects was 56.3 years , excluding 1 male patient whose age was not definitely stated. The underlying diseases of 255 subjects were as follows: HIV infection .Table P = 0.000 and 0.048, respectively). In addition, both JCV and herpesvirus DNAs were detected in the CSF specimens from 6 patients with either HIV infection or hematologic diseases.The clinical data of 29 herpesvirus DNA-positive patients were analyzed. The patients comprised 23 males and 6 females. The mean age of all except 1 patient was 52.5 years . There were no statistically significant differences in the age and sex ratios between the herpesvirus DNA-positive and -negative patients. The underlying diseases of the patients who provided herpesvirus DNA-positive CSF specimens are summarized in Table P = 0.049 and 0.004, respectively).In the final set of analyses, the clinical features of the patients with herpesvirus-positive CSF were compared to those of PML patients. Table The present study aimed to comprehensively assess the prevalence of six human herpesviruses in the CSF specimens from patients diagnosed with or suspected of having PML. Since the aim of this study was the detection of herpesviruses DNA using PCR from the CSF samples of the patients diagnosed with or suspected PML associated with several immuno-suppressive underlying diseases, the detection of virus DNA indicates the existence of these viruses, but the data do not always indicate the main contribution to brain damage.One of the most important findings is that EBV was present in the CSF of approximately 16% of the confirmed PML patients. Although the detection of EBV as well as JCV in the CSF has been reported previously -20, the It is of interest to note that HSV-1/2, CMV, and HHV-6 were not detected in the CSF specimens from any PML patient. Two previous reports demonstrated that JCV was concomitantly detected with CMV or HSV-1 in the CSF ,21. The In some herpesvirus-positive cases, a combination of two herpesviruses, such as CMV and HSV-1, CMV and EBV, and VZV and EBV, were detected in the CSF, which is consistent with previous reports describing the co-detection of herpesviruses in CSF ,20,22,23Currently, no specific treatment has been established for PML. Restoration of the immune system, either by combination antiretroviral therapy for patients with AIDS or by moderating the immunosuppressive therapies for non-AIDS patients, is the only treatment option for the management of PML, although several experimental treatments are being investigated . In contIn summary, as herpesviruses can contribute to CNS disorders in a significant proportion of patients suspected of having PML, comprehensive testing for the herpesviruses as well as for JCV is required for the accurate diagnosis and treatment of CNS diseases in patients diagnosed with or suspected of having PML.PML: Progressive multifocal leukoencephalopathy; CNS: Central nervous system; JCV: JC virus; CSF: Cerebrospinal fluid; HSV: Herpes simplex virus; VZV: Varicella-zoster virus; CMV: Cytomegalovirus; EBV: Epstein-Barr virus; HHV-6: Human herpesvirus 6; MRI: Magnetic resonance imaging; NIID: National Institute of Infectious Diseases; CWM: Cerebral white matter.The authors declare that they have no competing interests.KN and MS conceived of the study. KN and NI carried out real-time PCR testing, and KN created the database of patients. KN analyzed the clinical data and drafted the manuscript. TS performed the statistical analyses. MS, NI, TS, IK, and CKL participated in the study design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/13/200/prepub


Where and how does the brain code reward during social behavior? Almost all elements of the brain's reward circuit are modulated during social behavior. The striatum in particular is activated by rewards in social situations. However, its role in social behavior is still poorly understood. Here, we attempt to review its participation in social behaviors of different species ranging from voles to humans. Human fMRI experiments show that the striatum is reliably active in relation to others' rewards, to reward inequity and also while learning about social agents. Social contact and rearing conditions have long-lasting effects on behavior, striatal anatomy and physiology in rodents and primates. The striatum also plays a critical role in pair-bond formation and maintenance in monogamous voles. We review recent findings from single neuron recordings showing that the striatum contains cells that link own reward to self or others' actions. These signals might be used to solve the agency-credit assignment problem: the question of whose action was responsible for the reward. Activity in the striatum has been hypothesized to integrate actions with rewards. The picture that emerges from this review is that the striatum is a general-purpose subcortical region capable of integrating social information into coding of social action and reward. The striatum is necessary for voluntary motor control. Research on its role in movement planning and execution uncovered its participation in cognition and reward processes. Rigorous experimentation demanded social isolation to properly study this neuronal circuit. However, action, rewards and cognition also occur in the company of conspecifics, in a social context. Social behaviors, those behaviors that occur in a social context, place an extra demand on cognition since others' behaviors are difficult to predict and they affect our own behavior. Therefore, to understand the properties of the striatum it is important to study it while the organism engages in social behavior. Recent studies highlight this brain structure during different social behaviors. Among these studies, we found that the striatum contains neurons that signal the social action that will result in own reward. We place these new findings within the context of previous findings on the known role of this area in movement and reward coding in the brain. The question that guides the review is as follows: “does the striatum serve a social function?” We conclude that the striatum is a general-purpose subcortical region capable of integrating and reflecting social information into its better known non-social functions.The striatum is the input module to the basal ganglia, a neuronal circuit necessary for voluntary movement control  expressing D1 receptors which mainly project to GABAergic neurons in the substantia nigra pars reticulata (SNr)  the trustee and vice versa. Thus, both players develop a model of the other's reputation  and reward given its anatomical connections and known role in action and reward coding. We recorded striatal neuron's activity while an animal performed a reward giving task with a conspecific in order to investigate the interaction of social action and reward . If a neuron is modulated by the social component of the task, then it should stop differentiating between actors during the “bucket test.” This test for social-specific coding indicated that close to 50% of social actor coding-neurons were indeed modulated by the social environment. This is, to our knowledge, the first direct test of a neuronal correlate of social behavior in single neurons.These experiments showed that there are multiple signals in the striatum relevant for social interactions. The data suggests an extension of the known role of the striatum in movement and reward processing into the social domain. Several questions arise from these findings.How are these signals formed? One possible mechanism is as follows: Striatal neurons receive biological motion information either directly from area STP  in the ventral striatum, systemic administration of d-amphetamine produces a significant increase in DA release compared to socially-reared rats  and molecular mechanisms (changes in DAT) are involved. This snapshot of studies on the relationship between social housing conditions, behavior and basal ganglia function suggest that this is not a simple relationship. Notwithstanding, it can be concluded that social isolation and social defeat result in changes in neurotransmission to the mesolimbic circuit.Sex is a primary reward and it is the basis of pair-bond formation in voles. The striatum is part of the neuronal circuitry underlying a remarkable pair-bond formation in which both partners remain monogamous. It is important to note that the role of the striatum extends beyond that of movement and reward. Studies on vole pair formation provide an interesting example of the interaction between social behavior and striatal function.Microtus ochrogaster) form life-long bonds with their first mate, remain monogamous and live in burrows with extended families; meadow voles (Microtus pennsylvanicus), in contrast, are a promiscuous species often living in solitary burrows  activation modulates partner preference in female prairie voles , which in turns transmits the individual's information to the extended amygdala and the central amygdala further transmits this information to striatum. VNO lesions in female voles disrupt pair formation (Curtis et al., Based on the studies reviewed here, we conclude that the striatum plays a role in computations that take place during social behavior. These computations revolve around social actions and social rewards. fMRI and neurophysiology studies show that neural activity in the striatum is modulated by social rewards and by learning in a social context Figure . By learOverall, these observations suggest that the striatum does not appear to have a particular “social” specialization; rather its neurons are capable of flexibly incorporating social information into their computations. Therefore, it is justified to speak of the striatum as containing a general purpose neuronal mechanism to associate actions or events with reward. Importantly, it can also associate—or reflect—other's actions to the rewards they lead to. Rewards are also coded in the activity of striatal neurons, and as social rewards are a sub-class of rewards, they are processed in the striatum. Importantly, a functional subdivision based on different types of social behaviors need to await further experimentation. In conclusion, the striatum plays a role in the computation of social behavior.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background. Disability in multiple sclerosis (MS) is related to neuroaxonal degeneration. A reliable blood biomarker for neuroaxonal degeneration is needed. Objectives. To explore the relationship between cerebrospinal fluid (CSF) and serum concentrations of a protein biomarker for neuroaxonal degeneration, the neurofilaments heavy chain (NfH). Methods. An exploratory cross-sectional (n = 51) and longitudinal (n = 34) study on cerebrospinal fluid (CSF) and serum NfH phosphoform levels in patients with MS. The expanded disability status scale (EDSS), CSF, and serum levels of NfH-SMI34 and NfH-SMI35 were quantified at baseline. Disability progression was assessed at 3-year followup. Results. At baseline, patients with primary progressive MS  and secondary progressive MS  were more disabled compared to patients with relapsing remitting MS . Serum and CSF NfH phosphoform levels were not correlated. Baseline serum levels of the NfH-SMI34 were significantly (P < .05) higher in patients with PPMS (2.05 ng/mL) compared to SPMS (0.03 ng/mL) and RRMS (1.56 ng/mL). In SPMS higher serum than CSF NfH-SMI34 levels predicted disability progression from baseline . In RRMS higher CSF than serum NfH-SMI35 levels predicted disability progression . Conclusion. Serum and CSF NfH-SMI34 and NfH-SMI35 levels did not correlate with each other in MS. The quantitative relationship of CSF and serum NfH levels suggests that neuroaxonal degeneration of the central nervous system is the likely cause for disability progression in RRMS. In more severely disabled patients with PP/SPMS, subtle pathology of the peripheral nervous system cannot be excluded as an alternative source for blood NfH levels. Therefore, the interpretation of blood protein biomarker data in diseases of the central nervous system (CNS) should consider the possibility that pathology of the peripheral nervous system (PNS) may influence the results. In multiple sclerosis, irreversible disability progression is anatomically associated with neuroaxonal degeneration –3. UsingThere is a need to better understand the relationship between CSF and serum protein biomarkers for neuroaxonal degeneration. The situation in the CSF is relatively straightforward, because any increase can with a considerable degree of confidence be associated with damage to the brain. Interpretation of serum data is more complex. One potentially confounding issue is that Nf are also expressed in the peripheral nervous system .The relationship between CSF and serum NfH levels in MS is not known. In this study, we hypothesized that the well-recognized neuroaxonal degeneration of the central nervous system would lead to a higher concentration in the CSF compared to the serum. To test this hypothesis, we quantified NfH heavy chain phosphoforms from the serum and CSF in a cohort of MS patients we have been published on before .n = 21), secondary progressive MS , or primary progressive MS  according to published criteria [This study was approved by the local ethics committee, and informed written consent was obtained from the patients. All patients with MS were from a previously published Dutch cohort  and werecriteria . Blood acriteria .Disability was recorded on the expanded disability status scale score (EDSS) . ProgresSMI34 and NfH captured by SMI35 as NfHSMI35. The precise binding epitopes of these antibodies are not known. Binding of SMI34 is phosphate dependent which is correlated with but not identical to the degree of NfH phosphorylation. SMI35 binds more specifically to phosphorylated NfH. Nonmeasurable samples were reported as 0 ng/mL.CSF and serum Nf levels were measured using a sensitive sandwich ELISA which allows to quantify various NfH phosphoforms by exchanging the capturing monoclonal antibodies . This ELBecause of non-Gaussian data distribution the median and interquartile range (IQR) are shown. Nonparametric statistics were used for comparison throughout. We used general linear models for comparison of three variables. The concentration of NfH was compared between CSF and serum for each patient individually. If the concentration of NfH was higher in the CSF compared to the matched serum sample, this was indicated as “C > S”, otherwise as “S ≥ C”. The relationship between higher CSF or serum NfH levels with clinical progression on the EDSS was analyzed using the Kruskall-Wallis test. Correlation analyzes were performed using Spearman's R. The Bonferroni method was used to correct for multiple correlations. All statistical analyzes were performed in SAS (version 9.1).F2,48 = 4.87, P < .05), EDSS , and disease duration . None of the biomarkers was correlated with age, age at onset, disease duration, or the EDSS in the MS groups (data not shown).The demographic data of the MS patients is summarized in SMI34 levels . Significance was missed for CSF NfHSMI34 (P = .62), CSF NfHSMI35 (P = .59), and serum NfHSMI35 (P = .83).The only biomarker distinguishing the MS groups were serum NfHSMI35 or NfHSMI34 concentration in any of the MS groups (data not shown). The post hoc analysis showed that serum NfHSMI34 levels were higher in PPMS patients compared to SPMS patients (P = .0128).There was no correlation between either the CSF and serum NfHSMI35 was higher in the CSF compared to the serum in 8/9 (89%) of PPMS, 18/22 (82%) of SPMS, and 14/20 (70%) of RRMS patients. Surprisingly, for NfHSMI34 the CSF concentration was higher in 2/9 (22%) of PPMS, 11/22 (50%) of SPMS, and 5/20 (25%) of RRMS patients.The concentration for NfHThe dropout rate for the followup clinical assessment was 4/9 (44%) for PPMS, 4/22 (18%) for SPMS, and 9/20 (45%) for RRMS patients.F2,31 = 9.63, P < .001) and also for the individual progression on the EDSS .At 3-year followup most of the MS patients had progressed clinically on the EDSS scale . At follSMI35 and NfHSMI34 levels.Tables n = 5) precluded any meaningful statistical analyzes. Disability progression (ΔEDSS) appeared to be associated with higher CSF than serum levels for NfHSMI35 and higher serum than CSF levels for NfHSMI34 (The low number of patients with PPMS at followup (NfHSMI34 .SMI34 predicted disability progression (ΔEDSS) from baseline if higher in the serum compared to the CSF  from baseline if higher in the CSF compared to the serum . Furthermore the absolute concentration of serum NfHSMI34 was highest in patients with PPMS  similarfunction . In viewAn important limitation of our study is that in absence of specific tests –28, therSMI34 are different to NfHSMI35. For NfHSMI34, serum levels were frequently higher than CSF levels compared to NfHSMI35. Trapp et al. reported changes of NfH phosphorylation, particularly dephosphorylation of demyelinated axons in the MS brain [SMI34 binds to a wider range of NfH phosphoforms than NfHSMI35, it may be that serum NfHSMI34 levels are more sensitive in detecting axonal damage in MS than serum NfHSMI35 levels. This argumentation would be consistent with the finding that a higher concentration of NfHSMI34 in the serum compared to the CSF was predictive of disease progression in SPMS. It is noted that the results for NfHMS brain . As MS pMS brain . Becauseinto the brain. It is now well established that assessment of BBB integrity requires quantification of compounds on both sides of the barrier (reviewed in [Could inflammation-related impairment of the blood brain barrier (BBB) function in MS explain higher blood than CSF NfH levels? We do not think so. Historically, the concept of the BBB originated from the observation that certain compounds did not diffuse freely into the central nervous system (CNS), but they would lead to dramatic symptoms if injected intracerebrally, intraventricularly, or intrathecally . For oveiewed in ). In faciewed in , 38, 39.iewed in , 38. TheCould the localization of MS lesion formation in the CNS influence whether products of damage are predominantly released into CSF or blood? This certainly is a possibility. The CSF flow dynamics are such that biomarkers released from cortical pathology are likely to diffuse through the cortical arachnoid villi into the blood stream and only a fraction may reach the lumbar CSF . In cont


Xenopus laevis tadpoles raised in normoxic water without access to air can routinely complete metamorphosis with lungs that are either severely stunted and uninflated or absent altogether. This is the first demonstration that lung development in a tetrapod can be inhibited by environmental factors and that a tetrapod that relies significantly on lung respiration under unstressed conditions can be raised to forego this function without adverse effects. This study compared lung development in untreated, air-deprived (AD) and air-restored (AR) tadpoles and frogs using whole mounts, histology, BrdU labeling of cell division and antibody staining of smooth muscle actin. We also examined the relationship of swimming and breathing behaviors to lung recovery in AR animals. Inhibition and recovery of lung development occurred at the stage of lung inflation. Lung recovery in AR tadpoles occurred at a predictable and rapid rate and correlated with changes in swimming and breathing behavior. It thus presents a new experimental model for investigating the role of mechanical forces in lung development. Lung recovery in AR frogs was unpredictable and did not correlate with behavioral changes. Its low frequency of occurrence could be attributed to developmental, physical and behavioral changes, the effects of which increase with size and age. Plasticity of lung inflation at tadpole stages and loss of plasticity at postmetamorphic stages offer new insights into the role of developmental plasticity in amphibian lung loss and life history evolution.Contrary to previous studies, we found that  Xenopus laevis, starts using lungs soon after hatching, the American bullfrog Rana catesbeiana (Lithobates catesbeianus) and other ranids do not inflate theirs until mid-tadpole stages, and bufonids, megophryine pelobatids and Scaphiopus delay inflation until metamorphosis , and the external skin, which can be specialized into respiratory papillae, folds, and expanded tail fins . Skin caorphosis . This vaXenopus tadpoles obtain 17% of their oxygen from air  usually breathe air, lung respiration is generally not considered essential for tadpole survival . Xenopusfrom air  and Ranan uptake . Lung inn uptake . Lung re feeding . Lung re feeding . Xenopuslung use . Whetherlung use .Rana catesbeiana tadpoles raised in hypoxic water developed oversized lungs  tadpoles can routinely complete climax metamorphosis. Further, these animals appear fully viable with lungs that are either severely stunted and uninflated or absent altogether and will survive, grow and mature without breathing air for at least two years.In the one previous experiment on long-term air deprivation in frogs, the effects of air deprivation on developmental rate, survivorship and heart development and function were severe enough for the authors to conclude that lung respiration was obligatory in tadpoles . We repeThis is the first demonstration that lung development in a tetrapod can be inhibited by environmental factors and that a tetrapod that relies significantly on lung respiration under unstressed conditions  can be rTo assess the effects of air deprivation and air restoration, it is first necessary to describe normal lung development during the stages affected by these treatments. Early NF 46 marks the onset of breathing as determined by the onset of spontaneous swimming towards the surface, quick departures from the surface followed by the release of bubbles, and the switch from sinking to floating when specimens are put in fixative. At NF 46 before the first breath , the traRana (Lithobates) pipiens , the smooth muscle in the lung wall is arranged in bands of densely staining fibers  and the rest were 0.5–0.75 PCL; the shorter lungs were in specimens ≤NF 55. Of the 58 lungs scored in dissected frogs, 86% were 0.75–1 PCL and the rest were 0.5–0.75 PCL; the shorter lungs were in recently metamorphosed specimens that had yet to accumulate fat bodies. Of the 10, 26 and 34 lungs observed through the skin of albino tadpoles, metamorphs and frogs, 90, 88 and 74% respectively were 0.75–1 PCL and the rest were 0.5–0.75 PCL . On the AD lungs at NF 46 were cylindrical and of normal length (0.5–1.0 PCL), and most had thick walls and no sign of a lumen . Two of AD lungs at NF 49–51 were 1.5–2 times longer than at NF 46, but at 0.25–0.5 PCL were shorter than normal lungs . They weIn AD specimens at NF 56–57 , the broOne AD frog at two months post NF 66 exhibited small, pigmented cylinders of undifferentiated tissue with continuous lumens in place of bronchi and bronchial diverticula and no lung tissue inside the cavity . The lunOf the 225 lungs scored in AD specimens , none shAR lungs were either stunted and uninflated or showed some degree of recovery to normal lung size and inflation. Stunted AR lungs resembled AD lungs of the same stages in shape, size, position, lack of conspicuous BrdU labeling, and the development of lumens, blood vessels, cartilages, smooth muscle and epithelial folds . RecoverThree tissue-filled lungs were found in two frogs that had been returned to air for 1 and 30 days. The former specimen had two tissue-filled lungs of unequal length and the latter appeared to lack one lung entirely. Given the short period of air restoration for the former specimen and the occurrence of partially filled AD lungs , it is lAR tadpoles at NF 55–58 tended to recover lungs of normal length . RecoverOf the 128 lungs scored in AR tadpoles and frogs, 43 or 34% had recovered to a normal length. Of these 43, 84% were fully inflated, 12% were inflated proximally but not at the distal tip, and two were tissue-filled. Of the 85 lungs that were shorter than normal length, 45% appeared to be absent, 15% were fully inflated, 13% were curled, and one was tissue-filled. Of the 62 AR specimens with two scoreable lungs, 87% had two straight lungs, 8% a curled left lung and straight right one, and 5% two curled lungs. Of the 11 specimens with differently sized lungs, 67% had longer right lungs.In Experiment 1, the percentage of time spent in normal feeding increased from zero immediately after transfer to 30–55 on day 1 . The tadIn Experiment 1, the 8-week old AR frogs did not exhibit consistent trends in floating behavior  or breatExperiment 3 involved five 11-week old frogs transferred to a nonaerated circular tank (depth or D = 6 cm). Immediately after transfer, the frogs stayed submerged and took 18 breaths per hour; a frog sampled after the observation period had no lungs. After week 1 with aeration, three frogs floated continuously and the fourth spent 83% of its time submerged and did not breathe while submerged; one floating frog had 0.5–0.75 PCL, inflated lungs and the mostly submerged frog had 0.25 PCL, uninflated lungs. At week 4, the two remaining frogs continued to float in nonaerated water, but stayed submerged in aerated water and took 20 breaths per hour; these frogs had no lungs.Experiment 4 involved three 21-week old frogs transferred to a filled fishbowl (D = 13 cm) with aeration, three others transferred to a filled fishbowl without aeration and three similarly sized control frogs transferred to a filled fishbowl without aeration. Immediately after transfer, the AR frogs with and without aeration floated 88 and 97% of the time respectively and did not breathe while submerged. The controls floated 5% of the time and appeared to take 165 breaths per hour, though their continuous activity made it difficult to distinguish between breaths and efforts to escape the bowl. POS values, which had been 99 and 91 for bowls with and without aeration before transfer, dropped to 98 in the aerated bowl with AR frogs, 87 in the nonaerated bowl with AR frogs, and 86 in the nonaerated bowl with control frogs after the 30-minute observation period. The three AR frogs in the nonaerated bowl died less than 24 hours later and had no sign of lungs. On day 1, the surviving AR and control frogs, whose fishbowl POS values had dropped to 93 and 57 respectively, were transferred to aerated rectangular tanks (D = 8 cm). Their breath frequencies were respectively 19 and 25 at week 2 and 25 and 16 at week 4. At 29 days, the AR frogs were moved to a similar tank without aeration, the POS value of which dropped from 97 before transfer to 83 at ten hours and then leveled off. At day 30, these frogs had one normal length, inflated lung and five absent lungs.Experiment 5 involved three 26-week old frogs and three similarly sized control frogs transferred to nonaerated rectangular tanks (D = 8 cm). On days 0 and 1, the AR frogs floated 21 and 71% of the time and did not breathe while submerged. One AR frog sampled on day 1 had 0.5 and 0.75 PCL, inflated lungs and the other two were missing lungs. The control frogs were too active to measure floating or breathing frequency; older untreated frogs that were acclimated to their tank stayed submerged and breathed 18 and 27 times per hour on two consecutive days.To summarize these results, with the exception of Experiment 4, AR frogs tended to stay submerged immediately after transfer, but once acclimated to the new tank tended to float in nonaerated water and stay submerged in aerated water. In Experiment 4, all frogs were transferred initially to full fishbowls, the surface area-to-volume ratio (0.04) of which was much lower than for the half-full fishbowls and tanks used in other experiments (0.13–18); the AR frogs that received no aeration died. Lung recovery in all experiments did not correlate with age, aeration or changes in breathing or swimming behavior as the behavior of the few frogs with one or two recovered lungs was not consistently different from that of cohorts with stunted or absent lungs.Xenopus laevis tadpoles in normoxic water without access to air . Hence, we raise all Xenopus tadpoles in our lab in a salt solution buffered to maintain a pH of 7.0–7.4. We also raise untreated siblings in adjacent tanks to check for fast developers reaching NF 58 by 4–5 weeks. A minimum age of 4–5 weeks for NF 58 is consistent with Nieuwkoop and Faber's average age of 6 weeks and Hilken et al.'s minimum age of 5–7 weeks for NF 66 ; females were preinjected with 50–100 units of pregnant mares serum gonadotropin (Sigma G4877) four days previously. Specimens were reared under natural lighting at room temperature (22–24°C) in 0.1× Marc's Modified Ringers (MMR) and fed every 1–2 days with a tadpole chow suspension or sinking frog pellets (Xenopus Express). Partial water changes were done every 2–3 weeks. Specimens were staged using the Nieuwkoop Faber (NF) staging system for Xenopus laevis  values for water, which were measured outside the cages using a YSI 556MPS, ranged from 92 to 97. Specimens were removed from cages by inserting a dip net through a closable hole in the plastic roof of each cage. AR tadpoles and metamorphs were transferred to half-filled drum fishbowls  without aeration. Depending on the experiment, AR frogs were transferred to a half-filled or filled drum fishbowl  or a circular or rectangular tank  with or without aeration. All air restoration experiments were done at densities of 2–4 individuals per container to score the swimming and breathing behaviors of individual specimens; tadpoles at these densities did not exhibit schooling behavior  to petri dishes that were filled and lidded to exclude air. AD specimens ≥NF 47 were produced by raising five batches of animals in polypropylene screen cages. For each batch, 30–40 embryos at 3 days 4 hours pf (NF 41–42) were transferred into two cages  that were fully immersed in a 5-gallon tank . The tank was equipped with four air stones for aeration and a pump that circulated water for four 1.5-hour periods every day. The cage screen had an opening size of 0.65 mm and thread diameter of 0.29 mm. Videotape of animals being raised in cages can be viewed in behavior .Untreated specimens were sampled directly from a tank, AD specimens directly from a petri dish or cage, and AR specimens after varying intervals following transfer to a fishbowl or tank. Some specimens in each category were treated prior to sampling with bromodeoxyuridine  to analyze cell division in lung tissue. Embryos and tadpoles younger than 1 week pf (≤NF 46) were immersed in 0.3% BrdU in 0.1× MMR for 6, 12, 24 or 72 hours before sampling. Older tadpoles were injected intraperitoneally with 4–6 µl of 0.05% BrdU in deionized water 24 hours before sampling. When handling AD specimens, care was taken to minimize the time spent in air and to keep netted specimens submerged during injection and euthanization. All specimens were euthanized by immersion for 15 minutes in 0.1% MS222 (Sigma A5040).Specimens were prepared as whole mounts, histological sections or both. Most whole mounted specimens were fixed in 10% neutral-buffered formalin for 48 hours, stored in 70% ethanol, and staged, dissected and photographed under a Zeiss Stemi SV 11 dissecting scope. Some were stained with a Cy3 antibody for α-smooth muscle actin (Sigma C6198) and photographed under a Leica MZ 16 F fluorescent stereomicroscope. Histological specimens were staged, wax-embedded, sectioned at 6 or 10 µm in frontal, transverse and sagittal planes, and stained with hematoxylin and alcian blue and direct red for connective tissues. Some slides were stained with DAPI and the Cy3 antibody for α-smooth muscle actin, and photographed under a Leica DM 2500 fluorescent compound scope. Slides of BrdU-treated specimens were processed with standard immunohistochemical techniques using the G3G4 antibody for BrdU , peroxidase-diaminobenzidine labeling, and hematoxylin counterstaining.To test for long-term effects of air deprivation on frog survivorship and lung development and recovery, 12 AD frogs were transferred to an 11-liter, coarse-mesh cage immersed in a 16-liter tank with aeration at 4 months of age. Water was changed once a week and the cage walls were scrubbed to remove debris when necessary. Seven frogs were removed from the cage at 10–15 months and transferred to normal holding tanks without aeration. One AD and 4 AR frogs were sampled at 19 and 14.5 months respectfully, 2 AR frogs were lost to containment problems at 17 months, and 3 AD and 2 AR frogs have been alive for 24 months at the time of publication.supplementary material Table S1 for a breakdown by preparation method and stage). To quantify the effects of air deprivation and restoration on lung development, the unstained lungs of untreated, AD and AR whole mounts at NF≥49 were scored for the following features: length relative to the length of the pleuroperitoneal cavity; anterior or dorsal curling of the posterior tip of the lung; inflation, which was indicated by transparent lung surfaces bulging outwardly from a lattice-like network of opaque partitions that correspond to primary alveolar septa  was similar to depths used in other experiments on Xenopus tadpole behaviors , at rest except for tail beating and feeding/ventilation movements of the head , not sinking or rising but possibly drifting (meaning a slow forward movement with or without changing depth), and either in the water column or just above the bottom; this category accords with Gradwell's description of phasic water pumping . The secAR frogs were scored for breathing frequency and percentage of time spent floating. Breaths were recognized as rapidly protruding the snout above water before submerging, a series of rapid snout protrusions lasting less than 30 seconds, and floating with the snout above water for less than 30 seconds. Frog floating and submerged times were tallied from timed observations or 30 second clips of videotape, and only periods of continuous floating that lasted longer than 30 seconds were counted. Breathing frequencies were calculated using the total length of submerged time.