In mild infection, cell cycle perturbations are fewer and are integrated with an efficient apoptotic program. Importantly, the loss of integration between cell cycle perturbations and apoptosis marks the transition from a mild viral illness to a severe, life threatening infection. Our findings suggest that circulating immune cells may play a significant role in the evolution of the host response. Further study may reveal alternative host response factors previously unrecognized in the current disease model of influenza.Influenza A infection is a global disease that has been responsible for four pandemics over the last one hundred years. However, it remains poorly understood as to why some infected individuals succumb to life threatening complications whilst others recover and are relatively unaffected. Using gene-expression analysis of circulating leukocytes, here we show that the progression towards severe influenza A infection is characterised by an abnormal transcriptional reprogramming of cell cycle and apoptosis pathways. In severely infected humans, leukocyte gene-expression profiles display opposing cell cycle activities; an increased aberrant DNA replication in the G Disease progression remains poorly understood in influenza A infection. Each year, millions of individuals worldwide are infected with influenza virus A better understanding of how host response determines the progression of influenza infection is critically important for two reasons. First, a greater insight into the mechanisms that modulate host response may lead to the development of new therapeutic agents. Second, clinical manifestation of influenza infection is highly variable making it difficult to identify at-risk individuals. Discovering new markers that indicate a decompensated host response will assist clinicians in identifying individuals who are more likely to progress to a more severe infection. Such a risk stratification approach will allow clinicians to deliver prompt treatment to at-risk individuals and hence reduce the fatality rate from influenza-related complications.in vitro and animal studies Current understanding of influenza infection is limited by the lack of an appropriate human model. Data supporting the established model of influenza infection are predominantly from To identify the unique pathways that characterized progression from mild to severe illness, we performed a meta-analysis of five microarray data sets. This analysis compared pathway data between different categories of human influenza virus infection, with each category representing a different stage of immune activation . These cTLR7 (Toll-like receptor 7), RIG-1 and MDA-5. In the Severe and Symptomatic groups, these genes are highly expressed whereas in the Post-vaccination or Asymptomatic groups, there is minimal expression of these genes , cyclin A (S-phase progression), cyclin B (G2–M phase transition), CDK1 and CDK2. Furthermore, this up-regulation is accompanied by an extensive activation of DNA replication machinery, including the pre-replication complex assembly, MCM complex and Cdt1 . In contrast, mild infection shows considerably fewer changes and asymptomatic infection shows no changes at all. These differences may reflect shifting changes in immune cell populations during different stages of the influenza infection. For example, in the early stage of influenza infection, CD4+ cells differentiate into T-helper 1 cells (Th1) and a bias towards Th1 cells development protects host from severe infection The above observations also reveal important differences between severe and mild infection. In severe infection, host circulating leukocytes undergo extensive transcriptional reprogramming  is associated with reduced lung inflammation and better survival Our findings also reveal a critical role of apoptosis in influenza infection. While apoptosis has been widely reported in studies of influenza infection, its implication on disease progression has not been well understood. Conflicting evidence exist as to whether apoptosis is harmful or beneficial to the host during influenza infection Put together, our findings reveal a systematic loss of control by the host leukocytes over key cellular functions, including DNA synthesis, mitotic exit and self-repair response. As infection resolved, these perturbations subsided and were accompanied by a recovery in host response including lymphocyte, monocyte and neutrophil cell counts . LeukocyPrevious studies have focused predominantly on the role of immune cells in lung parenchyma and local lymphoid tissue in driving disease progression. It is commonly believed that disease progression occurs when influenza virus replication overwhelms the local defence mediated by immune cells in the lung. However, such a model fails to explain why viral loads assayed from airway samples in infected human are similar regardless of disease severity, a finding consistently observed in the recent pandemic H1N1 influenza infection There are limitations in our study. First, different influenza virus strains were used in the study. Ideally, each group being infected with the same virus will allow a more valid comparison between the groups. Second, an inherent limitation with observational study is that groups differ not just in the phenotype of interest (severe infection vs. mild infection), but also in other characteristics as well . These other characteristics could have confounded our findings. Third, two different microarray platforms were used in generating data for this study. The difference in technology may have introduced artifectual differences that may potentially dilute the real biological signals.In conclusion, our study extends the established influenza model to include a key role for circulating leukocyte response to infection. The discovery of a significant pathology in circulating leukocytes provides an additional perspective from which to interrogate the role of host response in influenza infection. It also provides an opportunity to develop new diagnostic and therapeutic approaches. Importantly, the discovery of infection progression signature markers may help address the prevailing challenge in an influenza pandemic, namely, to distinguish high risk individuals from a vast number of uncomplicated, self-limited infected cases. An improved ability to stratify, select and protect high risk individuals has major public health implications, as we anticipate the arrival of the next influenza pandemic.Critically ill patients with severe infection were enrolled in our study. Severe infection is defined as infection where there is at least one major organ failure (e.g. respiratory failure) that requires critical care intervention . Viral infection (n = 4) was confirmed using PCR and bacterial infection (n = 6) by microbiological cultures. Healthy volunteers (n = 18) were enrolled from a local influenza vaccination program. Whole blood samples were drawn from all subjects. For critically ill patients, sampling coincided with their peak clinical symptoms. These critically ill patients were followed up for a further four days to assess their recovery profiles. A previously published report provide the gene-expression data of 17 subjects with mild seasonal influenza infection Subjects from the Symptomatic and Asymptomatic groups were infected with the seasonal H3N2 influenza virus. Subjects from the critically ill viral infection group were infected with the pandemic H1N109 influenza virus.TM Blood RNA kit - Qiagen, Germany). Extracted RNA was then stored at minus 80 degrees Celsius until required for amplification and labelling using Illumina TotalPrep Amplification kit. Prior to sample amplification and labelling, RNA quality was analysed using Agilent Bioanalyser and all samples obtained a RIN of greater than 6.5. Amplification and labelling was carried out 24 samples at a time. 200ng of Total RNA was used as the starting quantity for amplification and labelling of all samples. Once the amplification and labelling was completed, the amplified cRNA was also assessed using the Agilent Bioanalyser, to ensure satisfactory amplification. The samples were then immediately hybridised on the HT-12 beadchips. 750ng of each sample was loaded on to the arrays. The hybridisation and washing procedure was identical for each set of arrays processed and after normalisation, no significant batch effects were identified.Samples were collected into PAXgene tubes. Upon collection, the samples were immediately stored at minus 20 degrees Celsius. RNA extraction was performed in batches of 12–24 samples at a time. Samples were first incubated at room temperature for 3 hours before following the standard RNA extraction protocol for the PAXgene RNA extraction kit , in accordance with minimum information about a microarray experiment (MIAME) standards.Five data sets were analysed . AnalysiWhen generating differentially expressed genes, the diseased group was compared to the healthy controls within the same cohort. Hence each patient group was compared to its own control group on the same microarray platform (e.g. Affymetrix), ensuring that the comparison between groups was not confounded by the difference in technology (e.g. Affymetrix vs. Illumina).To undertake pathway analysis, the generated differentially expressed genes were uploaded into the GeneGO™ MetaCore™ . MetaCore is an integrated software suite for functional analysis of gene-expression data. The software is based on an extensively curated database of protein structures and molecular interactions, and is substantially more comprehensive than the knowledge base provided by KEGG and Biocarta. Using MetaCore, pathway analysis and network analysis were performed in each data set. Pathway analysis involves matching a list of pre-specified genes onto canonical pathways and calculating the statistical relevance of the matches found. Each canonical pathway represents the current consensus knowledge of a specific biological process including intracellular cell signalling, regulatory processes and metabolic processes. Results are presented as pathway maps with overlaying gene-expression data. A false discovery rate of 5% is used as the cut-off to determine if a pathway is statistically significant. Network analysis was performed within GeneGo using pre-specified genes as root objects and then subsequently expanded based on known biological relationships and protein/gene interactions.To identify the cellular sources of the gene-expression signals, we performed cell tagging analysis using ImmGen. ImmGen is a public data gene-expression repository consisting of whole-genome microarray datasets for nearly all characterized cell populations of the adaptive and innate immune systems We performed qPCR on selected cell cycle and apoptosis genes. The findings from qPCR show that the gene-expression level correlates well with those from microarray experiments. These findings are presented in All microarray data has been deposited in GEO (record number - GSE20346).Figure S1Schematic representation of study design and bioinformatic workflow.(TIF)Click here for additional data file.Figure S2Hierarchical clustering of global gene expression using average linkage and centred correlation. (A) Heatmap of data sets assayed on the Illumina Platform. (B) Heatmap of data sets assayed on the Affymetrix platform.(TIF)Click here for additional data file.Figure S3PKR-dependent apoptosis in mild (A) and severe (B) influenza A infection. Pale blue lines indicate direct interaction with PKR. Coloured circles above individual genes indicate up (red) or down (blue) regulation.(TIF)Click here for additional data file.Figure S4Interferon and Inflammatory response in mild and severe influenza A infection. (a) Expression level of interferon related genes in mild and severe influenza A infection. Day 1 samples are shown for the severe group. (b) Expression level of inflammatory cytokine genes. Day 1 samples are shown for the severe group. (c) Heatmap of interferon related genes during recovery in severe influenza A infection.(TIF)Click here for additional data file.Figure S5DNA replication and Cell cycle pathways and leukocyte response in severe influenza A infection.(a) Pathway diagram for start of DNA replication in early S phase. Red bars indicate up-regulation and blue bars indicate down-regulation. Bars labelled 1 refer to severe influenza A infection and bars labelled 2 refer to mild influenza A infection. A detailed description of this map can be found at http://www.genego.com/map_705.php. (b) Pathway diagram for transition and termination of DNA replication. Red bars indicate up-regulation and blue bars indicate down-regulation. Bars labelled 1 refer to severe influenza A infection and bars labelled 2 refer to mild influenza A infection. A detailed description of this map can be found at http://www.genego.com/map_707.php. (c) Pathway diagram for the role of APC in cell cycle regulation. Red bars indicate up-regulation and blue bars indicate down-regulation. Bars labelled 1 refer to severe influenza A infection and bars labelled 2 refer to mild influenza A infection. A detailed description of this map can be found at http://www.genego.com/map_472.php. (d) Pathway diagram for role of ATM/ATR regulation of G1/S checkpoint in DNA damage. Red bars indicate up-regulation and blue bars indicate down-regulation. Bars labelled 1 refer to severe influenza A infection and bars labelled 2 refer to mild influenza A infection. A detailed description of this map can be found at http://www.genego.com/map_426.php. (e) Leukocyte response to severe infection. Leukocyte response on day 1 in subjects with severe viral pneumonia and bacterial pneumonia. Data is based on subjects from Severe group (n = 4), Bacterial group (n = 6) and healthy volunteers (n = 18).(TIF)Click here for additional data file.Table S1Validation of representative genes by polymerase chain reaction (PCR).(DOC)Click here for additional data file.


PSPHL) is expressed at significantly higher levels in breast tumors from African American women (AAW) compared to Caucasian women (CW). How overexpression of PSPHL contributes to outcome disparities is unclear, thus, molecular mechanisms driving expression differences between populations were evaluated.Phophoserine phosphatase-like  and CW . Gene expression levels were evaluated by qRT-PCR using RNA isolated from tumor tissue and blood. Data were analyzed using chi-square analysis and Mann–Whitney U-tests; P < 0.05 was used to define significance. Gene expression levels correlated with deletion status: patients homozygous for the deletion had no detectable expression of PSPHL, while heterozygous had expression levels 2.1-fold lower than those homozygous for retention of PSPHL. Homozygous deletion of PSPHL was detected in 61% of CW compared to 6% of AAW with invasive breast cancer (P < 0.0001); genotype frequencies did not differ significantly between AAW with and without breast cancer (P = 0.211).PCR was used to detect deletion of 30-Kb of chromosome 7p11 including the first three exons of PSPHL, is significantly higher in CW compared to AAW, suggesting that this 30-kb deletion and subsequent disruption of PSPHL may be a derived trait in Caucasians. The similar frequency of the deletion allele in AAW with and without invasive breast cancer suggests that this difference represent population stratification, and does not contribute to cancer disparities.Thus, deletion of 7p11, which prevents expression of  PSPHL [GenBank AJ001612.1]) gene was identified over a decade ago as a gene upregulated in fibroblasts from patients with Fanconi Anemia (FA) [PSPHL results in an altered coding region. Microarray analysis of the ocular disease pterygia identified PSPHL as one of three genes that could predict disease recurrence; immunohistochemical analysis revealed PSPHL is expressed at the superficial epithelium of the primary but not recurrent pterygia, suggesting that decreased PSPHL is correlated with increasing alterations in the basement membrane [PSPHL gene has also been associated with susceptibility to bipolar disorder [PSPHL remains unknown.The phosphoserine phosphatase-like  [PSPHL were also found to be significantly higher in endometrial tumors from AAW compared to Caucasian women (CW) [Microarray analysis has revealed significantly higher sian men ,5. Higheen (AAW) ,7. Expremen (CW) ,9.PSPHL has been in sex-hormone derived tumors, which have been shown to have less favorable outcomes in African American compared to Caucasian patients [PSPHL in FA suggests that PSPHL may influence rates of cellular proliferation [PSPHL is an 841 bp gene spread over four exons on chromosome 7p11.2. The promoter and first three exons of PSPHL are located within a 30 Kb region that is not represented in the human reference sequence, thus individuals harboring this deletion would not express PSPHL. If increased expression of PSPHL drives pro-tumorigenic properties, this may contribute to higher mortality rates in AAW. In contrast, baseline expression of PSPHL is heritable [PSPHL may reflect different minor allele frequencies in patients of European compared to African ancestry. To determine whether PSPHL is involved in tumorigenesis or reflects population stratification, the association between retention or loss of the 30 Kb region on chromosome 7p11 and expression levels of PSPHL were investigated in African American and Caucasian populations.This detection of significantly higher levels of patients . Alteredferation , which mGenomic DNA was available from 199 AAW and 589 CW with invasive breast cancer, and 360 AAW and 364 CW controls. Genotypes were successfully generated for all available specimens. All populations were in Hardy-Weinberg equilibrium. Genotype frequencies were significantly different between AAW and CW with breast cancer (Table PSPHL were generated by qRT-PCR from 116 tumor specimens (33 AAW and 83 CW) and 49 blood samples, 13 of which overlapped with the tumor samples. Patients with the del/del genotype had no detectable expression in either tissue (n = 49) or blood (n = 23). Expression levels in breast tissue , while tissue from patients expressing one or two copies of PSPHL were significantly higher than those patients homozygous for the deletion allele (P < 0.00001). When analyzing blood RNA, expression of PSPHL was significantly higher in carriers of the insertion allele (ins/ins or ins/del) compared to patients homozygous for the deletion allele (<P < 0.005), although PSPHL levels did not differ significantly in blood RNA between heterozygotes (n = 17) and those with the ins/ins genotype (n = 9), which may be attributable to small sample size.Expression levels for To determine whether expression of PSPHL was associated with aggressive tumor characteristics or survival, the loss (del/del) or retention (ins/del or ins/ins) of the 30 Kb of chromosome 7p was evaluated within each population for a number of clinical factors Table . AlthougPSPHL in tumors from African Americans coupled with its putative role in processes such as cellular proliferation may be contributing to more aggressive tumor phenotypes and less favorable outcomes in African Americans. Moreover, if altered expression of PSPHL does have a role in tumorigenesis, it becomes a molecular target for the development of new, tailored treatments for the African American population.AAW have the highest breast cancer mortality rates in the United States with 32.4/100,000 deaths compared to CW who have a mortality rate of 23.9/100,000 . These dPSPHL. The allele frequency of retention of this region of chromosome 7p is high in AAW, and only 6% of AAW were homozygous for the deletion and thus did not express PSPHL. In contrast, the retention allele frequency was only 21% in CW and 62% of Caucasian cases expressed no PSPHL. These data thus provide a functional explanation for expression differences across individuals and between populations. Previous data from our laboratory demonstrated that while within populations, expression levels of PSPHL did not differ significantly between tumor and non-malignant stroma, between populations, PSPHL levels were significantly higher in both tumor tissues as well as non-malignant stroma. In addition, higher expression levels of PSPHL in non-malignant tissues from AAW were not associated with higher rates of tumor development [PSPHL in a variety of tumor types, the surrounding tumor stroma and non-malignant breast tissues, significantly higher expression of PSPHL was also detected in blood endothelial cells from healthy African Americans compared to Caucasians [Our data demonstrates that the deletion of 30 kb from chromosome 7p11, including the promoter and the first three of four exons of PSPHL, effectively eliminates expression of ucasians . TogethePSPHL provides an example of how population stratification may confound identification of genes associated with health care disparities. Given the higher expression levels of PSPHL in breast and prostate tumors from African Americans, a two-gene predictor including PSPHL and CRYBB2 has been proposed to effectively distinguish between tumor epithelia from African American and European Americans [PSPHL is expressed at higher levels in breast (and other) tumors from African Americans, it is also expressed at significantly higher levels in non-tumor tissues as well as blood. In addition, the frequency of the deletion variant which determines expression levels of PSPHL is significantly higher in Caucasians but does not differ between African American cases and controls. Thus, differential expression of PSPHL caused by retention of a 30 Kb region of 7p11 in individuals of African Ancestry suggests that this difference represent population stratification, and does not contribute to cancer disparities.Differential expression of mericans , howeverFor inclusion in the Clinical Breast Care Project (CBCP), all patients must have met the following eligibility criteria: 1) adult over the age of 18 years, 2) mentally competent and willing to provide informed consent, and 3) presenting to participating breast centers with evidence of possible breast disease or for routine mammographic screening. Tissue and blood samples were collected with approval from the Walter Reed National Military Medical Center Human Use Committee and Institutional Review Board. All subjects enrolled in the CBCP voluntarily agreed to participate and gave written informed consent. Clinical information was collected for all CBCP samples using questionnaires designed by and administered under the auspices of the CBCP.http://www.oege.org/software/hwe-mr-calc.shtml). RNA was isolated from tumor specimens (n = 116) after laser microdissection as previously described [Genomic DNA  was isolated from blood clots using the Gentra Clotspin and Puregene DNA purification kits . The del/ins polymorphism was assessed using the primer sets described in [t method [For qRT-PCR, RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription kit . qRT-PCR was performed using TaqMan gene expression assay Hs00863464_m1 . Amplification was performed in duplicate using TaqMan Universal PCR Master Mix . GAPDH was used as the endogenous control for normalization of all assays. Relative quantification of gene expression levels was determined using the Comparative Ct method .rxc Contingency Tables. For qRT-PCR data, the medians of the 2-ΔΔCt values were compared using a Mann–Whitney U test to determine if the relative fold change was significantly different (two-tailed) between genotypes. Significance was determined using P < 0.05.Allele and genotype frequencies were compared between AAW with and without invasive breast cancer and between AAW and CW with invasive breast cancer by chi-square analysis using The authors declare that they have no competing interests.SR developed protocols for testing the insertion/deletion, generated gene expression data and participated in the writing of the manuscript, CP genotyped the ins/del variant, CDS oversaw collection of all patient samples and reviewed the manuscript; REE designed the project, performed data analysis and wrote the manuscript. All authors read and approved the final manuscript.


The second law [of thermodynamics] is one of the all-time great laws of science, for it illuminates why anything—anything from the cooling of hot matter to the formulation of a thought—happens at all.Peter Atkins.Four laws that drive the universeIt seems already like a long time ago that we used to think about heart failure as a problem in which mitochondria and energy were not involved. Energy has been for so long granted in the mind of mainstream electrophysiologists, but not anymore. Although energy can be taken for granted under normal conditions, this is not the case in heart failure where dysfunctional mitochondria, lack of substrate, and downstream effects become dominant factors. In the last decade, this unrealistic assumption has dramatically changed. Increasing experimental evidence supports the role of mitochondrial malfunction in the origin of arrhythmias  is dissipated or ATP synthesis inhibited. Alternans—a well-established risk factor for ventricular and atrial dysrhythmias—occur in cardiac failure and during myocardial ischemia. Hindering either of the two main components of the energy transducing machinery of mitochondria: the energy flow (ATP) and its main driving force (ΔΨm), trigger ailleurs Ca2+ alternans, or the beat-to-beat variations in Ca2+ transient amplitude at constant stimulation frequency. Further enhancement of alternans also occurred after inhibition of respiratory complexes or the Ca2+-sensitive dehydrogenases from the tricarboxylic acid cycle. Together, these data suggested that factors diminishing mitochondrial Ca2+ sequestration generate favorable conditions for Ca2+ alternans. Affecting mitochondrial Ca2+ handling by decreasing either uptake through the uniporter or extrusion through the Na+/Ca2+ exchanger also increased alternans. Then, Florea and Blatter made the following intriguing observation: alternans may happen in the absence of effects on the sarcoplasmic reticulum (SR) Ca2+ replenishment, and despite impaired mitochondrial energetics and Ca2+ dynamics. They suggested that under these conditions cytosolic ATP levels are still sufficiently high to allow for normal sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) activity and/or ATP for SERCA is supplied by glycolysis.A push forward of this idea came in 2010, when 2+ alternans even in the presence of selective inhibition of either glycolytic or mitochondrial ATP supply, both enhancers of alternans occurrence. Only severe deficit of energy supply given by simultaneous inhibition of both catabolic pathways prevented β-adrenergic stimulation-mediated abrogation of Ca2+ alternans. The authors suggest that enhanced Ca2+ sequestration together with β-adrenergic-mediated effects on the ryanodine receptor appear to act in tandem to protect against pacing-induced Ca2+ alternans. In fact, all cellular and transport processes involving subcellular organelles that contribute to clear cytosolic Ca2+ protect against alternans, and that conditions that impair cytosolic Ca2+ sequestration promote alternans. Impairment of mitochondrial Ca2+ uptake during excitation-contraction (EC) coupling can potentially have an impact on alternans incidence, although its quantitative importance is still a matter of debate. In this scenario, beta adrenergic receptor stimulation triggers a tug of war between protective and enhancing effects on alternans, the former through increase in Ca2+ sequestration and the latter by augmenting SR load and fractional release.In more recent work, Florea and Blatter  investigm depolarization that under critical conditions can scale up to the whole heart, thereby producing fatal arrhythmias (Aon et al., 2+ ATPase to sequester cytosolic Ca2+ back into the SR or its efflux from the cell (Aggarwal and Makielski, Mitochondrial redox and energetic functions are inextricably linked, and both are liable in heart dysfunction in chronic diseases such as obesity and diabetes (Bugger and Abel,


Recent studies have demonstrated that following learning tasks, changes in the resting state activity of the brain shape regional connections in functionally specific circuits. Here we expand on these findings by comparing changes induced in the resting state immediately following four motor tasks. Two groups of participants performed a visuo-motor joystick task with one group adapting to a transformed relationship between joystick and cursor. Two other groups were trained in either explicit or implicit procedural sequence learning. Resting state BOLD data were collected immediately before and after the tasks. We then used graph theory-based approaches that include statistical measures of functional integration and segregation to characterize changes in biologically plausible brain connectivity networks within each group. Our results demonstrate that motor learning reorganizes resting brain networks with an increase in local information transfer, as indicated by local efficiency measures that affect the brain's small world network architecture. This was particularly apparent when comparing two distinct forms of explicit motor learning: procedural learning and the joystick learning task. Both groups showed notable increases in local efficiency. However, a change in local efficiency in the inferior frontal and cerebellar regions also distinguishes between the two learning tasks. Additional graph analytic measures on the “non-learning” visuo-motor performance task revealed reversed topological patterns in comparison with the three learning tasks. These findings underscore the utility of graph-based network analysis as a novel means to compare both regional and global changes in functional brain connectivity in the resting state following motor learning tasks. The combination of resting state neuroimaging methods with motor learning paradigms has ushered in a new era to the investigations of adult brain plasticity. Until recently neuroimaging paradigms examining motor learning were almost exclusively investigated during the execution of a learning task. This has generated a wealth of data showing rapid neural changes occurring during the execution of the learning task. Although the vast majority of these studies were investigated with fMRI, other techniques such as diffusion weighted imaging have shown that long term motor practice can induce structural changes in both gray  or implicit (task 2) version of the serial reaction time task . In all functional scans the TR = 2800 ms, TE = 35 ms, and flip angle = 85°. An 8 channel (SENSE factor 2) head coil was used. EPI volumes consisted of forty-nine 96 × 96 axial slices of 2.5 × 2.5 × 3 mm voxels. Using an FOV of 240 × 147 × 240 mm, the entire cerebral cortex and cerebellum were covered.For all 4 experiments, scans were conducted at the Birmingham University Imaging Centre (BUIC), University of Birmingham, Birmingham, UK; the experiments were approved by the University's local ethical panel, and all participants gave written informed consent. The MRI unit was a 3 Tesla Philips Achieva scanner . Each participants had a high-resolution T1-weighted structural scan where the All data were motion-corrected and normalized to a standard template using the statistical parametric mapping software  for each subject . For a given region this is defined and computed as the sum of weights wij (connection density) of all the connections of a region/node i, providing information on the total level of weighted pair-wise correlations of the region/node. In mathematical terms:Another measure of global connectivity is strength S of the graph was computed as the mean of Si for all nodes  and vi (similar to N). Ei_loc discloses how efficient the communication is between node i and its neighbors. The mean local efficiency of a graph, is merely the mean of the local efficiency of all the nodes in the graph  is the total sum of shortest paths linking nodes f and j that go through node i.Measures founded on the notion of betweenness centrality in a complex network  and between-group differences (corrected of the four groups) across all network measures , while the explicit task showed a greater difference (p < 0.01). There was also a marked difference between the performance of these two groups, with significantly reduced reaction times for the explicit group (p = 0.01). This is an expected outcome due to the more rapid sequence acquisition of participants with explicit awareness .For the SRTT both groups showed procedural learning following the training exposure phase of the SRTT task. Comparing pre- and post-training for the implicit task, there was a significant reduction in reaction times . Given the different nature of visuo-motor rotation tasks (task 3 and 4) we expected different network responses; indeed, in the learning group (task 3) the most significantly affected nodes were the amygdala and the hippocampus (p < 0.01), while for the performance group, there were no significant effects in these brain areas.For the sequence learning tasks (task 1 and 2), the global changes in strength measures of association between regions, we also measured the topological properties of the binary (unweighted and undirected) graphs derived by thresholding the Pearson's correlation coefficient of the individual functional connectivity matrices. At each threshold we compared the observed values of degrees, correlation coefficients, and path length in brain networks to their distributions in comparable random graphs with the same number of nodes and degree distributions.To complement these results based on analysis of continuous degrees after learning, at all costs or threshold levels  this also indicates a large effect size. This was followed by the visuo-motor learning group (task 3), while the implicit sequence group (task 2) showed the least significant increase across costs among the three learning conditions. In contrast, the visuo-motor performance group (task 4) showed a significant decrease across all 4 cost levels , which also showed significant increases in strength. However, unlike strength, for degrees, the right superior partietal gyrus also showed a significant increase in the implicit SRTT task (task 2). Although a similar overall pattern of global increase in degrees was seen in the visuo-motor learning task  the most pronounced local effects were in entirely different regions. The significantly affected nodes include the right amygdala and left cerebellum (lobule III) (p < 0.01) while the visuo-motor adaptation performance group (task 4) showed a significant decrease over the left cerebellum and basal ganglia (p < 0.01).For task 1 the main nodes showing significant increases in local efficiency showed similar post-learning increases across all costs in the learning groups .The measure of s Figure . Howeverlocal efficiency (p < 0.05) in the frontal orbital regions and the right angular gyrus and the right medial temporal cortex while the implicit group (p < 0.05) (task 2) showed increases in the left precentral gyrus, SMA and the thalamus  were also evident for this measure of local efficacy: the learning group (task 3) revealed significant increase the right cerebellum (p < 0.05) (lobule 9) , while the performance group (task 4) revealed significant decreases in the right inferior parietal  showed significant increases in ) lobule  , while the performance group (task 4) showed a significant increase (p < 0.001) across all thresholds except at the threshold r = 0.6 which showed a more subtle increase (p < 0.05).Another measure that showed significant decreases across all costs for all learning conditions was path length in the orbital frontal regions, left inferior triangular gyrus, right post central gyrus, left middle occipital cortex, right basal ganglia, and right cerebellum crus II (p < 0.05). The implicit SRTT group (task 2) showed increased effects path length in the left hippocampus and the left parahippocampus (p < 0.05). The visuo-motor learning group (task 3) showed decreases in the precunus, the left amygdala, and the cerebellum while there was also a single increase in the left inferior opercular frontal cortex (p < 0.05). The performance group (task 4) did not show any significant changes in path length at the node level despite a significant overall increase.Here the explicit SRTT group (task 1) showed significant and widespread regional decreases in In order to calculate the small worldness coefficient, sigma, we also calculated the clustering coefficient for all the four tasks this produced near identical results to the local efficiency measure (see above). An additional measure that is required for the calculation of small worldness is path length (see above).Small worldness did not show any significant change  while showing a global increase in the performance group .betweenness centrality were seen in the explicit SRTT group (task 1) including decreases in the left precental gyrus, the right angular gyrus, left thalamus and right cerebellum crus I, while the implicit SRTT group (task 2) only showed decreases in the left post central gyrus and left caudate (p < 0.01). As for the visuomotor learning group (task 3), they also showed a general decrease in the right inferior triangular gyrus and right middle occipital gyrus, cerebellum crus II left (p < 0.01), while the performance group (task 4) showed overall increases for this measure in the right precental gyrus and right SMA and a decrease in the cerebullum (p < 0.01).More specific significant nodal changes for global efficiency showed a non-significant increase for all the learning groups  over all costs while the performance group (task 4) showed a non-significant decrease (p = 0.42).Overall modularity in the form of Q value . This showed opposite effects to global efficiency, with non-significant decreases for the learning groups and a non-significant increase for the visuo-motor performance group (p > 0.05). However, modularity exposed a different network distribution between the two SRTT tasks and the two visuo-motor tasks  showed widespread cortical increases over frontal, parietal, visual and sub-cortical regions while the implicit SRTT (task 2) increased over sensory motor and sub-cortical regions (p < 0.05). The visuo motor learning group (task 3) also showed increases in the frontal cortex, precuneus, temporal gyrus, and multiple areas in the cerebellum, while the performance group showed no significant changes (p > 0.05).Table Our graph analytic results highlight regular patterns in the changes across four resting state functional connectivity data sets. In each case we tested for between pre- and post-motor task changes, showing comparable global topological patterns following the three motor learning tasks, although the different tasks affect different nodes and sub-networks. Moreover, the group performing a “non-learning” visuo-motor task revealed a different global topological pattern in comparison with the three learning groups. The current graph theoretic analysis also emphasizes that motor learning leads to rapid functional reorganization that is maintained during post-learning resting state activity as indicated by emergence of new functional network relationships as a result of training.Our resting state BOLD results followed an analogous pattern, showing identical changes in all of the key measures aspects of the network topology in comparison to Heitger et al.  graph thAlthough behavioral differences existed between the tasks, performance differences due to task duration are unlikely to have affected the outcome of the graph analysis results as all the tasks lasted ~10 min.Task differences showed the expected differential local network changes. Generally a large number of network measures showed that the explicit tasks i.e., task 1 and task 3 affected the prefrontal cortex. These effects were not seen in the implicit condition (task 2). This dissociation between implicit and explicit conditions has also been shown in task based imaging data  for rest conditions. However, the decrease in these Q values following learning is likely to be due to the increase in the number of nodes participating in a greater number of modules, as indicated by the participation coefficient. Intra-modular connectivity therefore showed a significant increase in the number of connector nodes following motor learning  in the fronto-parietal and hippocampal networks, while the performance group (task 4) showed very minor decreases. Hub measures for all three learning tasks were also significantly increased. Among the motor learning tasks the explicit serial reaction time task showed the greatest difference in connectivity degree, betweeness centrality, mean path length, and connection strength. This was followed by the visuo-motor adaptation task and finally the implicit serial reaction time task. The visuo-motor learning task (task 3) was difficult and very obvious to the participants. Hence it may have considerable explicit components. This suggests that this hub outcome could be partly due to the additional areas recruited by these two different explicit tasks (the sequence task 1 and the visuo-motor learning task 3). This is then analogous to the results of Heitger et al. (Due to these regional increases in density within the same module compared to random graphs of the same size and connection density, there was a positive r et al.  who alsoefficiency and shorter path length of functional links between the nodes of a neural network will probably lead to more rapid transmission times and reduced noise degradation. This increased efficiency also implies that these strengthened functional connections form new “virtual” networks, reducing the need for the equivalent dedicated structural networks, and thus also avoiding the added incremental metabolic costs in terms of modifying physical connections. As such, this may underlie a general brain optimization strategy that may support consolidation of these motor memories, as the brain areas affected following immediate task based changes also play a role in consolidation (Ma et al., Greater This work has used a number of graph theoretical methods to assess functional connectivity changes in resting state networks following motor learning. Our findings of changes in resting state activity following motor learning tasks are consistent with prior observations of changes in graph metrics that were based on task-related BOLD recordings. This adds further credence to the growing view that resting state network analysis can identify changes in functional connections that are both task-relevant and likely to support longer-term consolidation of these motor memories. An additional finding is that we show for the first time using graph analysis a clear distinction between network changes in groups challenged with motor learning compared to a motor performance group.local efficiency these results imply that motor learning results in more direct information transfer across the relevant networks, while motor performance alone either decreased or maintained the status quo.Taken together with the other network measures like 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.


Objective. The aim of this study was to compare the shear bond strength (SBS) and adhesive remnant index (ARI) scores of no-primer adhesives tested with two different bracket bases. Materials and Methods. 120 bovine permanent mandibular incisors were divided into 6 groups of 20 specimens. Two brackets (ODP) with different bracket bases (anchor pylons and 80-gauge mesh) were bonded to the teeth using a conventional adhesive (Transbond XT) and two different no-primer adhesive  systems. Groups were tested using an instron universal testing machine. SBS values were recorded. ARI scores were measured. SEM microphotographs were taken to evaluate the pattern of bracket bases. Statistical analysis was performed. ANOVA and Tukey tests were carried out for SBS values, whereas a chi-squared test was applied for ARI scores. Results. Highest bond strength values were reported with Transbond XT (with both pad designs), Ortho Cem bonded on anchor pylons and Heliosit on 80-gauge mesh. A higher frequency of ARI score of “3” was reported for Transbond XT groups. Other groups showed a higher frequency of ARI score “2” and “1.” Conclusion. Transbond XT showed the highest shear bond strength values with both pad designs. Orthodontic brackets have to deliver an optimal orthodontic force, must be able to withstand the masticatory loads, and should be easily removed at the end of the treatment with no or minimal damage to the tooth surface , 2.In order to enhance speed of orthodontic bonding procedure no-primer adhesives have been introduced. Previous studies showed similar  or slighIn fact masticatory loads can be very variable, as often some occlusal zones are differently and asymmetrically stressed during chewing. It has been demonstrated that the symmetry of surface electromyography activity is not related to symmetry of occlusal contacts , so duriBond failure of brackets is frustrating for the practitioner, affects appliance efficiency, has an economic impact on the practice, and can significantly delay the treatment progress , 8. One To our knowledge, there are no studies that compared shear bond strength of different no-primer adhesives tested in combination with different bracket pads. It would be interesting to evaluate various no-primer orthodontic adhesives tested in combination with different bracket bases in order to appreciate if some combinations are particularly favourable for bonding.Accordingly, the aim of the present investigation was to measure and compare shear bond strength and adhesive remnant index score of one conventional and two different no-primer adhesives tested in combination with two different bracket pad designs. The null hypothesis of the study was that there is no significant difference in shear bond strength values and debond locations among the various groups.One hundred and twenty freshly permanent extracted bovine mandibular incisors were collected from a local slaughterhouse and stored in a solution of 0.1% (wt/vol) thymol. The criteria for tooth selection included intact buccal enamel with no cracks caused by extraction and no caries. The teeth were cleansed of soft tissue, and cold-curing, fast-setting acrylic resin  was funneled inside a stainless steel cylinder (15 mm diameter). Vaseline was used as separating medium between the metal cylinder and the acrylic resin. Subsequently, the root of the tooth was inserted in the resin so that the vestibular portion of the crown was parallel to the metal cylinder. After resin hardening, each tooth resulted oriented so that its labial surface was parallel to shearing force.Two different bracket mesh pad designs were investigated Figures : metal bThree different hybrid adhesive systems were tested : a conveBefore bonding, the labial surface of each incisor was cleaned for 10 seconds with a rubber on low-speed handpiece with a mixture of water and fluoride-free pumice . The enamel surface was rinsed 10 seconds with water and then dried with an oil-free air stream.Enamel etching was conducted with 37% phosphoric acid gel  for 30 seconds, followed by thorough washing and drying. In control groups (1 and 4) a thin layer of primer  was applied on the etched enamel, and then the brackets were bonded with a resin  near the center of the facial surface of the teeth (etching + priming resin + composite resin). In experimental Ortho Cem (2 and 5) and Heliosit (3 and 6) groups, the composite resin was applied on bracket base and bonded directly after etching, without primer application, following manufacturers' instructions (etching + composite resin).Subsequently brackets of all groups were squeezed with sufficient pressure to express excess adhesive, which was removed with a scaler from the margins of the bracket base. Brackets were then light-cured with a visible light-curing unit  for 10 seconds on the mesial side of the bracket and for 10 seconds on the distal side . After bonding, all samples were stored in thymol at room temperature for 24 hours and then tested in a shear mode on a universal testing machine .Specimens were secured in the lower jaw of the machine so that the bonded bracket base was parallel to the direction of the shear force.Specimens were stressed in an occlusogingival direction (crosshead speed of 1 mm per minute) as in previous studies . The maxAfter bond failure, both enamel surfaces and bracket bases were examined under an optical microscope  at 20x magnification, and the adhesive remnant index (ARI) score was recorded to assess the amount of adhesive left on the enamel surface . The ARIpost hoc. The chi-square test was used to determine significant differences in the ARI scores among the different groups. Significance for all statistical tests was predetermined at P < 0.05.Statistical analysis was performed with Stata 9.0 software . Descriptive statistics including the mean, standard deviation, median, and minimum and maximum values were calculated for all groups. Normality of the data was assessed with Kolmogorov and Smirnov test. An analysis of variance (ANOVA) test was applied to determine whether significant differences in debond values existed among the groups. The Tukey test was used as P < 0.0001). Post hoc test showed that when testing brackets with anchor pylons, Transbond XT (Group 1) and Ortho Cem (Group 2) showed no significant difference between them (P > 0.05), and both exhibited significantly higher shear bond strength values (P < 0.05) than Heliosit (Group 3). On the other hand, when testing brackets with 80-gauge mesh base, Transbond XT (Group 1) and Heliosit (Group 3) presented no significant difference between them (P > 0.05), and both expressed significantly higher shear bond strength values than Ortho Cem (Group 2), as reported in Descriptive statistics for the shear bond strength (MPa) of the different groups are presented in P = 0.0013). In particular, when testing Transbond XT and Heliosit no significant differences were found when comparing shear bond strength of the two different bracket bases (P > 0.05), whereas for Ortho Cem a significant reduction in bond strength values (P < 0.001) was reported when testing brackets with 80-gauge mesh base. When considering the two different bracket bases tested , bracketP < 0.05) that showed no significant difference between them (P > 0.05). Other groups reported a higher frequency of ARI score “2”  and “1” (Group 2).The results of ARI scores are presented in SEM microphotographs of the two different pad designs  showed tThe null hypothesis of the study has been rejected. Brackets in the oral cavity are subjected to variety of forces. Bond strength is influenced by various factors like the surface area, conditioning procedures, type of adhesive used, bracket base design, the treatment of the bracket base, and protocol followed during bonding . When evIn particular, in the present investigation, when testing Transbond XT and Heliosit, no significant differences were found when comparing shear bond strength of the two different bracket bases, whereas for Ortho Cem a significant reduction in bond strength values was reported when testing brackets with 80-gauge mesh base, thus suggesting that also adhesives played an important role in determining shear strength. Resin type might be of great effect, but also other factors should be considered, such as type, size, and distribution of particles. In fact in the present investigation three different adhesive systems were tested. The compositions are illustrated in Brackets with anchor pylons pad performed higher shear strength with Transbond XT conventional adhesive system (with priming) and with Ortho Cem no-primer adhesive, whereas the lowest values were reported with Heliosit no-primer adhesive, thus indicating that with these devices the filler would not to be too low.On the other hand brackets with 80-gauge mesh base presented higher shear strength with Transbond XT conventional adhesive system (with priming) and with Heliosit no-primer adhesive, thus indicating that with these devices adhesion is not influenced by the filler content but by resin type.Transbond XT (with primer) and Heliosit (no-primer) have been extensively tested , 21, butThe analysis of the shear bond strength indicates that all the adhesive systems and both pad designs tested in the present investigations provided clinically acceptable bond force levels (6–8 MPa) suggested by Reynolds .In the present study bovine teeth were used. Although these teeth differ from human ones in size and geometry, previous reports showed that bovine and human enamel are similar in their physical properties, composition, and bond strengths. Therefore permanent and deciduous bovine enamel has been demonstrated to be a reliable substitute for human enamel in bonding studies –25. Finally the ARI scores were recorded. Groups 1 and 4  presented a higher frequency of ARI score of “3.” Other groups (no-primer adhesives) reported a higher frequency of ARI score “2”  and “1” (Group 2), thus showing that ARI scores seem not to be influenced by pad design but mainly from adhesive type. In the literature previous reports showed contradicting results. Different previous studies showed an insignificant , 27 or sFurther studies are needed to complete and deepen the preliminary results of the present report about shear bond strength and ARI scores of no-primer adhesives.When testing brackets with anchor pylons, Transbond XT and Ortho Cem showed no significant difference between them, and both exhibited significantly higher shear bond strength values than Heliosit. When testing brackets with 80-gauge mesh base, Transbond XT and Heliosit presented no significant difference between them, and both expressed significantly higher shear bond strength values than Ortho Cem.When testing Transbond XT and Heliosit no significant differences were found when comparing shear bond strength of the two different bracket bases, whereas for Ortho Cem a significant reduction in bond strength values was reported when testing brackets with 80-gauge mesh base.A higher frequency of ARI score of “3” is reported for Transbond XT groups that showed no significant difference between them. Other groups (no-primer adhesives) reported a higher frequency of ARI score “2” and “1.”


MBL2 genotypes may have increased risk for MRSA co-infection.Low-producing  MBL2 genotypes had a 7-fold increased risk for invasive methicillin-resistant Staphylococcus aureus (MRSA) co-infection compared with fatal cases with high- and intermediate-producing MBL2 genotypes . Limited analysis of 2 genes important to the innate immune response found no association between genetic variants and fatal influenza infection. Among children and young adults who died of influenza, low-producing MBL2 genotypes may have increased risk for MRSA co-infection.We compared the prevalence of 8 polymorphisms in the tumor necrosis factor and mannose-binding lectin genes among 105 children and young adults with fatal influenza with US population estimates and determined in subanalyses whether these polymorphisms were associated with sudden death and bacterial co-infection among persons with fatal influenza. No differences were observed in genotype prevalence or minor allele frequencies between persons with fatal influenza and the reference sample. Fatal cases with low-producing  It is unknown why some apparently healthy persons become severely ill after influenza infection while others infected by the same strain remain asymptomatic or become only mildly ill. The presence of neutralizing antibody to a specific influenza strain is protective, and certain chronic medical conditions increase the risk for severe outcomes of influenza infections, but the risk factors for influenza-associated deaths among previously healthy persons remain largely unknown  convened a meeting of experts in 2007 to solicit opinions on how to explore the role of host genomics in public health activities for influenza conducted by the agency. A study of host genomic factors related to severe influenza outcomes in children was recommended as an activity that CDC was well positioned to pursue. This article reports the findings of the study implemented in response to that recommendation.TNF) and the mannose-binding lectin gene .We conducted a hypothesis-generating pilot study to examine if host genetic variants were associated with fatal influenza virus infection by comparing prevalence of selected host genetic variants among children and young adults who died of influenza with population-based prevalence estimates. We focused on 8 single-nucleotide polymorphisms (SNPs) in 2 candidate genes important in the innate immune response to influenza infection and for which national prevalence estimates were available: the gene for tumor necrosis factor superfamily, member 2  and young adults (18–40 years old) residing in the United States were reported to CDC for the 1998–99 through 2007–08 influenza seasons; of these, 105 cases with laboratory-confirmed influenza infection had sufficient tissue specimens available for DNA extraction and constitute the analytic dataset for this study. Fatal influenza cases were considered laboratory confirmed if a positive test result for influenza by viral culture, immunohistochemical analysis, or reverse transcription PCR (RT-PCR) had been documented. These represented 1) fatal pediatric cases reported to CDC during the 2003–04 influenza season when CDC conducted surveillance for influenza-associated pediatric deaths as part of an emergency response effort; 2) fatal pediatric cases identified through national surveillance since 2004 when pediatric influenza–associated death was made nationally notifiable in the United States; or 3) fatal cases of influenza among young adults at any point in time or among children before 2003 whose case reports and specimens were received by the CDC Infectious Diseases Pathology Branch on a case-by-case basis.http://snp500cancer.nci.nih.gov). Each 25-μL real-time PCR consisted of 12.5 μL of TaqMan Universal PCR Master Mix , 900 nmol of assay-specific primer, 200 nmol of assay-specific probe, and 5 μL of DNA. All controls  and unknown samples were assayed in duplicate. Thermal cycling conditions consisted of 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, and 50 cycles of 92°C for 30 s and 60°C for 1 min. Data were collected during the annealing plateau.To obtain DNA for genotyping, a 10-μm section from blocks containing formalin-fixed, paraffin-embedded tissues was deparaffinized with xylene and washed twice with absolute ethanol. After residual ethanol evaporated, tissues were digested overnight at 56°C in 200 µL Buffer PKD with 20 µL proteinase K . Extraction of the supernatant was completed with an EZ1 DNA Tissue Kit or a MagAttract DNA Mini M48 Kit (QIAGEN), with DNA eluted into a final 100-µL volume. DNA quality was assessed with a human RNase P real-time PCR in 25-μL volumes by using Agilent Brilliant II QPCR Master Mix as described , −238G>A (rs361525), and −555G>A (rs1800750) , B , and C ; the wild-type is A  and −221  encode variants that mediate MBL2 expression. Case-patients were classified as low, intermediate, or high producers of MBL on the basis of their structural and promoter variants (referred to as a “truncated haplotype”)  III CDC–National Cancer Institute Collaborative Genomics Project databank .Cases were stratified by presence or absence of any chronic medical conditions in the patients known to increase the risk for influenza-associated complications   cases had available autopsy specimens with sufficient DNA for genotyping. Case-patient characteristics are summarized in Compared with case-patients who were not genotyped, the 105 case-patients with DNA available for genotyping were slightly older , less likely to have had a preexisting medical condition , and less likely to have been vaccinated for influenza during the season of death . Case-patients genotyped were more likely to have experienced sudden death  and to have died before reaching medical care . It is not surprising that case-patients with sudden death were more likely to have undergone autopsy and, hence, to have had tissues available for DNA extraction. Genotyped case-patients were less likely to have had pneumonia evident on chest radiograph  and about equally likely to have had invasive bacterial co-infection , but differences in these characteristics are difficult to interpret because genotyped case-patients were less likely to have received medical care for their illnesses (presumably because of a greater frequency of sudden death).Genotype and minor allele frequencies among case-patients are summarized in TNF variants with all case-patients examined together or with black and white racial groups examined separately. Length of illness was shorter among case-patients with the TNF rs1800750 AG genotype than among those with the GG genotype ; no case-patients had the AA genotype. The estimated odds ratio for sudden death was 15.0 for case-patients with the AG genotype compared with case-patients with the GG genotype (p = 0.04 by Fisher exact test). No significant associations were found between any TNF variant examined and bacterial co-infection.No statistically significant differences were observed in minor allele frequencies or genotype prevalence between the case-patients and the NHANES reference sample for the 3 MBL2 SNPs examined . Among low producers of MBL, we observed an estimated odds ratio of 7.1  for invasive methicillin-resistant Staphylococcus aureus (MRSA) co-infection compared with case-patients with high or intermediate MBL production, according to MBL2 genotype . This finding is consistent with results from previous studies that found associations between MBL insufficiency (defined by genotype) and respiratory infection in children , some of which (including TNF rs361525) have been associated with increased TNF serum levels. Therefore, it is possible that the observed association may be due to linkage disequilibrium with unmeasured polymorphisms that are the causal variants, and more exhaustive analysis of TNF variants is worthy of future study.Among persons with fatal cases, we observed an increased risk for sudden death in carriers of the variant allele of A strength of this study is its use of a cohort of case-patients particularly well-suited for investigation of potential host genetic risk factors—these case-patients died with active influenza infections, yet were predominantly children and young adults without severe preexisting medical conditions. In such a group, other factors associated with severe influenza are less likely to obscure possible genetic associations. An additional strength was access to postmortem lung tissue for immunohistochemistry and/or RT-PCR confirmation of influenza infection.MBL2, haplotype information for TNF was unavailable. Despite these shortcomings, the possibility that specific variants of the MBL2 gene known to influence serum MBL levels appear to be associated with severe bacterial co-infection is an intriguing finding deserving of additional study, especially given the prevalence of co-infection among case-patients who died of pandemic (H1N1) 2009 virus infection . Interest in association studies of rare variants, the availability of new sequencing technologies that dramatically decrease the cost of sequencing, and access to reference human sequence data suggest that investigating rare variants in candidate genes (including MBL2 and TNF) and their functional effects may be a promising avenue of research. Large-scale genotyping of a sample of case-patients to look for common variants by using methods such as genomewide association studies may be possible if a network of collaborators capable of pooling a sufficient number of case-patients is developed. Recent initiatives such as the Genome-based Research and Population Health International Network (www.graphint.org/ver2) are aimed at encouraging such networks. Given the rapid acceleration in laboratory technologies, enhancement in bioinformatics methods and capacity, and trends toward collaborative research within large consortia, exploration of the role of host genomic factors in serious illness associated with influenza and other viral pathogens is increasingly feasible. We believe that host genomics is a promising area for future research regarding who is at risk for severe complications of acute infectious diseases, including influenza.Our findings suggest several opportunities for additional influenza-related research. An obvious next step is examination of all functional variants of the


A receptors (eGABAAR) and augmented “tonic” GABAA inhibition in thalamocortical neurons have been demonstrated across a range of genetic and pharmacological models of absence epilepsy. Moreover, evidence from monogenic mouse models (stargazer/lethargic) and the polygenic Genetic Absence Epilepsy Rats from Strasbourg (GAERS) indicate that the mechanism underlying eGABAAR gain of function is nonneuronal in nature and results from a deficiency in astrocytic GABA uptake through the GAT-1 transporter. These results challenge the existing theory that typical absence seizures are underpinned by a widespread loss of GABAergic function in thalamocortical circuits and illustrate a vital role for astrocytes in the pathology of typical absence epilepsy. Moreover, they explain why pharmacological agents that enhance GABA receptor function can initiate or exacerbate absence seizures and suggest a potential therapeutic role for inverse agonists at eGABAARs in absence epilepsy.It is well established that impaired GABAergic inhibition within neuronal networks can lead to hypersynchronous firing patterns that are the typical cellular hallmark of convulsive epileptic seizures. However, recent findings have highlighted that a pathological enhancement of GABAergic signalling within thalamocortical circuits is a necessary and sufficient condition for nonconvulsive typical absence seizure genesis. In particular, increased activation of extrasynaptic GABA In the electroencephalogram (EEG), human absence seizures are typified by the appearance of generalized, synchronous, and bilateral “spike (or polyspike) and slow wave discharges” (SWD) occurring at frequencies between 2.5–4 Hz . Th. Thin vi animals . Up to p animals  [36]. Intar rats .A currents in TC neurons of genetic absence models is, however, not due to increased vesicular GABA release, overexpression of δ-subunit containing eGABAARs, or misexpression of synaptic GABAARs but results from a dysfunction of GABA re-uptake by the transporter GAT-1 . In. Inγ-hydic rats, , 36] an anγ-hydrneurons,  [36]. Thin vivo,  52]. Mo. Moγ-hydGP55845, . In factcontrol, , respect−/−) mouse ..−/−) mouA inhibition through direct activation of extrasynaptic GABAARs and indirect facilitation of extrasynaptic GABAARs via activation of GABABRs. In summary; therefore, a GAT-1 malfunction in thalamic astrocytes of mouse and rat genetic models leads to an increase in ambient GABA in the sensory thalamus, which in turn elicits an enhancement in tonic GABAin vivo, the strong converging corticothalamic input that result from cortical volleys during SWDs produces bursts of excitatory postsynaptic potentials (EPSPs) that trigger T-type Ca2+-channel-mediated LTS and bursts of action potentials. In contrast, TC neurons receive both monosynaptic excitation directly from corticothalamic inputs and disynaptic inhibition via the NRT. In vivo intracellular recordings made in GAERS have shown that during ictal activity TC neurons typically receive sequences of one EPSP plus four to six IPSPs arriving in phase with each EEG spike and that action potential firing is rare [ARs and that the corresponding increase in tonic current contributes to the observed downregulation of TC neuron output during ictal activity.As previously described, SWDs of typical absence epilepsy appear to be initiated in deep layers (V/VI) of the cortex where intracellular recordings show rhythmic paroxysmal depolarisations occurring in phase with the EEG spike –63. The  is rare , 64. Thi is rare  and the  is rare , 64. ThuA current of TC neurons might have in the expression of absence seizures, experiments in freely moving animals are required. Under these conditions, both the behavioural and EEG components of the seizures can be assessed, and data are not confounded by the concomitant use of anaesthetics and/or analgesics. Thus, unrestrained GAT-1 KO mice (GAT-1−/−), which have not undergone any pharmacological treatment and whose TC neurons display enhanced tonic GABAA currents in vitro express ethosuximide-sensitive typical absence seizures , stargazer , lethargic (Ca2+ channel β4 subunit), SSADH−/− and GAT-1−/− mice all display SWDs characteristic of typical absence epilepsy, whereas in δ−/− mice drugs that commonly produce SWDs are ineffective. Importantly, because powerful GABAA IPSPs can be recorded in the vast majority of TC neurons during absence seizures in vivo [ARs of TC neurons are inherently flawed. Augmented tonic GABA in vivo , 66, theA inhibition in TC neurons of genetic absence models shifts the emphasis from a neuronal to an astrocytic aetiology for this type of nonconvulsive epilepsy. Impaired GAT-1 activity in GAERS is not caused by decreased thalamic or cortical expression of GAT-1 mRNA or protein levels. Also, no genetic variants are present in GAT-1 cDNA from GAERS, stargazer, or lethargic mice nor are the mutations responsible for absence seizures in stargazer and lethargic mice present in GAERS. Future studies, therefore, may investigate whether GAT-1 is unable to reach the outer astrocytic membrane and/or whether there are abnormalities in its phosphorylation processes. The discovery of a malfunction in GAT-1 as the underlying abnormality that produces increased tonic GABABR agonists and can be blocked by selective GABABR antagonists, applied either systemically or intrathalamically. Because about 50% of the tonic GABAA current observed in TC neurons of GAERS, stargazer, and lethargic and SSADH−/− mice is abolished by a GABAB antagonist [B drugs on typical absence seizures can no longer be simply explained by the ability of these drugs to affect GABAB IPSPs and/or presynaptic GABABRs but should also take into account the positive modulation by GABABRs of the tonic GABAA inhibition in TC neurons.Experimental typical absence seizures can be elicited or aggravated by selective GABAtagonist , 60, thereduction of tonic GABAA inhibition in thalamic neurons presents perhaps the best possible therapeutic intervention. Intriguingly, a recent study demonstrated that excessive tonic GABAAergic inhibition is also a feature of cortical neurons surrounding the infarct site (peri-infarct) after induction of stroke in experimental models  [ARs largely contain α5 and δ subunits. The α5 selective benzodiazepine inverse agonist L655,708 produced a significant reduction in the tonic current amplitude in peri-infarct neurons of slices from poststroke animals as well as improving the performance of animals in an in vivo motor task [ARs in typical absence seizures provides compelling preclinical data for the development of inverse agonists selective for α4-δ subunit containing GABAARs which may have potential therapeutic value in this type of nonconvulsive epilepsy.From a clinical perspective, it is important to stress that all the results reviewed above provide a mechanistic explanation for the aggravation of absence seizures that is observed in humans and experimental animals following either systemic or intrathalamic administration of drugs that increase GABA levels, including tiagabine, a GABA uptake blocker, and vigabatrin, a GABA transaminase blocker –70. Thusbserved) . In the tor task . In a si


Langerhans cell sarcoma is a rare and aggressive high grade hematopoietic neoplasm with a dismal prognosis. It has a unique morphological and immunotypic profile with a CD1a/ langerin/S100 + phenotype. T cell lineage markers except for CD4 in Langerhans cell sarcoma have not been documented previously. We report a case of 86 year-old male of Caucasian descent who presented with an enlarging right neck mass over 2 months with an underlying unknown cause of anemia. Computed tomography scan of the neck, chest and abdomen revealed generalized lymphadenopathy and mild splenomegaly suspicious for lymphoma. Diagnostic core biopsy performed on right neck mass revealed a possible T cell lymphoma with expression of T cell lineage specific marker CD3 but conclusive diagnosis could not be made due to insufficient core biopsy sample. Further excisional biopsy performed on a left inguinal node showed a hematopoietic neoplasm with features of Langerhans cell sarcoma with a focal cytoplasmic CD3 expression in 30-40% of the tumor cells. PCR for T cell receptor (TCR) gene rearrangement failed to demonstrate a clonal gene rearrangement in the tumor cells arguing against a T cell lineage transdifferentiation, suggesting an aberrant CD3 expression. To the best of our knowledge, this case represents the first report of Langerhans cell sarcoma with an aberrant cytoplasmic CD3 expression.http://www.diagnosticpathology.diagnomx.eu/vs/2065486371761991 According to the most recent WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues (2008) , LangerhClinically, LCS is often an extranodal tumor with skin and bone involvement, but it may present with multi-organ involvement including lymph node, lung, liver and spleen. 11% of the reported cases patients had pancytopenia. Compared to more commonly known Langerhans cell histiocytosis (LCH), also a clonal neoplastic proliferation of Langerhans cells, LCS has a much higher degree of cytological atypia and prominent proliferation rate with an aggressive clinical course. Both LCH and LCS have a similar immunophenotype including CD1a/langerin/S100+ ,3 but wi9/L . He also had a neutrophilia with WBC 19.8 x 109/L . LDH was mildly elevated with a value of 240 u/L. CT of neck/thorax/abdomen demonstrated diffuse lymphadenopathy involving the neck, mediastinum, lung, abdomen and pelvic and mild splenomegaly. Two previous right neck core biopsies at another hospital suggested a peripheral T cell lymphoma based on the expression of T cell specific marker CD3 with negative CD20 and cytokeratin AE1/3. Definitive diagnosis could not be made due to insufficient tissue. An excisional left inguinal node biopsy was performed at the Ottawa Hospital.An 86 year old Caucasian male who had been relatively healthy with medically controlled diabetes on insulin, treated temporal arteritis and myocardial infarction in 1987 without ongoing angina was referred to hematology/Oncology service for possible T cell lymphoma at Ottawa Hospital in July 2011. The patient had a short history of a rapidly growing right neck mass, an anemia of unknown cause, mild weight loss and generalized weakness. Laboratory investigations showed a bicytopenia with a normocytic anemia, Hb 89 g/L  and thrombocytopenia, PLT 86 x 10Microscopic evaluation of formalin-fixed, paraffin-embedded lymph node tissue revealed diffuse architectural effacement of the lymph node with replacement by a population of large hematolymphoid cells with very distinctive nuclear features. The nuclei were slightly elongated and many of them showed prominent longitudinal nuclear grooves reminiscent of Langerhans cells Figure  A. TheseThe immunohistochemical profile of the case is summarized in Table Interestingly, specific monoclonal antibody CD3 against epsilon chain of human CD3 complex  showed a focal cytoplasmic staining in 30-40% of the tumor cells, comparing to the strong staining of admixed normal T lymphocytes Figure  A, B. ThLangerhans cells are one subset of hematopoietic cells, thought to derive from monocyte-macrophage lineage. They are specialized dendritic cells in skin or mucosal sites devoted to antigen presentation to T cells upon activation. They are then thought to migrate to lymph node through lymphatics. Physiologically, Langerhans cells can respond in a non-clonal fashion to certain reactive stimuli such as smoking, in the lung ,9. AlterLCH and LCS have been believed to originate from myeloid stem cells -16 ratheIn our case, because of the expression of cytoplasmic CD3 and CD4, we performed T cell receptor (TCR) gene arrangement studies for gamma, delta and beta chains by PCR to rule out the possibility of T cell lineage transdifferentiation or lineage plasticity. The results failed to demonstrate a clonal rearrangement pattern, suggesting that the LCS tumor cells had an aberrant cytoplasmic CD3 expression rather than a T cell transdifferentiation phenomenon.As a rare subtype of sarcoma, LCS has a poor prognosis . AlthougAmong the reported LCS cases , patientIn summary, we reported the first case of Langerhans cell sarcoma with an aberrant cytoplasmic CD3 expression, which initially with insufficient immunophenotyping created diagnostic difficulty, especially about lineage assignment. Recognizing the cytomorphologic features characteristic of Langerhans cells prompted use of markers enabling the correct diagnosis.Written informed consent was obtained from the patient for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.The manuscript was prepared by ZX under the supervision of BFB. BFB and ZX were directly involved in diagnosis and interpretation of histology and immunohistochemistry results. RP were responsible for interpretation of flow cytometry results and paper discussion. CF and LD were responsible for the treatment of the patient. All authors read and approved the final manuscript.


CHRNA5-A3-B4 gene cluster , the ANKK1 gene (rs1800497), and the D2 dopamine receptor gene  from 104 smokers of European ancestry in a smoking cessation trial. Prior to treatment, we recorded ERPs evoked by emotional (both pleasant and unpleasant), neutral, and cigarette-related pictures. Smokers were assigned to two groups (IRS+/IRS−) based on the amplitude of the late positive potential (LPP) component to the pictures, a neural marker of motivational salience. Smokers (n = 42) with blunted brain responses to intrinsically rewarding (pleasant) pictures and enhanced responses to cigarette pictures were assigned to the IRS− group, while smokers (n = 62) with the opposite pattern of LPP responding were assigned to the IRS+ group. Carriers of the protective minor T allele  of the CHRNA3 rs578776 were less likely to be members of the IRS− group than those homozygous for the at-risk C allele (C/C). The CHRNA3 rs578776 polymorphism did not differ on questionnaires of nicotine dependence, depressed mood, or trait affective disposition and did not predict abstinence at 6 months after the quit date. These results suggest that polymorphisms of genes influencing nAChR expression are related to an endophenotype of reward sensitivity in smokers.A compromised brain reward system has been postulated as a key feature of drug dependence. We examined whether several polymorphisms of genes found to regulate nicotinic acetylcholine receptor (nAChR) and dopamine expression were related to an intrinsic reward sensitivity (IRS) deficit we previously identified among a subgroup of smokers using event-related potentials (ERPs). We examined genetic polymorphisms within the  A compromised brain reward system has been postulated as a key feature of drug dependence. Volkow and Colleagues –3 proposRecently, using event-related potentials (ERPs), we found that smokers who demonstrated IRS− were more likely to relapse following a smoking cessation intervention than smokers with “normal” intrinsic reward sensitivity . We ideOur findings suggest that a large portion of smokers have blunted brain responses to intrinsically rewarding stimuli, but it is unclear why certain smokers have this presumed deficit while others do not. Genetic factors, particularly those that influence and regulate DA-mediated reward signaling, are likely targets. While nicotine dependence involves numerous neuroadaptations, the binding of nicotine to nicotinic acetylcholine receptors (nAChRs) on dopaminergic neurons in the ventral tegmental area (VTA) is thought to be key to nicotine’s effects on motivation . Nicotin2 DA receptor (D2R) density. A model that links striatal D2R deficits, D2 dopamine receptor (DRD2) (and ANKK1) genes, and substance abuse is the “reward deficiency syndrome” (RDS) model by Blum and Colleagues  and are prone to substance dependence to increase DA levels and hedonic tone. The RDS model predicts that those with D2R deficits should experience greater enjoyment of drugs that stimulate DA release, which has been largely supported. For example, among drug-naive individuals, those with the lower striatal D2R density were more likely to report enjoying the novel effects of the psychostimulant methylphenidate , and DRD2 rs1079597 (TaqI-B). The ANKK1 rs1800497, formerly known as the DRD2 TaqI-A, is a protein kinase gene located 9.5-kb downstream from the DRD2 locus on chromosome 11 , the CHRNA3 rs578776 (3′ UTR), the CHRNA3 rs1051730 (a synonymous SNP in exon 5), and the rs8034191  are located in a region of strong linkage disequilibrium (LD) and have been found to be related to smoking and smoking heaviness , the ANKK1 gene (rs1800497), and the D2 receptor gene , from 104 smokers of European ancestry in a smoking cessation trial . We hypWe recruited smokers from the community who were seeking to quit smoking. Inclusion criteria included being aged 18–65 years, smoking five or more cigarettes per day, producing a baseline expired carbon monoxide (CO) level ≥6 ppm, having fluency in English, and having a working telephone. Conditions that excluded participants included taking psychotropic medication, having a current psychiatric disorder , being involved in any smoking cessation activities  except those provided by the clinical trial, having contraindications for either bupropion or varenicline, or having any uncontrolled medical illness. Psychiatric disorders were assessed using the Mini-International Neuropsychiatric Interview . Of the ad libitum before the baseline laboratory session. At the baseline laboratory session, participants provided an expired carbon monoxide (CO) sample, provided a buccal sample, and completed questionnaires including the Fagerström Test for Nicotine Dependence  as a between-subjects factor, picture category  as a within-subjects factor, and mean LPP amplitude (between 400 and 700 ms after picture onset) as the dependent variable. We found a significant main effect of picture category, F = 20.98, p < 0.0001  = 15.52, p < 0.0001 , than in IRS+ smokers . LPPs to CIG pictures were somewhat larger in IRS−  than in IRS+ smokers , but the difference was not statistically significant (p < 0.13). The two IRS groups did not differ on LPPs to either neutral or unpleasant pictures. Covarying expired CO did not alter the significant findings.To verify the differential responsivity by IRS group to cigarette and pleasant pictures in the present sample, we conducted an ANOVA on the 104 smokers of European ancestry using IRS group  and increased the AUC to 0.76. These results show that the CHRNA3 rs578776 predicted the IRS endophenotype, and that this prediction may be augmented by adding other SNPs associated with smoking behavior. Including the other polymorphisms did not significantly increase the AUC.To determine whether the at-risk alleles have an additive impact on the prediction of IRS group membership, we examined the incremental impact of the other polymorphisms we evaluated on the analysis . As repoIn the original sample of 180, we found that IRS group predicted abstinence outcome  between genotype were found for any of the baseline questionnaires.To determine whether the participants differed on smoking or affective disposition by genotype, we analyzed our baseline questionnaires by the dominance models for the polymorphisms that differed by IRS group, the ANKK1 rs1800497 T (A1) allele or the at-risk CHRNA5 rs16969968 A allele were more likely to be among those in the IRS− group.These results support the hypothesis that genes associated with nAChR activity are related to a novel neural marker of reward sensitivity in smokers. Carriers of the protective minor T allele of the CHRNA3 rs578776, a SNP in the CHRNA5-A3-B4 gene cluster on chromosome 15, were significantly less likely to be among those in the IRS− group. Though not statistically significant after correcting for multiple comparisons, carriers of the at-risk CHRNA3 rs578776 polymorphism is associated with IRS+ smokers is consistent with previous findings concerning the relationship between that allele and nicotine dependence. The protective minor T allele of the CHRNA3 rs578776 has been found to be associated with reduced risk for nicotine dependence in European-Americans , 3-months, and 6-months post-quit. These findings are not necessarily inconsistent, because an endophenotype such as IRS group membership is likely determined by multiple genetic and environmental factors, as is shown by our ROC analysis.There were several null findings that were unexpected. First, some of the polymorphisms most commonly associated with smoking behavior, the ANKK1 rs1800497 polymorphism.Previous results largely support models of addiction that postulate that chronic drug use leads to overvaluing drug-related stimuli and undervaluing intrinsically reinforcing stimuli due to the large differential in DA enhancement between the two reinforcers –3. HowevANKK1 rs1800497 and the CHRNA5 rs16969968, produced results suggesting a link between their at-risk variant and blunted brain responses to the intrinsically pleasant pictures that were statistically non-significant, after a correction for multiple comparisons. However, this limitation will likely confront any researcher who seeks to examine drug use endophenotypes that are more complex than smoker/non-smoker or nicotine dependence status. Second, we did not include a control group and only measured ERP during conditions of relative nicotine satiation, at baseline. We were thus unable to determine whether the reward system alterations we observed were specific to drug dependence or whether they would be exacerbated by protracted withdrawal in this study, as is predicted by recent addiction models [e.g., Ref.  (SLC6A3)   and DA t(SLC6A3)  genes.CHRNA3 rs578776 was found to predict membership in clusters that varied by reward sensitivity to natural and cigarette stimuli and that predicted smoking cessation outcome. These results suggest that an endophenotype comprised of ERP measures of motivational salience can be a valuable tool for studying the interplay between drug dependence, genetics, and brain alterations thought to maintain dependence and as a diagnostic marker for identifying smokers who are at greater risk for relapse and who may need cessation interventions tailored to address their hyposensitivity to intrinsically reinforcing stimuli.In conclusion, these results suggest that polymorphisms of genes influencing nAChR expression are related to a neural marker of reward sensitivity in smokers, a marker that may be a clinically useful endophenotype for identifying smokers at risk for relapse. In particular, the This study was sponsored by the National Institute on Drug Abuse through grant R01 DA017073 to Paul M. Cinciripini. Other support included a NIDA sponsored K23 DA024697 to Jason Robinson, and a faculty fellowship from The University of Texas MD Anderson Cancer Center Duncan Family Institute for Cancer Prevention and Risk Assessment to Francesco Versace and the National Institutes of Health through MD Anderson’s Cancer Center Support Grant (P30 CA016672). Dr. Paul M. Cinciripini served on the scientific advisory board of Pfizer Pharmaceuticals, conducted educational talks sponsored by Pfizer on smoking cessation (2006–2008), and has received grant support from Pfizer. Dr. Karam-Hage has conducted educational talks sponsored by Pfizer Pharmaceuticals, and has participated as study physician and co-investigator in two studies funded by Pfizer Pharmaceuticals. In 2011, Dr. Versace received an independently reviewed competitive grant supported by Pfizer . The other authors declare no conflict of interest.


Pharmacological agents, such as bezafibrate, that activate peroxisome proliferator-activated receptors (PPARs) and PPAR γ coactivator-1α (PGC-1α) pathways have been shown to improve mitochondrial function and energy metabolism. The mitochondrial DNA (mtDNA) mutator mouse is a mouse model of aging that harbors a proofreading-deficient mtDNA polymerase γ. These mice develop many features of premature aging including hair loss, anemia, osteoporosis, sarcopenia and decreased lifespan. They also have increased mtDNA mutations and marked mitochondrial dysfunction. We found that mutator mice treated with bezafibrate for 8-months had delayed hair loss and improved skin and spleen aging-like phenotypes. Although we observed an increase in markers of fatty acid oxidation in these tissues, we did not detect a generalized increase in mitochondrial markers. On the other hand, there were no improvements in muscle function or lifespan of the mutator mouse, which we attributed to the rodent-specific hepatomegaly associated with fibrate treatment. These results showed that despite its secondary effects in rodent’s liver, bezafibrate was able to improve some of the aging phenotypes in the mutator mouse. Because the associated hepatomegaly is not observed in primates, long-term bezafibrate treatment in humans could have beneficial effects on tissues undergoing chronic bioenergetic-related degeneration. Many recent studies have linked mitochondrial dysfunction with aging and aging-associated diseases Surf1 knock-out (KO) mice, bezafibrate also induced markers of fatty acid oxidation and hepatomegaly while increasing the expression of PPARs Bezafibrate is a pharmacological agent, used in clinical practice to treat hyperlipidemia, which activates the PGC-1α/PPAR pathway In an attempt to compensate for mitochondrial dysfunction in the Mut mouse, we placed them on a bezafibrate diet (BD) for 8 months. Here we show that bezafibrate did not increase several tested markers of mitochondrial content/function; however, it delayed hair loss and drastically improved the skin and spleen phenotype of Mut mice. These results suggest that activation of PPARs can have beneficial effects in certain tissues undergoing mitochondrial stress.To improve mitochondrial function in all tissues, 2 month-old male Mut mice were placed on a standard mouse diet containing 0.5% bezafibrate (BD). This mouse group is heretofore referred to as MutBD. To ensure that the changes observed in MutBD mice were due to bezafibrate, we also placed 2 month-old WT mice on BD (heretofore referred to as WTBD). Mice on the BD were compared to same aged male Mut and WT mice that were fed a standard mouse diet . These mice will be referred to as MutSD and WTSD respectively. The animals were fed their respective diets for 8 months and then analyzed at 10 month-old to uncover the effects of long-term bezafibrate administration on physical phenotype, organ structure/function and mitochondrial function in a model of mitochondrial aging.The Mut mouse was previously shown to have decreased body weight compared with WT mice In addition, we monitored changes in bone mineral density and content (BMD and BMC respectively), body area and body fat and lean mass of Mut and WT mice on BD and SD by DEXA scanning. Mut were previously shown to have decreased BMD, BMC and body fat at 10 month-old Because the Mut has decreased lifespan compared to WT mice As part of their premature aging phenotype, Mut mice develop grey hairs and start showing signs of hair loss at about 6 to 7 months of age To further characterize the effects of bezafibrate on hair loss and graying, dorsal and ventral skin were collected from 10 month-old WTSD, MutBD and MutSD mice. Paraffin embedded skin sections were stained with hematoxylin and eosin (H&E) to determine structural changes. We observed that dorsal skin sections from MutSD mice were thinner and lacked the distinct layers that were present in WTSD mice, while MutBD mice had a skin structure more similar to WTSD mice . SpecifiBecause collagen and elastin fibers are the primary functional components of the dermis/connective tissue layer of the skin The transforming growth factor beta 1 (TGF-β1)/Smad3 signaling pathway was shown to regulate a number of collagen gene promoters in human dermal fibroblasts Mut mice were previously shown to have enlargement of some organs, one of which was the spleen Because MutSD mice had pronounced atrophy in the spleen, we performed immunohistochemistry for cleaved caspase-3 in paraffin embedded spleen sections from 10-month-old mice. After quantifying the number of positive cells, we found that compared with WT mice, MutSD mice had increased cleaved caspase-3 . HoweverIt was reported that Mut mice develop anemia, decreased red blood cells (RBC), which is a clinical feature in aging humans PPARγ and PGC-1α. Both genes are known targets of bezafibrate, and PPARγ has been shown to be involved in regulating anti-inflammatory responses PPARγ mRNA levels were elevated in the spleen of MutSD compared with WTSD mice and its levels were restored in MutBD mice  to determine the expression level of tBD mice . PPARγ lafibrate . These fafibrate . To deteafibrate . HoweverPGC-1β mRNA levels compared with WTSD mice and these levels were also not increased in MutBD mice  (Mut) were previously characterized MtDNA mutator mice , Verhoeff’s Van Geison (EVG) and Masson’s trichrome stain at the University of Miami Dermatology Pathology Core. The stained sections were analyzed using a light microscope. For spleen and liver analysis, deeply anesthetized mice were perfused with cold PBS and the liver and spleen were collected and stored in formalin for at least 24 hours. Fixed tissues were then paraffin embedded, sectioned, stained for H&E and analyzed for structural changes at the University of Miami Comparative Pathology Laboratory.Paraffin embedded spleen sections were warmed then deparaffinized and rehydrated. Sections were incubated in 70% formic acid to retrieve antigen before being permeabilized with 0.2% Triton. Endogenous peroxidases were quenched then sections were blocked for 1 hour with normal goat serum (KPL). Sections were incubated overnight in 1∶250 dilution of primary antibody for cleaved caspase-3 . Secondary antibody, biotinylated goat anti-rabbit (KPL), was used then sections were treated with streptavidin peroxidases (KPL) for 30 minutes before being incubated in Diaminobenzidine (DAB). Slides were dehydrated in alcohol then mounted in xylene with aqueous mounting medium and viewed with a light microscope. The number of positive (brown) staining was counted in 4 field of view from each section and averaged.Western blot was performed as previously described 5′-CTGCGGGATGATGGAGACA, 5′-AGCAGCGAAAGCGTCACA), PGC-1β  and RNeasy Mini kit (for liver and spleen) (Qiagen). cDNA was synthesized from 1µg of RNA using the iScript cDNA synthesis kit (BIO-RAD). Quantitative real-time PCR, with specific primers for PGC-1α (5′- TGGGCCTCTTTCAGTAAGCT), PPARα , PPARγ , PPARδ , ACOX , CD36 , CPT1 , SCAD  and β-Actin  was performed using SsoAdvanced SYBR Green (BIO-RAD). MtDNA copy number was quantified as previously described Citrate synthase (CS) activity was determined spectrophotometrically in total homogenate from the quadriceps of mice as previously described The treadmill test was performed as previously described Mice were fasted overnight and the following day anesthetized and blood was collected by cardiac puncture. Blood was sent to the University of Miami Comparative Pathology Laboratory for analysis of complete blood cell count (CBC) and for a hepatic blood panel to measure markers of liver damage/inflammation.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Miami (Permit Number: 12-094). All efforts were made to minimize suffering.