Fmr1 KO mice. Thus, the EEG abnormalities seen in FXS patients support the model of neocortical hyper-excitability in FXS, and may provide useful translational biomarkers for evaluating novel treatment strategies targeting its neural substrate.Sensory hypersensitivities are common, clinically distressing features of Fragile X Syndrome (FXS). Preclinical evidence suggests this abnormality may result from synaptic hyper-excitability in sensory systems. This model predicts reduced sensory habituation to repeated stimulus presentation. Fourteen adolescents and adults with FXS and 15 age-matched controls participated in a modified auditory gating task using trains of 4 identical tones during dense array electroencephalography (EEG). Event-related potential and single trial time–frequency analyses revealed decreased habituation of the N1 event-related potential response in FXS, and increased gamma power coupled with decreases in gamma phase-locking during the early-stimulus registration period. EEG abnormalities in FXS were associated with parent reports of heightened sensory sensitivities and social communication deficits. Reduced habituation and altered gamma power and phase-locking to auditory cues demonstrated here in FXS patients parallels preclinical findings with  Disorganized and hyper-excitable networks can have increased cortical ‘noise' or desynchronous firing in the gamma frequency range that reduces stimulus selectivity and lowers the impact of local network output on postsynaptic circuits.9Animal models of FXS point to neuronal network hyper-excitability as a possible cause of sensory hypersensitivity. Local circuit hyper-excitability in 10 The most common finding in FXS EEG studies has been enhanced early-sensory (N1) event-related potential (ERP) responses to auditory stimuli, which is consistent with neocortical hyper-excitability models of FXS.13Electroencephalography (EEG) can be used to characterize auditory processing abnormalities and cortical hyper-excitability in FXS. To date, few EEG studies in FXS have been conducted, especially studies of evoked sensory responses.15 In typically developing subjects, reduced N1 suppression during sensory gating tasks have been associated with increased attention to irrelevant sounds16 and sensory avoidance on the Adolescent and Adult Sensory Profile,17 suggesting a link between N1 habituation and auditory hypersensitivity.One approach for using EEG to assess sensory hypersensitivity is to utilize gating-type paradigms to determine whether there is a reduced N1 response to repeated stimulus presentations, reflecting reduced neural habituation in sensory cortex. The mechanism for sensory or short-term N1 habituation is believed to be related to refractoriness of cell populations involved in basic sensory processing systems rather than more complex cognitive processes exerting top–down control of sensory cortex.18 However, previous habituation studies in FXS utilized an auditory oddball paradigm, a task in which rare novel  tones are presented in the context of a majority of standard (repeated) tones. Adding this probe of expectancy effects adds cognitive complexity and variable attentional influences to the assessment of basic auditory processing.21 Second, stimuli in prior studies were separated by relatively long inter-stimulus intervals, which can diminish habituation effects, as refractory circuits significantly complete their recovery after intervals longer than 500 ms.23 When tones are presented in sequence with a shortened inter-stimulus interval and without novel distracters, cognitive factors are limited and habituation effects seen in N1 reduction to repeated tones are increased. Limiting cognitive factors is particularly important when assessing sensory processing in groups with cognitive deficits such as FXS, where group differences in cognitive processing could obscure results. Thus, habituation might be better assessed using shorter inter-stimulus intervals (500 ms ISI) in a passive listening paradigm.There is preliminary EEG evidence that individuals with FXS do not habituate to repeated sounds.24 Time–frequency analyses of evoked EEG data to our knowledge have not been conducted in FXS, but may be useful to investigate neural correlates of local circuit hyper-excitability, particularly in the gamma range where deficits have been observed in Fmr1 mice and proposed to underlie sensory processing abnormalities.5Time–frequency analyses have revealed a relationship between increased power in the gamma frequency range and N1 amplitude abnormalities during sensory gating for other disorders with habituation deficits. These have been related to regulation of inhibitory interneuron circuits in sensory cortex, which act to bind stimulus features.Fmr1 mouse slice preparations,5 and if alterations in gamma activity were related to abnormalities in N1 habituation deficits as might be expected due to the role of gamma oscillations in maintenance of synchronous population activity in local cortical circuits. Electrophysiological abnormalities were correlated with parental reports of sensory and behavioral deficits in FXS patients to evaluate their relationship to sensory hypersensitivities and other behavioral problems associated with FXS.The present study investigated ERP and time–frequency components of short-term habituation to repeated tones in auditory cortex in individuals with FXS and healthy matched controls. Gamma activity was investigated to determine if there were similar increases in power in this frequency band in response to tones as has been reported in somatosensory cortex in 18 Healthy controls had no known prior diagnosis or treatment for psychiatric illness. Exclusion criteria included history of seizures and current use of anticonvulsant medications including benzodiazepines, or novel potential treatments for FXS (minocycline). Four patients were receiving atypical antipsychotics, and three antidepressants, all on a stable dose for at least 4 weeks. Medicated patients did not differ on primary EEG measures.Fourteen adolescents and adults with full mutation FXS  and 15 age-matched controls  participated in the study. Previous studies support this sample size as sufficient to detect group differences.25 Social and Communication Questionnaire (SCQ), Achenbach Adult and Child Behavior Checklists (ABCL), and Aberrant Behavior Checklist (ABC) were completed for FXS participants by their primary caregiver. Intelligence quotient (IQ) of FXS participants was assessed with the Stanford–Binet Intelligence Scale 5th Edn,26 which characterizes intellectual ability across a broad ability range. IQ of controls was estimated using the briefer Wechsler Abbreviated Scale of Intelligence (WASI).27 All participants provided written informed consent  prior to participation, as approved by the UT Southwestern Institutional Review Board.Clinical questionnaires including the Adolescent and Adult Sensory Profile,Stimuli consisted of 150 trials; each trial consisted of a train of four 1000 Hz tones (50-ms duration). Tones within each train were separated by a 500-ms inter-stimulus interval; trials were separated by a 4000-ms interval. Tones were delivered at 65 db through headphones, while participants underwent dense array EEG. Participants watched a silent movie to facilitate compliance.28 All sensors were referenced to a monopolar reference feedback loop connecting a driven passive sensor and a common mode sense active sensor, both located on posterior scalp.EEG was continuously recorded and digitized at 512 Hz, with a 5th-order Bessel anti-aliasing filter at 200 Hz, using a 128 channel BioSemi ActiveTwo system  with sensors placed according to the International 10/10 system.M=137.6, s.d.=8.8; Control M=144.3, s.d.=4.3).Raw data were visually inspected offline. Bad sensors were interpolated using spherical spline interpolation implemented in BESA 6.0 . Data were digitally filtered from 0.5 to 55 Hz  and re-referenced to average reference. Eye movement, cardiac and muscle movement (EMG) artifacts were removed blind to group using independent components analysis (ICA) implemented in EEGLAB 11 ref.  in Matla31 Component weights were multiplied by each subject's average data, summed across sensors and divided by the sum of the component weights, reducing waveforms from one for each sensor to one waveform per component with a defined spatial distribution across the scalp.To take advantage of the dense electrode array and integrate data from every sensor, spatial principal components analysis (PCA) was implemented on the grand average ERP.32 wavelets were calculated using a linearly increasing cycle length from 1 cycle at the lowest frequency (2 Hz) to 10 cycles at the highest (55 Hz). Single trial power (STP) and inter-trial coherence (ITC) measures were obtained to evaluate amplitude of response at each frequency and how stable or phase-locked responses were across trials, respectively.29 STP and ITC values were averaged over trials for each individual and transformed into time–frequency plots downsampled to 300 time-bins.33 Group differences were calculated using two-sided t-tests at each point in the time–frequency matrix. In particular, based on mouse model studies8 we predicted that FXS participants would show non-stimulus-specific increases in gamma STP due to hyper-excitable or ‘noisy' local cortical networks.Spectral analyses using Morlet wavelets with 1 Hz resolution were conducted on epoched single trial data weighted by the average PCA component topographies. To balance time resolution in lower frequencies with stability in higher frequencies,33 To maintain a family-wise α of 0.01, a minimum of three sequential time-bins and three adjacent frequencies were required to be significant at a nominal threshold of P<0.05.Control for multiple comparisons was achieved using time–frequency clustering techniques and Monte Carlo simulations.Amplitude and latency of the N1 component to each tone was measured for each individual at the peak, defined as the most negative-going waveform deflection between 50 and 150 ms post stimulus, and verified through visual inspection.34 The exponential decay function is not well-suited to datasets with such a limited number of time points. Therefore habituation of N1 amplitude across repeated tones in a trial was quantified as percent change from the first tone in each trial, with a focus on comparing fast decay (habituation to the first repeated tone) with asymptote (habituation to the fourth tone) to quantify habituation effects. Percent change across repetitions was analyzed using a repeated measures analysis of variance comparing repetition number in sequence  by group (FXS vs control).34 All repeated measures tests included Greenhouse–Geisser correction. Latency and amplitude were measured for the P2, defined as the largest positive-going peak between 150 and 250 ms post stimulus. The N2 component was less easily visualized in some participants, so it was quantified as the average amplitude over 30 ms centered on the N2 peak amplitude in the grand average and latency was not calculated.We limited the number of repetitions to 3 as prior habituation literature suggests that the majority of habituation effects are present by that time.ρ. To examine whether such an abnormal increase in high frequency ‘noise' was associated with abnormalities in habituation, correlations between gamma STP, gamma ITC, low frequency ITC, N1 amplitude and N1 percent change were conducted using Spearman's ρ. Clinical scales were prioritized based on their applicability to sensory and social abnormalities . Clinical correlations are presented as exploratory, not corrected for multiple comparisons.Clinical correlations were examined with N1 amplitude to the initial tone, average N2 amplitude across all four tones, percent change in N1 amplitude, gamma STP and all significant time–frequency clusters using Spearman's FXS patients showed significantly lower full scale IQ than controls . Most oft(27)=1.7, P=0.09. In a repeated measures analysis of variance on percent change across repetitions, there was a main effect of group, F=6.9, P=0.014, indicating that FXS showed less habituation of the N1 ERP than healthy controls =2.54, P=0.17,  but no difference in habituation between repetitions 2 and 3, indicating asymptote of habituation by the fourth stimulus in the trial, consistent with findings in typically developing individuals.35 The repetition number by group interaction was not significant, indicating no difference in rate of habituation between groups. This result suggests that additional repetitions do not result in eventual normalization of N1 habituation by allowing FXS to ‘catch up' with more gradual habituation of sensory responses. Gender was entered as a covariate but was not significant, so was removed from the final model. There was no group difference in N1 latency or P2 amplitude or latency for any of the four stimuli.Spatial PCA revealed a single spatial component representing 93.0% of the variance that was consistent with known auditory N1/P2/N2 topography . Subsequcontrols . There wt(27)=3.7, P=0.001, with FXS participants showing significantly reduced N2 amplitude  relative to healthy controls .N2 amplitude did not show a significant habituation effect, so amplitude was collapsed over all four stimuli. There was a significant group difference in overall N2 amplitude, t(27)=3.36, P=0.002; t-tests on time–frequency plots for ITC, STP and baseline-corrected STP (corrected for multiple comparisons) revealed 18 time–frequency clusters with significant differences between FXS and controls =13. 23, Significant correlations with clinical measures in FXS participants are presented in ρ=−0.61, P=0.02), and increased gamma STP to the first repetition was correlated with less habituation to the second repetition . Absolute increases in gamma STP throughout the trial were significantly correlated with decreases in gamma ITC to the initial stimulus , which in turn was correlated with increased N1 ERP to the same stimulus . Increased gamma power in FXS, both absolute increases relative to healthy controls and further increases above an already elevated baseline, can be thought of as an increase in background neural ‘noise'. This increase in non-specific high frequency neural activity contributed to a decreased ability to synchronize gamma frequency activity when necessary , which was associated with an overgeneralized excitatory response (increased N1 amplitude), as well as lowered ability to habituate to repetition.For FXS, increased gamma STP above the elevated baseline to the initial stimulus was correlated with less habituation to the first repetition  and inhibitory  activity, then composite variables were created by averaging these rankings in each category. Composite variables were plotted for FXS and control participants to characterize the relative imbalance in excitatory vs inhibitory activity . Control38 and abnormalities in electrodermal activity to repetitive stimuli,39 however, neural correlates for these findings have not been elucidated. We believe the current study provides the first direct clinical evidence for a contribution of gamma band abnormalities, suggesting an imbalance of excitatory vs inhibitory activity, an effect found in mouse models of FXS, and the first evidence that these are clinically relevant by virtue of their relationship to parental reports of sensory sensitivities in FXS patients.Sensory processing abnormalities are common and clinically distressing features of FXS. Inability to habituate to sensory events and ongoing background noise may contribute to hypersensitivity to sounds and overstimulation. Behavioral and physiological findings have suggested deficits in short-term habituation in FXS, including reduced prepulse inhibition of the startle response13 N1 amplitude to the initial non-habituated stimulus did not differ between groups (though there was a non-significant trend) and it was not significantly correlated with habituation , suggesting potentially dissociable mechanisms between neural excitation 11 and inhibition (habituation).14The current study also provides the first direct evidence of short-term local circuit-based habituation deficits in FXS patients. FXS patients showed a significant decrease in N1 suppression (both ERP amplitude and low frequency phase-locking) to repeated stimuli. This alteration was also associated with parental reports of auditory hypersensitivity and social problems in FXS participants. N1 habituation did not normalize across additional repetitions of the stimulus, suggesting a fundamental deficit in organization of local networks representing basic auditory stimulus properties. Although overall N1 amplitude has been the focus of previous work,18 increased N1 amplitude and increased low frequency ITC during the N1 time period that contributes to formation of the N1 ERP,40 were correlated with increased abnormalities on the Sensory Profile, suggesting that individuals with high ERP amplitudes may have more severe sensory symptoms. Previous studies finding N1 differences used younger18 and/or smaller11 samples, and their level of sensory sensitivities is unknown. The current finding of decreased N2 amplitude in FXS is consistent with findings of some11 but not all prior studies.13 The long inter-stimulus interval single stimulus task employed by Knoth et al.13 may have decreased N2 amplitudes and their utility for detecting deficits. The N2 ERP is commonly associated with incorporation of frontal generators in stimulus identification and maintenance of auditory memory traces.41 Decreased amplitude of N2 was associated with increased scores on the SCQ, Sensory Profile and ABC Irritability component, suggesting a broad sensory and behavioral impact of this alteration.While N1 amplitude to the initial non-habituated stimulus did not differ between groups as in some previous studies,Using time–frequency analyses of evoked EEG for the first time in FXS, the current study provides evidence for both increased gamma ‘noise' (increased desynchronous high frequency firing) and decreases in ability to organize inhibitory networks to synchronize gamma band activity in the brain. Broad increases in excitation of gamma circuits without corresponding increases in synchronization of those circuits are consistent with presence of heightened neocortical excitability in FXS, and may represent a mechanism underlying sensory hypersensitivities in this disorder and the observed neural habituation deficit.42 Decreased phase-locking during the gamma spike was associated with greater N1 amplitudes, suggesting an overall increase in neural background noise that contributes to both hyper-excitability (increased N1) and disorganization (decreased ability to ‘lock in' or synchronize gamma response to the stimulus). Transient increases in gamma power above the already elevated baseline in response to each stimulus were associated with poorer habituation response to the following stimulus, suggesting that this increase in power is not beneficial or compensatory, but rather reflects an over-recruitment of local networks, which generally oscillate in the gamma range.43Higher ongoing or background gamma power in FXS relative to controls was not locked in time to stimulation. Individuals with elevated background gamma activity showed decreased ability to coherently organize and phase-lock gamma activity during the ‘gamma spike' period, a short burst of phase-locked gamma activity that normally occurs during early-stimulus processing.14 Less synchronized and more widely excitable local synaptic networks would mean that the same circuit of synapses may not be excited in the same way for each repetition, preventing proper functioning of the refractory system. Long-term depression, which weakens synaptic connections in a neural circuit, is enhanced in Fmr1 knockout mouse models of FXS;44 weaker connections between neurons in a given local circuit coupled with increased firing of unrelated neurons may also account for that circuit's inability to reliably activate and thus habituate to a repeated stimulus. Importantly, these findings suggest not only a promising avenue for investigating brain dysfunction in FXS, but a promising translational strategy for integrating rodent and patient studies and developing mechanistically important biomarkers useful for advancing drug discovery.In a normally functioning local circuit, refractory properties of the network prevent the same circuit from activating as strongly to repeated stimuli presented in close succession, creating short-term habituation.Fmr1 knockout mice has been related to decreased excitatory drive on fast-spiking inhibitory interneurons, which is associated with decreased synchronization or phase-locking in the gamma range.5 Decreased activation of fast-spiking inhibitory interneurons that operate in the gamma range and drive the local neural oscillatory response could account for both local circuit hyper-excitability and reduced neural habituation. Reduced inhibitory drive onto pyramidal cells in auditory cortex from fast-spiking interneurons would lead to increased cell firing and less coherent organization and tuning to sensory stimuli. In the current study, increases in non-phase-locked gamma power were correlated with decreases in phase-locked gamma activity, suggesting a hyper-excitable but disorganized system consistent with these circuit dynamics.Gamma hyper-excitability in The decrease in phase-locked gamma was also correlated with increased N1 amplitudes, suggesting a connection between local circuit hyper-excitability and increased recruitment of neural generators for sensory evoked potentials. Decreased habituation to a given repetition was associated with increased gamma power to the preceding stimulus, suggesting a direct relation between increased background high frequency ‘noise' activating extraneous neural circuits during early-stimulus processing that could disrupt refractory-based circuit inhibition to the next repetition of that stimulus. Of note, N1 amplitudes were heterogeneous and suggest a subset of individuals with abnormally high N1, but habituation and gamma function were more consistently abnormal and associated with clinical measures.46 though individuals with Down syndrome generally show decreased or normal ongoing and evoked high beta and gamma power,48 which is opposite of that observed here in FXS. Second, FXS participants were taking various medications, and their potential impact on the data, while not apparent in statistical analyses, cannot be ruled out. Third, clinical correlations were performed on an exploratory basis only, and require validation via replication in additional samples. Last, although no gender differences were found, we had a limited number of FXS female participants.Certain limitations in this study need to be considered. First, we cannot determine the degree to which our findings represent FXS-specific factors or alterations associated with developmental or intellectual disabilities. For example, there is evidence that individuals with Down syndrome may also show abnormal habituation to repetitive auditory stimuli,49 partially reduces aberrant N1 amplitudes and improves N1 habituation,18 suggesting that circuit dynamics associated with habituation may be good translational targets for drug development. Gamma abnormalities in particular may help track and explain hypothesized excitatory/inhibitory imbalances in FXS, and may contributed to lower seizure thresholds. Concurrent work with EEG in FXS mouse models50 will further elucidate molecular mechanisms underlying these electrophysiological alterations and their potential use as translational biomarkers for new targeted treatments for FXS. Such work might also lead to new treatment options for neural hyper-excitability and sensory sensitivities in a subgroup of patients with idiopathic autism.To the best of our knowledge, this study presents the first comprehensive analysis of short-term auditory habituation in FXS. Our findings are consistent with a model that abnormalities in local circuit function leads to a hyper-excitability of sensory cortex that contributes to altered sensory experiences in FXS and other behavioral problems. Given the proposed linkage of circuit-based abnormalities in FXS with sensory processing abnormalities, reduced habituation and gamma abnormalities may represent promising candidate translational biomarkers for FXS. In FXS mice, minocycline treatment reduces audiogenic seizures,


Many aspects of autoimmune disease are not well understood, including the specificities of autoimmune targets, and patterns of co-morbidity and cross-heritability across diseases. Prior work has provided evidence that somatic mutation caused by gene conversion and deletion at segmentally duplicated loci is relevant to several diseases. Simple tandem repeat (STR) sequence is highly mutable, both somatically and in the germ-line, and somatic STR mutations are observed under inflammation.TPO), phogrin (PTPRN2) and filaggrin (FLG), include STRs that are among the eleven longest STRs spanned by protein-coding genes. This association of primary autoantigens with long STR sequence is highly significant (Protein-coding genes spanning STRs having markers of mutability, including germ-line variability, high total length, repeat count and/or repeat similarity, are evaluated in the context of autoimmunity. For the initiation of autoimmune disease, antigens whose autoantibodies are the first observed in a disease, termed primary autoantigens, are informative. Three primary autoantigens, thyroid peroxidase (The results support the hypothesis that many autoimmune diseases are triggered by immune responses to proteins whose DNA sequence mutates somatically in a coherent, consistent fashion. Other autoimmune diseases may be caused by coherent somatic mutations in immune cells. The coherent somatic mutation hypothesis has the potential to be a comprehensive explanation for the initiation of many autoimmune diseases. I have previously provided evidence that somatic gene conversion and/or deletion in sequence harboring long segmental duplications is correlated with disease HP) has been observed in vivo in humans HP2 allele have a segmentally duplicated 1.7kb sequence fragment within the gene that includes two additional exons beyond the shorter HP1 allele. In an HP2 homozygote, Asakawa et al HP2 homozygotes subsequently tested, a small but measurable concentration of the shorter sequence was identified. Asakawa et al. argued that rare but regular somatic deletion events occur in vivo. In the mouse, a similar kind of somatic mutation has been observed in vivo at a longer 70 kb segmental duplication HP in humans, presumably due to both the longer duplicon and the fact that phenotypic measurement was performed in gene-expressing tissues where mutations would be more common, rather than in blood cells Coherent somatic mutation of the haptoglobin gene , including microsatellites and minisatellites that are highly mutable in germ-line cells, are also mutable in somatic cells Other STRs occur within introns, where changes in repeat counts can change splicing behavior Additionally, repeat mutations are often accompanied by significant changes in methylation Yet another reason to focus on somatic repeat mutations in autoimmune disease is the observation that somatic tandem repeat mutations can be induced by inflammation typical of an immune or autoimmune response Human STR sequence is overabundant near telomeres While somatic and germ-line microsatellite mutation patterns appear similar Autoimmune diseases have overlapping features, including shared susceptibility loci The most prominent phenotype of autoimmune disease is the presence of specific antibodies  and 2. WA B cell epitope does not have to be from the same protein molecule as the T cell epitope in order for the B cell to be activated by a CD4+ (helper) T cell. A B cell that endocytoses a protein complex by binding to one of its proteins can be activated by a CD4+ T cell specific to another protein in the complex. Such a mechanism has been used to explain anti-TG2 antibodies in celiac disease, where a TG2-specific B cell is activated by a CD4+ T cell specific to gliadin after endocytosis of a TG2-gliadin complex peri-antigen to mean an in-vivo binding partner of an autoantigen. A peri-antigen can potentially function as a CD4+ T-cell target supporting B cell specificity to the autoantigen.Thus, a protein is a candidate CD4+ T cell target either if it elicits antibodies itself, or if an in-vivo binding partner of the protein elicits antibodies. B cell specificities (and thus antibodies) to multiple proteins can be supported by a single CD4+ T cell epitope. I use the term I sought data to test the hypothesis that autoimmune disease is associated with mutable repetitive sequence. Because of its construction from long contigs primary autoantigen is one whose antibodies have been shown, in at least a subset of cases, to be the first disease-associated antibodies to appear. A test of the coherent somatic mutation hypothesis can be formulated as follows: Is there a statistical link between primary autoantigens (and/or their peri-antigens) and genes containing highly mutable sequence?Antibodies that develop early in disease progression provide the strongest evidence for a causative role for the corresponding antigen. A Once such a statistical link is established, a subsequent test of the comprehensiveness of the coherent somatic mutation hypothesis would consider other mutable  sequence. To what extent could somatic mutation at these loci explain other autoimmune phenomena?NSUN6, TTC34, and ANKRD36C each contain multiple long repeats, and thus appear more than once. As previously discussed, high repeat length, high repeat count, and high repeat identity are markers of repeat mutability. Additionally, for intronic repeats, longer repeats are more likely to induce long mutations that in turn are more likely to alter methylation and splicing. At this scale, all repeats are minisatellites with intermediate to long repeat units.Using the Tandem Repeat Finder TPO); protein-tyrosine phosphatase, receptor-type, n, polypeptide 2 (PTPRN2); and filaggrin (FLG). TPO encodes a primary autoantigen in both Hashimoto's Thyroiditis (HT) and Graves' Disease (GD); PTPRN2, and FLG encode primary autoantigens in Type-1 Diabetes (T1D) and Rheumatoid Arthritis (RA) respectively  initiation factor that binds to RNAP-III BRF1 thus encodes a peri-antigen for SSc.Additionally, the tenth ranked gene, HP, I looked for examples of long tandem duplications with at least 96% identity occuring within protein coding genes. Since the tandem repeat finder algorithm limits repeat units to 2000 bp, and its coverage of some longer units appears to be incomplete , such repeats may have been overlooked in the earlier analysis. The segmental duplications track of the UCSC database Motivated by known somatic mutation of CR1) in SLE and multiple sclerosis (MS); pepsinogen 4, group I (PGA4) in pernicious anemia (PA); titin (TTN) in myasthenia gravis (MG); interferon-gamma-inducible protein 16 (IFI16) in RA, SLE, SSc and SJ; and HP in celiac disease (CEL) (se (CEL) . The preEL)  see .CR1  and has a population frequency of about 15%, while the shorter CR1-F allele has two repeats and a frequency of 83% CR1-S allele is the high-risk variant CR1 plays an important immunological role in various cell types Copy number variations in the 54.7 kb STR of CR1  have beePGA4 is one of three genes in the human reference genome coding for highly similar  versions of pepsinogen A, an autoantigen in PA. Low levels of pepsinogen A are specific in diagnosing PA ATP4A/ATP4B  in several primate species, but not in more distantly related species whose genomes have been sequenced Ttc34 start site Ttc34 repeat is the fifth longest (The long  longest . The indTTC34 repeat mutates under inflammation TTC34 expression and/or PPP4C activity would be adaptive under inflammation. PPP4C depletion makes T cells resistant to apoptosis If the LPA encodes a protein that binds to ApoB-100 in LDL particles to form Lp(a) lipoprotein particles containing lipids, phospholipids and cholesterol LPA encodes a peri-antigen for CAD. Under the coherent somatic mutation hypothesis, rare but regular somatic mutation to LPA would occur, analogously to that observed for HPLPA is central to CAD pathenogenesis, since an elevated plasma Lp(a) lipoprotein level predicts stroke and vascular disease, particularly in men LPA have the largest known effect on CAD risk ABCG8 contains a long (10.8 kb) intronic repeat, part of a larger compound repeat separated by a LINE insertion  and Crohn's disease (CD) PTPN22 rs6679677 C/A polymorphism that is in high linkage disequilibrium with the rs2476601 C/T polymorphism associated with many autoimmune diseases PTPN22 association in terms of alternative responses to somatic mutation. UC would be caused by an autoimmune response against the mutant protein, while CD would be caused by the failure of the mutant protein's function, in the absence of a direct immune response against that protein. This interpretation is consistent with a clear role for MHC alleles in UC but not CD A critical clue is provided by the NOD2 polymorphisms NOD2 variation modulates adaptive immune responses to microbial antigens DMBT1 expression in CD DMBT1 tandem repeat that encode fewer bacterial recognition sites are overrepresented in CD but not UC DMBT1 -coded protein binds to pancreatic amylase DMBT1 encodes a peri-antigen for CD.CD and UC have opposite risk alleles for MUC4 is an interesting somatic mutation candidate because its expression pattern in the eye, vagina, ectocervix, trachea, and salivary gland MUC5B is expressed in many of these tissues In Sjogren's sydrome, a primary initiating change is the dysregulation of mucins KIR3DL1 encodes an inhibitory receptor expressed on natural killer (NK) cells and T cells KIR3DL1KIR3DL1 is protective for AS KIR3DL1 could reduce inhibition of NK cells and/or T cells, leading to selective activation and clonal expansion.NKG2-E locus overlaps the genes KLRC1, KLRC2 and KLRC3. Copy number variation at NKG2-E (manifested as a deletion of KLRC2) is associated with psoriasis susceptibility KLRC2 expression in T cells is observed in PSO KLRC2 on CD4+ T cells is observed in MS KLRC1 encodes a critical receptor on NK cells, regulating the elimination of autoreactive CD4+ T cells in animal models of MS KLRC1 plays a critical role in tolerization by regulatory T cells The segmental duplication at the KIR3DL1 and KLRC1 encode NK cell receptors. NK cells and their receptors regulate autoimmunity in MS HCAR1 repeat identified in HCAR2 and HCAR3. HCAR2 codes for a niacin receptor that is expressed on antigen presenting cells and functions in a tolerization pathway for T cells The segmental duplication within the long PTPN22 gene  the population of mutant cells increases, concurrently with immune system stimulation.In individuals with impaired tolerance or with sensitive B-cell or T-cell activation thresholds, reactions against mutant cells occur.Inflammation caused by immune reponses induces new coherent mutation in neighboring cells, and creates a cycle of autoimmunity.PTPN22 gene polymorphism  on chromosome 17 resides in a region characterized by complex segmental duplications with identity ranging from 92% to 96%. This cluster is a hot-spot for germ-line gene conversion GH1 codes for human growth hormone, and growth impairment is observed in celiac disease in conjunction with anti-pituitary antibodies A five gene cluster  Cancer studies provide valuable information about coherent somatic mutation in vivo. Many cancers elicit antibodies that are also found in autoimmune disease CD5 expression on B cells is a common feature of both RA and CLL IKZF1 mutations that consistently occur in ALL also occur in RA B cells, possibly followed by clonal expansion, then aberrant glycosylation would be explained because IKZF1 appears to be critical for proper IgG glycosylation IZKF1 could be adaptive, because it would lead to RF production and potentially enhanced clearance of immune complexes IKZF1 expression has been associated with SLE If the RAG-dependent ENO1VIMFGBVIM mutations induced by oxidative stress influence antigenicity Cigarette smoking is mutagenic, and appears to be selectively associated with antibodies to the primary autoantigens encoded by TSHR gene are relatively common Somatic mutations in the A pathogen-expressed protein that binds with an endogenous protein complex could serve as a CD4+ T cell target, providing help to B cells generating antibodies to proteins in the protein complex. A pathogen-modified endogenous protein could behave in a similar fashionRheumatic Fever (RHF) is a condition characterized by autoimmune attack against cardiac muscle, usually associated with group A streptococcal infections The RHF autoantigens vimentin, myosin, and tropomyosin  form parAdaptive immune reponses require the joint participation and mutual activation of CD4+ T cells and antigen-presenting cells such as B cells. B cells become anergic under chronic low-level exposure to antigen with limited costimulation An additional potential mechanism of coherent somatic mutation is retrotransposition. Retrovirus In SSc, the presence of one antibody type is generally exclusive of the others A similar neoantigen-creating role for sumoylation in a subset of patients with primary biliary cirrhosis (PBC) has previously been proposed Schizophrenia and autism have prominent immunological features, including HLA associations, comorbidity with autoimmune diseases, and associations with viral triggers and maternal infections during pregnancy . ImmunolA clue that somatic repeat mutation may contribute to schizophrenia comes from a twin study in which a genomewide measure of somatic trinucleotide repeat mutation was obtained NBPF family genes are among the top twelve in NBPF genes in NBPF genes contain many copies of the DUF1220 element; DUF1220 copy number is closely related to brain size, and humans have many more copies than other primate species DUF1220 copy number correlates with macrocephaly, and low copy number correlates with microcephaly Four IL3RACACNG7IL3RA encodes a receptor for IL3 that is expressed in neurons, and IL3 expression is correlated with brain volume Other schizophrenia-associated genes among those in ASMT encodes the last enzyme in the melatonin biosynthesis pathway, low melatonin expression is observed in autism spectrum disorders, and rare ASMT mutations are associated with autism MGAM is a gene involved in starch metabolism, with dysregulated mRNA expression in autism KATNAL2 have been associated with autism Several autism-related genes appear in Additional autism related genes appear in There is a high concentration of autism-related genes among a relatively small set of putatively mutable genes. In light of the autoimmune features of autism , this coANKRD36C, ANKRD36, AHNAK2, NSUN6) do not have a link with an autoimmune disease, an autoimmune-associated mental disorder, or atherosclerosis. These relatively uncharacterized genes are promising candidates for future study.A satisfying feature of the coherent somatic mutation hypothesis is that it provides a parsimonious yet comprehensive account of autoimmunity. The initiation of most diseases is attributed to a single mutable locus. A handful of diseases having several known subtypes include more than one corresponding mutable locus. Only four of the top sixteen genes in The most prominent prior theory of autoimmunity is molecular mimicry, the hypothesis that peptides similar to host proteins are expressed by host-resident microbes, sometimes inducing an autoimmune reaction against the host proteins. The attractive feature of molecular mimicry has been that it provides a plausible explanation for the known link between infection and autoimmunity PTPRN2) leads to an organ-specific disease (T1D), while a widely expressed protein complex (TTC34/PPP4C as proposed in this report) leads to a systemic disease (SLE).Autoimmune diseases have historically been categorized as organ-specific or systemic, with some diseases hard to categorize The incidence of each of several autoimmune diseases has been rising in recent years FLG gene in some cases FLG, reinforced by local inflammation, could contribute to the pathogenesis of these conditions. An accumulation of somatic mutations in PTPRN2 (without autoimmunity) could lead to glucose intolerance Several non-autoimmune diseases may also be caused by somatic mutation of highly mutable repeat sequence in the context of inflammation. Atopic dermatitis and icthyosis vulgaris are inflammatory skin conditions caused by inactivating germ-line mutations of the MUC1CR1, LPA, HP and DMBT1. Nevertheless, primary autoantigens whose genes contain long repeats were identified in a presumably healthy random individual, suggesting that, at least for those genes, all humans have some degree of somatic mutation and risk for disease.Our study is limited by its reliance on a single human genome for long repetitive sequence. Some reference alleles are much shorter than those typically observed in the population  and long STR sequence was established. Additional autoantigens and peri-antigens were identified among genes spanning long STR sequence, and among genes with other known markers of somatic mutation. The work presented here could lead to a partial resolution of the mystery of why particular proteins are targets of autoimmune destruction Genome coordinates use the GRCh37 (hg19) sequence. Gene names use HGNC approved nomenclature. Queries were submitted to the UCSC MySQL database server Query 1 was submitted to obtain genes containing long or frequent repeats. The output from this query was edited as follows:MGC39584/AC018692.2 on chr4_gl000193_random) had length over 5 kb and none had a repeat count over 100.Genes not on the reference chromosomes were removed. Only one such gene  the TMRF entry was replaced by one with a shorter repeat unit  and a higher repeat-unit count, retaining the degree of identity from the longer sequence. Examples include VPS53 (in which a 96 bp repeat is itself made of 3 instances of a 32 bp repeat), MUC4 (in which a 96 bp repeat consists of two consecutive instances of a 48 bp repeat), and MAL (with an 8 bp AGTGAGTG repeat).When TMRF generated a consensus repeat unit that was itself repetitive  where the contiguous repeat records were combined into a single longer 12.6 kb repeat.In a small number of cases, TMRF generated multiple essentially contiguous repeats with the same period and consensus sequence. The only such case where the repeat was either more than 5 kb long or contained more than 600 repeat units was To see whether the output was dependent on the source of the gene annotations, I reformulated the query as Query 2 using RefSeq NBPF genes. The NBPF repeat sequences were the same, with the exception of one NBPF10 repeat (see below).There was some discrepant labeling of the ANKRD36C/49539; FAM230A, USP1/7516; PLEKHB2/6521; ANKRD36C/6410; FAM182B/6292.The following genes/repeat-lengths were identified by GENCODE but not RefSeq: NBPF10/15997; ANKRD36B/25486; MUC19/8607.The following genes/repeat-lengths were identified by RefSeq but not GENCODE: A large majority of repeats were common to the two annotations, with the differences mentioned above largely due to differences in the labeling of a gene transcript as protein coding.MUC19 transcript identified by RefSeq may have immunological significance given the association of MUC19 with Crohn's disease and ulcerative colitis The differences between the two annotations appear to be small. The MUC5ACMUC5AC exon 31 sequence reported by Guo et al Genes that span gaps in the human assembly where the gaps are presumed to include repetitive sequence , the records for the gene were combined into a single record for the longer compound tandem repeat. When multiple segmental duplications overlapped (TTC34) only the longer duplication was retained.Query 3 was used to identify a preliminary set of segmental duplications occuring within protein-coding genes, using the segmental duplication track Query 4 was used to identify long self-alignments (score at least 60) within protein-coding genes, using the self-alignment track NSUN6 repeats . For the purposes of determining a Determining the significance of a set of autoantigens within a gene list requires an estimate of the total number of autoantigens. Stadler et al. File S1(PDF)Click here for additional data file.


N-MeVal-4-CsA but not FK506 decreased Notch1 NICD cleavage. To examine the effect of CSA on vascular development and function, double transgenic Fli1-GFP/Gata1-RFP zebrafish embryos were treated with CSA and monitored for vasculogenesis, angiogenesis, and overall cardiovascular function. Vascular patterning was not obviously impacted by CSA treatment and contrary to the anti-angiogenic activity ascribed to CSA, angiogenic sprouting of ISV vessels was normal in CSA treated embryos. Most strikingly, CSA treated embryos exhibited a progressive decline in blood flow that was associated with eventual collapse of vascular luminal structures. Vascular collapse in zebrafish embryos was partially rescued by global Notch inhibition with DAPT suggesting that disruption of normal Notch signaling by CSA may be linked to vascular collapse. However, multiple signaling pathways likely cause the vascular collapse phenotype since both cyclophilin A and calcineurin/NFAT were required for normal vascular function. Collectively, these results show that CSA is a novel inhibitor of Notch signaling and vascular function in zebrafish embryos.Cyclosporin A (CSA) suppresses immune function by blocking the cyclophilin A and calcineurin/NFAT signaling pathways. In addition to immunosuppression, CSA has also been shown to have a wide range of effects in the cardiovascular system including disruption of heart valve development, smooth muscle cell proliferation, and angiogenesis inhibition. Circumstantial evidence has suggested that CSA might control Notch signaling which is also a potent regulator of cardiovascular function. Therefore, the goal of this project was to determine if CSA controls Notch and to dissect the molecular mechanism(s) by which CSA impacts cardiovascular homeostasis. We found that CSA blocked JAG1, but not Dll4 mediated Notch1 NICD cleavage in transfected 293T cells and decreased Notch signaling in zebrafish embryos. CSA suppression of Notch was linked to cyclophilin A but not calcineurin/NFAT inhibition since  Cyclosporin A (CSA) is an immunosuppressant that binds to and suppresses cyclophilin A . CSA binN-MeVal-4-CsA) that does not block calcineurin activity maintains anti-angiogenic activity suggesting that cyclophilin A rather than NFAT/calcineurin is linked to angiogenesis [In addition to immunosuppression, CSA has also been shown to elicit a wide range of negative effects in the cardiovascular system including disruption of heart valve development, smooth muscle cell proliferation, and angiogenesis inhibition. In particular, CSA suppress angiogenesis in a variety of models including the chick CAM , 9, rat ogenesis . Based oThe original goal of this research was to investigate the ability of CSA to control Notch signaling, and to determine if CSA suppresses angiogenesis in zebrafish embryos. Our results show that CSA directly suppresses Notch signaling in response to Jagged1 but not Delta-like 4 and that CSA inhibition of cyclophilin A, but not calcineurin is linked to Notch inhibition. However, CSA did not appear to have a direct effect on angiogenesis in zebrafish embryos, but rather had widespread negative effects on cardiovascular function that were initiated by inhibition of both cyclophilin A and calcineurin.Previous results have suggested that CSA may interact with the Notch signaling pathway  althoughin vivo. To accomplish this, we monitored the affect of CSA on Notch activity in transgenic zebrafish expressing GFP from the Notch responsive TP1 element (i.e. Tp1bglob:eGFP) as previously described [in vitro and in vivo.These results suggested that CSA decreases Notch signaling in transfected 293T cells, but it was important to determine if CSA also controls Notch signaling escribed . FreshlyN-MeVal-4-CsA which blocks cyclophilin A but not calcineurin/NFAT signaling [N-MeVal-4-CsA decreased JAG1—Notch signaling while FK506 did not significantly effect Notch signaling  and smooth muscle cells [CSA treatment elicits a wide variety of effects on endothelial and smooth muscle cells in the vascular tree. Notch signaling has emerged as a major regulator in the vertebrate vascular system, serving roles in both endothelial  CSA analog was required for this effect.Previous results have shown that signaling through the calcineurin/NFAT pathway is required for endothelial response to VEGF , and forThese results suggested that both the calcineurin/NFAT and cyclophilin A pathways are required for proper maintenance of vascular lumen structures. Although both cyclophilin A and calcineurin/NFAT have been functionally linked to VEGF signaling in endothelial cells , 29, theOur results have shown that suppression of either cyclophilin A or calcineurin/NFAT leads to the vascular malfunction phenotype triggered by CSA treatment of zebrafish embryos. We also demonstrated that inhibition of cyclophilin A, but not calcineurin/NFAT decreased JAG1 (but not Dll4) mediated Notch activation in 293T cells. Based on these results, it was not clear if suppression of Notch was in any way functionally linked to the CSA vascular malfunction and we therefore attempted to determine if Notch suppression was implicated in this phenotype. We hypothesized that if the vascular defects were due solely to Notch inhibition, then suppression of Notch with the gamma secretase inhibitor drug DAPT should recapitulate the vascular defects induced by CSA treatment. As shown in All work with zebrafish was approved by IACUC committee at Indiana State University and Boise State University and performed according to Indiana State University (Protocol #11–08–2007:AA) and Boise State University (Protocol # 006-AC13–004) Institutional IACUC guidelines.fli-1:eGFP)/Tg(gata-1:RFP) zebrafish were donated by Stephen C. Ekker . Freshly laid eggs were incubated in 10μM CSA, 15μM DAPT, or an equivalent concentration of DMSO and imaged 24–48 hpf under a fluorescent dissecting microscope (Nikon). Transgenic Notch reporter zebrafish (Tp1bglob:eGFP) were donated by Dr. Steven D. Leach . Notch expression was monitored in 24 hpf embryos by measuring GFP fluorescence on a fluorescence multi-well plate reader.Transgenic  was used at a dilution of 1:250 to detect cleaved Notch1. The 9E10 monoclonal antibody (Santa Cruz) was used at a concentration of 1:1000 to detect myc-tagged Notch1, JAG1, and Dll4. The β-actin monoclonal antibody (Santa Cruz) was used at a dilution of 1:1000 to detect β-actin as a loading control. The anti-vinculin antibody (Santa Cruz) was used at a dilution of 1:1000


Streptomyces platensis, is an excellent breakthrough in recent antibiotic research with unique structural pattern and significant antibacterial activity. β-Ketoacyl-(acyl-carrier-protein (ACP)) synthase (FabF) whose Gram-positive bacteria need to biosynthesize cell membranes is the target of inhibition of platensimycin. So, isolation, retrosynthetic analysis, synthesis of platensimycin, and analogues of platensimycin synthesized till today are the objectives of this review which may be helpful to further investigate and to reveal untouched area on this molecule and to obtain a potential antibacterial lead with enhanced significant antibacterial activity.Emerging resistance to existing antibiotics is an inevitable matter of concern in the treatment of bacterial infection. Naturally occurring unique class of natural antibiotic, platensimycin, a secondary metabolite from These pathways share many chemical, mechanistic, and structural features, and, thus, fatty acid synthesis inhibitors recurrently inhibit polyketide synthesis . An organism producing a fatty acid synthesis inhibitor must retain not only a resistant form of the fatty acid synthetic enzyme but also a resistant form of the antibiotic-producing polyketide synthase. Furthermore, if several polyketides are needed for survival of the organism in its ecological niche, then resistant forms of each of these pathways would be prerequisite. Such precincts seem likely to severely narrow the opportunities to evolve fatty acid synthesis inhibitors and may account for the scarceness of such antibiotics. However, various Streptomyces produce analogues of thiolactomycin, which has not been reported to block polyketide synthesis, thus demonstrating that development of fatty acid synthesis inhibitors by this organism is conceivable [The majority of the “illness” lies in the fact that when immune system is defeated, it is in war with pathogens. Development starting from ethnic to modern synthetic approaches in drug discovery mainly provides better weapons to combat and survive against pathogens. In contrast to medical development, pathogens also have acquired protection called “resistance.” More or less all classes of antibiotics are resistant to bacteria; hence a novel process to the discovery of antibiotic with new mechanism of action is essential , 2. Micr4 μg/mL) . On the ceivable . S. platensis (MA7327 and MA7331) using SephadexLH20 liquid chromatography medium by reversed-phase HPLC chromatography [ Streptomyces platensis (MA7327), recovered from soil samples collected in Eastern Cape, South Africa [Platensimycin was isolated by Wang et al. in 2006 at concentration of 2 to 4 mg/L from fermentation broth oftography . It was h Africa .Platensimycin  consistsPlatensimycin consists of an aromatic acid conjugated with aliphatic moiety by an amide linkage. The effective synthetic strategy is to synthesize the aromatic and aliphatic parts separately and then combine them by amide linkage. Many synthetic methods are available for the synthesis of those two building blocks.Nicolaou et al. in the year 2006 first reported the total synthesis of platensimycin . 2 and the carboxylic acid 3 by disconnection of amide linkage. The carboxylic acid was further simplified to a simplified enyne in successive three retrosynthetic steps. This resulted in two target molecules to synthesize the tetracyclic carboxylic acid and the aromatic amine from simplified starting material.Using retrosynthetic analysis  they sep 9 [ 10 from 8. Reduction followed by acidic hydrolysis and reintroduction of the TBS ether produced enone 11 from enone 10 (yield 84%). Spirocycle 12 was generated by cycloisomerization of 11 [ 12 produced bis-enone 13 [ 6. Secondary alcohol 14 was prepared by addition of samarium(II) iodide in a dilute solution of aldehyde 6 HFIP in THF/HMPA followed by NH4Cl solution. Esterification of 14 with TEA resulted in the formation of cage-like structure 4 which on treatment with KHMDS and MeI followed by KHMDS and allyl iodide produced olefin 16. Vinyl pinacol boronate and 16 reacted in presence of the Grubbs second generation catalyst to produce vinyl boronate 19 which on reacting with trimethylamine N-oxide gave 20. Following Pinnick protocol 20 was converted to desired carboxylic acid 3.The simplified enone generated from 3-ethoxycyclohex-2-enone which was used as a starting material . Allylic 9  -(−)-carvone from 26 . 31 on reaction with Grignard's reagent 32 using CeCl3 [ 33. Enone 33 was treated with Hg(OAc)2 and later on with NaBH4 to generate approximately 1 : 1 mixture of exo- and endotertiary alcohol, which on dehydration using Martin's sulfurane reagent produced exocyclic alkene 35 which was treated with TMSCl and HMDS and followed by an electrophilic quench with PhSeCl and subsequent oxidative elimination (H2O2) to give enone 36. Enone 37 was treated with SmI2 for radical cyclization, and by adding mentioned reagent ( 26 was synthesized.(R)-(−)-carvone reagent  in subse 4. They expected that the transannular radical cyclization of 44 will produce enone 4. A four-step retrosynthetic analysis is producing enone 47 and siloxydiene 48. The key feature of plan is that all stereocenters in 4 are controlled by the stereochemistry presented in 47 . Diels-Alder reaction in between 47a and 47b and siloxydienes produced two stereogenic mixtures 46a, 46b and 46c, 46d. The mixture of two inseparable diastereomers (46b and 46d) was employed in the next step. Following Noyori's procedure [ 53 was given which upon catalytic oxidation using palladium(II) chloride and copper(II) acetate produced 54a and 54b in 10 : 1 ratio. Vinyl triflate 55 was produced from 54 and reduced to 56 which was transformed to 57. Transannular radical cyclization of monothioacetal 57 gave desired 4  and Enterococci (VRE), MIC < 1.0 μg mL−1) by selectively inhibiting cellular lipid biosynthesis [ S. aureus to the drug platensimycin confirms that FabF is the useful target for antibacterial action. Platensimycin exhibited an IC50 of 48 nM and 160 nM against FabF in S. aureus and E. coli, respectively. But the drug shows weak inhibition against FabH with an IC50 value of 67 mM. Further studies have shown that in vitro binding of platensimycin with FabF is relatively weak, which leads to the discovery of its binding with acyl-thioester intermediate of the FabF pathway. From a crystal structure of a Cys-163-Gln FabF mutant, which simulates acyl-thioester intermediate, it was found that platensimycin bind with the active site of FabF with the carboxylic acid group lying in the malonate-binding site coplanar with the amide side chain of Gln163 [Bacterial cell wall synthesis, protein synthesis, and DNA replication are the predominant targets for widely used antibiotics. But the emergence of resistance to antibiotics demands new antibacterial targets. Fatty acid synthase (FAS) pathway is now an attractive target for antibacterial agents because, as a new target, FAS inhibition will not suffer from bacterial resistance immediately and biosynthesis pathway of bacteria, plants, and parasites (FAS II in which component proteins are dissociated) is different from mammals  in subcellular organization of components which demonstrate a target specificity for the FAS II inhibitors. A general scheme for type II fatty acid biosynthesis is shown in ynthesis . The mecf Gln163 . Streptococcus agalactiae . The need for synthesized fatty acid by their own  reduces in the presence of exogenous fatty acid in both in vitro and in vivo conditions. Human serum has a high composition of fatty acids, so FAS II inhibitor may not affect at all Gram-positive pathogens in vivo. But the findings with S. agalactiae can be reasonably extended to all Gram-positive bacteria, which has started a vigorous debate. Also there is no clear explanation how bacteria incorporates exogenous fatty acids into their cell [ Escherichia coli (tolC), but not against wild-type E. coli, specifying that efflux mechanisms, and not compound specificity, limit the effectiveness of platensimycin in E. coli and possibly other Gram-negative bacteria [Though Brinster et al.  explaineeir cell , 62. Plabacteria . S. aureus [The major drawback to most natural products, including platensimycin, is poor pharmacokinetic properties and negligible oral bioavailability. Only the continuous infusion of a high platensimycin dose showed effectiveness in mice infected with. aureus . For theDevelopment of bacterial resistance to the existing antibiotics is an alarming situation in the 20th century. Finding new target for bacterial demolition is essential. Platensimycin serving as a potential antibiotic interacting with FabF may bypass bacterial resistance. It should be noted that starting from 2006 till now a huge number of scientists provided many synthetic strategies and derivatives which are very encouraging. Though some evidence like incorporation of exogenous fatty acid inside bacteria when supplied makes the hope regarding platensimycin is uncertain, overall, isolation of platensimycin as a selective FabF inhibitor, complex synthesis of tetracyclic cage, and enhancement of its pharmacokinetic properties by its derivative synthesis are the excellent works in the era of medicinal chemistry. It is expected that this review might be helpful for the medicinal chemist.


In addition, among patients with severe RSV bronchiolitis during infancy, we examined genetic associations with asthma, active asthma, persistent wheeze, and bronchial hyperreactivity (methacholine PC20) at age 6 years. We identified four rare nonsynonymous variants that were significantly associated with asthma following severe RSV bronchiolitis, including single variants in ADRB2, FLG and NCAM1 in European Americans , and NOS1 in African Americans (p = 2.3x10-11). One of the variants was a highly functional nonsynonymous variant in ADRB2 (rs1800888), which was also nominally associated with asthma (p = 0.027) and active asthma (p = 0.013) among European Americans with severe RSV bronchiolitis without including the ESP. Our results suggest that rare nonsynonymous variants contribute to the development of asthma following severe RSV bronchiolitis in infancy, notably in ADRB2. Additional studies are required to explore the role of rare variants in the etiology of asthma and asthma-related traits following severe RSV bronchiolitis.Severe infection with respiratory syncytial virus (RSV) during infancy is strongly associated with the development of asthma. To identify genetic variation that contributes to asthma following severe RSV bronchiolitis during infancy, we sequenced the coding exons of 131 asthma candidate genes in 182 European and African American children with severe RSV bronchiolitis in infancy using anonymous pools for variant discovery, and then directly genotyped a set of 190 nonsynonymous variants. Association testing was performed for physician-diagnosed asthma before the 7 Asthma is a complex disease caused by both genetic and environmental factors, and the interactions between them. Among thMore than 100 genes have been associated with asthma and asthma-related traits, however there is marked variability in replication attempts in independent studies. This mayth birthday (asthma) following severe RSV bronchiolitis in infancy using the ESP as controls to gain statistical power. Lastly, among patients with severe RSV bronchiolitis during infancy, we examined genetic associations with asthma, active asthma, persistent wheeze, and bronchial hyperreactivity (methacholine PC20) at age 6 years.The evolution of the common disease/rare variant hypothesis suggests that the majority of the missing heritability in common and complex phenotypes is instead due to rare or private DNA variants. PreviousIn the RSV Bronchiolitis in Early Life (RBEL) prospective cohort study, 206 infants (12 months of age or less) were enrolled from 1998 to 2001, of which 182 had DNA samples available and were included in the current study . Completphysician-diagnosed asthma  if the parent/guardian answered “Yes” to “Has your child ever been diagnosed with asthma by a physician?” at any time before the child’s 7th birthday. A persistent “No” response defined children without physician diagnosed asthma. We defined active asthma as physician-diagnosed asthma at any time along with parent-reported wheezing during the last year of follow-up between the child’s 3rd and 7th birthdays. We defined persistent wheezing as having at least one wheezing episode in first three years of life and at least one wheezing episode between the child’s 3rd and 7th birthdays. Methacholine bronchoprovocation was performed at age 6 as previously reported.[bronchial hyperreactivity as the provocation concentration of inhaled methacholine that caused a 20% drop in baseline forced expiratory volume in one second (methacholine PC20).A child was classified as having reported. We definThis study was approved by the Washington University School of Medicine Institutional Review Board (RBEL), and the Boston Children’s Hospital Institutional Review Board (BRASS). Written informed consent was obtained from parents or guardians.A detailed description of the Boston RSV bronchiolitis cohort (BRASS) is available elsewhere. Briefly,A total of 99 European Americans (EA) and 83 African Americans (AA) from the RBEL study were sequenced in four anonymous pools for variant discovery. These pools were “anonymous” in that DNA from a single individual was not barcoded prior to pooling DNA for sequencing, and thus the resulting variants could not be assigned to a single individual . Genomic DNA was extracted from whole blood using Wizard genomic DNA purification kit (Promega) and individually quantified by NanoDrop 2000 spectrophotometer (Thermo Scientific). Insufficient DNA yield samples (n = 65) were amplified using REPLI-g Mini Kit (Qiagen Inc) and subsequently purified using QIAamp DNA blood mini kit (Qiagen Inc). 200 ng DNA from each individual was combined into four separate anonymous pools, with separate pools for European and African Americans, and for asthma cases and asthma controls.frodo.wi.mit.edu/), and using in silico PCR on the UCSC Genome Browser[11), and concatenated overnight at 22°C with T4 DNA ligase and T4 polynucleotide kinase (New England Biolabs) in the presence of 15% (w/v) polyethylene glycol, MW8000 (Sigma-Aldrich).A total of 131 candidate genes were selected from published studies that demonstrated a prior, positive association with asthma . Coding e Browser,20 to coe Browser Briefly,Positive and negative control DNA amplicons were included in ligations to monitor base calling accuracy. After 10Sequence analysis and variant calls was performed using SPLINTER. A model https://icom.illumina.com/Custom/UploadOpaPrelim/). A total of 384 variants in coding exons were chosen for custom array design , which included 190 nonsynonymous variants. Genotyping was performed concurrently on all 182 individuals from the RBEL study (the discovery cohort), and 177 individuals from the BRASS study (the replication cohort).We performed custom genotyping of 384 variants using an Illumina GoldenGate array for all individuals within each of the four anonymous pools. A total of 710 unique nonsynonymous variants were identified through pooled sequencing, and scored as to their designability for the GoldenGate array  was performed according to the manufacturer’s Instructions (ref. GoldenGate Genotyping Assay Guide 15004065 B). An iScan System was used for chip scan and image acquisition. Genotypes were called using Genome Studio v.2009.1 software (Illumina Inc), and quality control was performed using PLINK. Subjects20, log transformed) among individuals with severe RSV bronchiolitis in infancy (excluding the ESP). Covariates examined included age at severe RSV bronchiolitis, sex, daycare attendance, maternal smoking during pregnancy and at time of enrollment, smoke exposure from other family members in home, and asthma status for bronchial hyperreactivity.Association testing at individual variants genotyped on the GoldenGate array was performed using logistic or linear regression within each ethnicity in PLINK. We testeGenotype counts from the ESP were downloaded from the NHLBI Exome Sequencing Project Exome Variant Server (ESP6500SI-V2), convertWe obtained a total of 695,366 base pairs (bp) of targeted sequencing at an average depth of 52–353x coverage/allele at variable sites in 182 individuals in the RBEL study . We obseA total of 384 rare and common variants, including 190 nonsynonymous variants were genotyped directly in the same samples included in the anonymous pools on a custom GoldenGate array. Of these, 264 variants passed stringent quality control filters. Of the 120 variants failing QC, 76 had call rates below 95%, 61 had Hardy-Weinberg p-values < 0.05 in the African American samples, and 38 had Hardy-Weinberg p-values < 0.05 in the European American samples. Not all variants identified through sequencing were identified as being polymorphic on the GoldenGate array, which may be due to the variants being absent  or difficulties in clustering carriers of rare variants . However, variants were confirmed, and carriers of minor/rare alleles were identified at 78 variants in the African American samples, and 49 variants in the European American samples. Individuals from the replication study  were genotyped using the same custom array using whole genome amplified DNA; 275 variants passed QC ; a total of 76 variants were found to be polymorphic in BRASS. Across both studies combined (RBEL and BRASS), a total of 92 variants were brought forward for association testing.NOS1 (Bonferroni threshold: α78 = 6.4x10-4) and three in European Americans in FLG, ADRB2, and NCAM1 (α49 = 1.0x10-3)  and active asthma (p = 0.013) among European Americans with severe RSV bronchiolitis in infancy, without including the ESP as controls , one carrier of rs35576001 in NCAM1 (a European American), and one carrier of rs76090928 in NOS1 (an African American).To have sufficient statistical power, we included 6,503 individuals from the Exome Sequencing Project (ESP6500) as controls, including 2,203 African Americans and 4,300 European Americans. Following additional QC, allele frequencies in asthma cases from the RBEL study were in general consistent with frequencies in the ESP . However.0x10-3) . All of controls . Rs35576ADRB2 was present in the BRASS study at similar frequencies .Replication of four variants associated with physician-diagnosed asthma was attempted in an independent study  as controls. This allowed us to identify four nonsynonymous variants that were significantly associated with asthma following severe RSV bronchiolitis for follow-up studies. All four of the nonsynonymous variants were low frequency or rare in the ESP (MAF < 5%), and were at higher frequency in individuals with asthma following severe RSV bronchiolitis in infancy (MAF from 1–6%).We identified over 700 nonsynonymous variants by sequencing the coding exons of 131 asthma-associated genes in 182 individuals who experienced severe RSV bronchiolitis in infancy. Following this, we genotyped a set of 190 nonsynonymous variants, and performed association testing for physician-diagnosed asthma before the 7ADRB2 (rs1800888) that showed a significant association with asthma following severe RSV bronchiolitis in European Americans when using the ESP as controls. Rs1800888 was also nominally associated with asthma and active asthma within individuals with severe RSV bronchiolitis in infancy, without including the ESP as controls. ADRB2 is a G protein-coupled receptor that along with other proteins forms a receptor-channel complex involved in smooth muscle relaxation and bronchodilation. Notably, rs1800888 has the most profound functional consequence of all known nonsynonymous variants in ADRB2, and is associated with a 5-fold reduction in sensitivity to beta-2-receptor agonist-mediated vasodilation resulting in increased vasoconstriction.[ADRB2 have been associated with both asthma and bronchodilator drug response.[We identified a nonsynonymous variant in triction. This varresponse.–30FLG and NCAM1 in European Americans, and NOS1 in African Americans. These associations were driven by a single carrier of each variant and thus require additional study. FLG encodes the filaggrin protein, which functions in differentiation of the epidermis and maintaining barrier function.[FLG have been associated with atopic dermatitis[NCAM1 is unknown, it is predicted to be probably damaging by PolyPhen2.[NOS1, which contains a hypoxia-responsive promotor and is only transcribed in hypoxic conditions.[NOS1 have been associated with atopy[NOS1 is due to an imbalance in the proportion of European admixture, rather than a true genetic association with asthma following severe RSV.We similarly observed an association with asthma following severe RSV bronchiolitis and rare nonsynonymous variants in function. Common vermatitis–34, eczeermatitis,35–38, aermatitis NCAM1 isolyPhen2. NOS1 is olyPhen2. Rs760909nditions. Variantsith atopy,42, totaith atopy,44, and ith atopy However,Given that all of our associations were with low frequency and rare variants, and that a severe form of RSV bronchiolitis in infancy is relatively uncommon (0.5% of RSV bronchiolitis cases in children < 24 months), we werIt is important to note two limitations of using the ESP as controls in our study. First, given the heterogeneous clinical characteristics of the ESP samples they cannot strictly be considered “population controls” as some individuals may have experienced asthma following severe RSV bronchiolitis in infancy. The goal of the ESP was to identify genetic variation relevant to heart, lung, and blood disorders in well-phenotyped populations, and contains individuals from clinical studies including 191 African Americans with asthma from the Severe Asthma Research Program (SARP). This could have lead to rare variants that contribute to the development of asthma following severe RSV in infancy being more common in the African American ESP as compared to healthy controls, however this would not have lead to false positive associations, and is not expected to have a large effect on our power to detect an association. FurthermADRB2. Additional studies are needed to confirm these associations and determine the functional consequences of these genetic variants.In conclusion, we identified a number of novel, potentially functional nonsynonymous variants through the sequencing of anonymous pools of asthma cases and controls following severe RSV bronchiolitis during infancy. Direct genotyping of a subset of these variants identified four nonsynonymous variants associated with increased risk of asthma following severe RSV bronchiolitis, including a highly functional rare variant in S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Cells showed good proliferative ability in culture condition. The cells successfully differentiated into the adipogenic and osteogenic lineages. Based on these findings, amniotic fluid can be considered as an appropriate and convenient source of human amniotic fluid stem cells. These cells provide potential tools for therapeutic applications in the field of regenerative medicine. To get a better understanding of crosstalk between Oct4/NANOG with osteogenesis and adipogenesis, we used network analysis based on Common Targets algorithm and Common Regulators algorithm as well as subnetwork discovery based on gene set enrichment. Network analysis highlighted the possible role of MIR 302A and MIR let-7g. We demonstrated the high expression of MIR 302A and low expression of MIR let7g in hAFSCs by qPCR.Human stem cells and progenitor cells can be used to treat cancer and replace dysfunctional cells within a tissue or organ. The objective of this study was to identify the appropriate cells type in regenerative medicine and targeted therapy. As an alternative to embryonic and bone marrow stem cells, we examined human amniotic fluid stem cells (hAFSCs), one of the potential source of multipotent stem cells isolated from both cell pellet (using single-stage method), and supernatant of human amniotic fluid. Source of isolation and unique property of the cells emphasize that these cells are one of the promising new tools in therapeutic field. Double sources for isolation and availability of the left over samples in diagnostic laboratory at the same time have less legal and ethical concerns compared with embryonic stem cell studies. Cells were isolated, cultured for 18 Over the past two decades, a great interest has been paid to stem cell therapy in cancer therapy , regenerAlthough autologous types of stem cells have some advantages and are not subjected to issues but the main barriers could be rare in the number and difficulty of isolation, purification and maintenance to reach the required number for transplantation. In order to avoid these problems and overcome to limitations, scientists have looked to other sources for pluripotent cells such as amniotic fluid stem cells. Amniotic fluid is well-known in diagnostic fields and comprise multiple cell types derived from the developing fetus , 19 as wHuman amniotic fluid stem cells (hAFSCs) possess many characteristics, which may identical to human ESCs, such as: expression of embryonic markers, the ability to maintain their telomeric length, potential to give rise to multi-lineage and capable to maintain in culture for many population doublings .When they are stimulated with different growth factors, molecules and medium, have potential to give rise to multiple cells, derived from all the three germ layers , 26. TheThere are two main methods for isolation of hAFSCs –28 whichWe used network analysis based on algorithms including Common Targets, Common Regulators, and Gene Set Enrichment to obtain a better view on the relationships between Oct-4 and NANOG with adipogenesis and osteogenesis.Five milliliters of human amniotic fluid samples were obtained from 10 patients undergoing amniocentesis for routine karyotype screening and evaluated in Al-Zahra hospital . Amniotic fluid samples were donated by mothers for this research after clarification and filling patient consent forms under the ethic guide line of Tabriz University of Medical Sciences. All participants provide their written informed consent to participate in this study; the ethics committee approves this consent procedure: (registered number 5.4.753 at ethic committee of TUMA). The Amniocentesis was performed under gynecologist supervision by sonographer guides using a 22G Needle. Dulbecco's Modified Eagle Medium Low Glucose) supplemented with 15% FBS , 10 ng/ml basic fibroblast growth factor .Amniotic cells were isolated from human amniotic fluid through centrifuging samples at 450 g for 10 min and preserved in 37°C prior to prepare culture condition. To optimize the best culture condition for cells, we used two types of media. AmnioMAX II Complete Medium , this medium is developed for the short term culture of human amniotic fluid cells and is premixed complete media. Type two media was DMEM- L (The resulting cell pellets were homogenized in both culture media separately and were seeded in 6 wells plates with defined medium (AmnioMAX or DMEM-L) and incubated at 37°C in a humidified gas environment of 5% CO2 in air. After one week of culture, there were no attached cells in plates containing DMEM-L media but cells were divided into two sets in AmnioMAX medium:Some cells which attached to the plate were kept in this plate for 3 weeks and cultured based on Single-stage method Other Cells which were detached and floating in media were collected and cultured based on Two-stage method. So the supernatant was collected, transferred and cultured in new plate (P0) .th passages during 6 months.After 3 weeks, the cells of both methods showed multilayers morphology, which had the 90% confluency in AmnioMAX medium. The medium were replaced two times per week. After 3 weeks, primary passage (P1) was performed through harvesting cells by trypsinization. Cells were transferred to T25 flasks containing type two media (Gibco) and incubated at 37°C with 5% CO2 atmosphere for expansion. Cells were able to reached 80–90% confluence in 5 to 7 days which were passaged. The putative hAFSCs were cultured for 18The goal of this experiment was to characterize established putative cell lines (randomly 4 of 10 samples) at 3th-5th passage after extended culture for pluripotency markers using RT-PCR and mesenchymal stem cells markers using FACS analysis.Pluripotency statuses of cell lines were evaluated at P3-5 before cryopreservation. Cells were checked by reverse transcription polymerase chain reaction (RT-PCR) for gene expression of pluripotent markers including Oct4 and NANOG which were visualized by gel electrophoresis. In addition, cells were prepared for FACS analysis to assess for mesenchymal stem cells markers such as CD90 and CD44 and hematopoietic markers such as CD31 and CD45.5 undifferentiated hAFSCs from 3th-5th passages, using RNX-Plus kit  according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from total RNA using Termo Scientific Revert Aid First Strand cDNA Synthesis Kit  with oligo-dT primer in a 20 μl reaction mixture which was performed in a Thermal Cycler (PeQLab). Primers used for amplification are listed in Total RNA was extracted from 5–6×10–ΔΔCt method, the amount of target, normalized to an endogenous reference and relative to a calibrator, where ΔCt = Ct target gene–Ct endogenous reference and ΔΔCt = ΔCt sample– ΔCt calibrator. All tests have performed in triplicate . Down regulation of ID2 governed by gown regulation of POUSF1 and NANOG can promote both ossification and adipogenesis.Interestingly, Common Targets algorithm highlighted the possible function of 2 microRNAs including MIR302A and MIRLET7G Figs  and 9 asThe ranked presentation of regulators which regulate both NANOG and Oct-4, adipogenesis and osteogenesis based on Common Regulators algorithm is presented in Interestingly, this network suggests the key regulatory effects of 3 receptors, including CASR , NOTCH, and IGFIR . CASR isThe gene set enrichment concept was employed to identify and report the key subnetworks. This approach calculates the enrichment/overrepresentation of different subnetworks by an imported list of genes/miRNAs/functions to determine the statistically enriched subnetworks at p≤0.01, based on various statistical measurements, in particular Fisher’s exact test.Downstream subnetwork discovery, based on gene set enrichment concept, using adipogenesis and osteogenesis, NANOG and Oct-4 as inputs resulted in identification of some central regulatory subnetworks which are presented at In this study, we isolated hAFSCs and characterized them.Cells presented ability to form colony and maintain for extended passages 15. In addition our cell lines maintained multi-potency after freezing/thawing. These cells were easily isolated from patient’s samples undergoing amniocentesis for routine karyotype screening. Amniocentesis is a moderately gentle modality applied for prenatal and karyotype screening . Our metThe pluripotency of hAFSCs has been demonstrated by expressing Oct-4 and NANOG genes and b-actin as housekeeping gene through RT-PCR and real-time PCR (qPCR). NANOG and Oct4 are two transcription factors necessary to sustain the pluripotency and self-renewal of embryonic stem (ES) cells , 56. In Over 90% -99% and 85% of hAFSCs were positive for mesenchymal stem cell surface markers CD90 and CD44, respectively which is quite good level of multi-potency of cells. The cells were negative for hematopoietic markers, CD 31 and CD 45 as presented by flow cytometry.Cells were positive for NANOG and Oct4 as shown by RT-PCR. HAFSCs have been also identified to express pluripotency markers of embryonic stem cells, such as Oct4, NANOG , 58 as wIn this study, we provide a cell bank of undifferentiated hAFSCs which in future may provide a convenient source to patients as therapeutics applications.HAFSs have potential as precursors to a wide range of differentiated cell linages , 67. LikFurthermore, it is incredible that hAFSCs were shown positive for Oct-4 and NANOG genes, characteristic of the embryonic and adult stem cells, thus proposing their potential pluri- multi-potentiality properties, they are between hESCs and hASCs , 68.The surface marker expression of hAFSCs and their potential for expression of the transcription factor Oct4 proposes that they characterize an intermediate stage between pluripotent ESCs cells , 67, 69 Network analysis using Common Targets algorithm and Common Regulators algorith provided a better picture of the molecular mechanisms which are possibly involved in ossification and adipogenesis differentiation in relation to NANOG and POUSF1. The constructed network suggests importance of transcription factors including AHR, SOX2, ID2, KLF4 and EMT1 as well as MIR 302A and MIR Let7g. The candidate genes/mechanisms from network analysis provide the required knowledge for future laboratory experiment in stem cell differentiation to osteogenic and adipogenic cells.It has been reported that Oct4 and Sox2 have binding sites on the promoter region of MIR 302A. MIR 302A is able to repress the key proteins such as cyclin D1and keep stem cell in G1 phase . It has In conclusion, our results show the elasticity of hAFSCs and their favorable potential as a multi-potent cell source for regenerative stem cell therapy and their capability to give rise to multiple lineages including osteoblasts and adipocytes. We validated the high expression of MIR 302A and low expression of MIR let7g in hAFSCs by qPCR. These microRNAs were central in gene network of hAFSCs.S1 Fig(PNG)Click here for additional data file.S2 Fig(PNG)Click here for additional data file.S3 Fig(PNG)Click here for additional data file.S1 Table(CSV)Click here for additional data file.S2 Table(CSV)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Papillomaviridae family, is the primary etiological agent of cervical cancer. Here, we report the complete genome sequences of four HPV16 Asian American variants and four European variants, isolated from cervical biopsies and scrapings in India.Human papillomavirus type 16 (HPV16), a member of the  Papillomaviridae family, is phylogenetically clustered within the Alphapapillomavirus 9 species group and is predominantly associated with cervical cancer (CaCx) (–Human papillomavirus type 16 (HPV16), a member of the r (CaCx) . Correspr (CaCx) –5, namelIn India, HPV16 has been found to be the most prevalent high-risk type associated with CaCx cases –9.–Here, we report the complete genome sequences of four viral isolates belonging to the AA variant lineage and four viral isolates belonging to the E variant lineage, isolated from cervical biopsies and scrapings in India.De novo assembly was carried out to form consensus sequences using the Geneious version 7.0.3 assembler  according to the manufacturer’s instructions. HPV screening was carried out using broad range GP5+/GP6+ primer pairs, and the presence of HPV16 was confirmed by quantitative E6 PCR . Viral gssembler . Whole-gssembler  from eacssembler  using HPWhole-genome sequences of all eight viral isolates have been deposited in GenBank using NCBI’s BankIt tool, and the accession numbers are listed in


Renal fibrosis is the final common pathway of chronic kidney disease (CKD). Moesin is a member of Ezrin/Radixin/Moesin (ERM) protein family but its role in renal fibrosis is not clear.Human proximal tubular cells (HK-2) were stimulated with or without TGF-β1. Moesin and downstream target genes were examined by real-time PCR and western blot. Phosphorylation of moesin and related signaling pathway was investigated as well. Rat model of unilateral ureteral obstruction (UUO) was established and renal moesin was examined by immunohistochemistry. Moesin in HK-2 cells were knocked down by siRNA and change of downstream genes in transfected HK-2 cells was studied. All animal experiments were reviewed and approved by the Ethics Committee for animal care of Ruijin Hospital.HK-2 cells stimulated with TGF-β1 showed up-regulated level of α-SMA and down-regulated level of E-Cadherin as well as elevated mRNA and protein level of moesin. In rat model of UUO, renal moesin expression increased in accordance with severity of tubulointerestital fibrosis in the kidneys with ureteral ligation while the contralateral kidneys were normal. Further study showed that TGF-β1 could induce phosphorylation of moesin which depended on Erk signaling pathway and Erk inhibitor PD98059 could block moesin phosphorylation. Effects of TGF-β1 on moesin phosphorylation was prior to its activation to total moesin. RNA silencing studies showed that knocking down of moesin could attenuate decrease of E-Cadherin induced by TGF-β1.We find that moesin might be involved in renal fibrosis and its effects could be related to interacting with E-Cadherin. Chronic kidney disease (CKD) has received increased attention not only for its socioeconomic burden but also for its impact on the global public health. Irrespective of the underlying causes, progressive CKD often leads to renal fibrosis which is characterized as glomerulosclerosis and tubulointerstitial fibrosis histologically, and end-stage renal disease (ESRD) that requires costly renal replacement therapy clinically Moesin is a member of Ezrin/Radixin/Moesin (ERM) protein family that acts as cross linker between plasma membrane and the actin cytoskeleton In our previous proteomic study All experiments involving rats were reviewed and approved by the Ethics Committee for animal care and use of research center for experimental medicine of Ruijin Hospital.2 as we previously described Human proximal tubular cells  obtained from ATCC were grown in keratinocyte serum-free media  supplemented with 0.05 mg/ml bovine pituitary extract  and 5 ng/ml epidermal growth factor  in a 37°C incubator with 5% COTotal RNA was isolated from HK-2 cells or kidney tissues with TRIzol reagent (Invitrogen) following the manufacturer's protocol. Reverse transcription was performed according to the manufacturer's protocols with standard reagent (Promega). Real-time PCR amplification was performed using SYBR Green master mix  and Real-Time PCR (ABI). Primers were synthesized by invitrogen and listed in HK-2 cells were lysed in sample buffer containing RIPA and phenylmethanesulfonyl fluoride (PMSF). Protein concentrations were determined by BCA protein assay kit (Pierce). Thirty micrograms of protein extract was separated by SDS-PAGE gel and transferred onto nitrocellulose membranes (Millipore). The blots were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) at room temperature for 1 hr, followed by incubation with antibodies against primary antibodies at 4°C overnight. After three washes, the membrane was then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Kirkegaard & Perry Laboratories) and visualized with ECL kits (GE Amersham). The primary antibodies that were used included anti-α-SMA antibody, anti-E-cadherin antibody , anti-moesin antibody , anti-Erk 1/2 antibody , anti-pErk 1/2 antibody , anti-pERM antibody  and anti-β-actin antibody .GCAAACTCAGCCTCAATAAGC 3′.We designed three pairs of shRNA-moesin sequences and shRNAs were synthesized by Shanghai GenePharma . We then incorporated them respectively into a lentiviral vector and generated viral particles using viral packaging technology. PC12 cells were transfected with the viral particles before RNA extraction for determining knockout efficiency. Infected HK-2 cells with the highest knockout efficiency were chosen for subsequent experiments. In the preliminary study, we identified the shRNA with the most optimal inhibitory effect and used for current study. The sequence of the shRNA used in current study was: 5′ n = 15) were under isoflurane anesthesia, a midline abdominal incision was made and the left ureter was dissected out. The ureter was ligated at approximately 1 cm below the renal hilum with 3-0 silk suture. The abdominal wound was then closed. Control rats (n = 15) underwent abdominal incision and approximation with no ligation of the ureter. Rats were maintained in the animal facility with free access to water and standard food. At indicated time , 5 rats from each groups were sacrificed under isoflurane anesthesia. Both left and right kidneys were used for further studies. The right kidney from the rats that had undergone left unilateral ligation was also used as another control.Unilateral ureteral obstruction (UUO) rat model was induced in male Sprague-Dawley (SD) rats  by ligation of the left ureter. Briefly, UUO rats . All bar graphs represent Masson positively stained areas processed by ImageJ software.Rat kidneys were fixed with 4% paraformaldehyde and embedded in paraffin. Four-micrometer sections were cut and blocked with 10% goat serum/PBS for 30 min at room temperature. The sections were then incubated with anti moesin antibody  at 4°C overnight. Followed by being washed and incubated with biotinylatedanti-IgG secondary antibody (Dako) for 20 min. Biotin was identified by using streptavidin coupled to horseradish peroxidase and was visualized with diaminobenzidine . Sections were viewed and imaged with a spot-cam digital camera (Carl Zeissy).Statistical analysis was performed using SPSS 11.0 for windows . Data were presented as mean ± SD, unless otherwise indicated. Differences among different groups were compared using one-way ANOVA. p value <0.05 was considered statistically significant.As shown in in vivo, we continued our study in rat model of UUO which was a well characterized animal model of renal fibrosis To further investigate role of moesin in vivo and in vitro, and phosphorylation of moesin could also be induced by TGF-β1 As it was shown that TGF-β1 could induce moesin In order to further investigate role of moesin in TGF-β1 stimulation, we constructed moesin shRNA to knockdown the expression of moesin. Our results Figure 4Despite of the initial causes, the progression of CKD would eventually lead to ESRD that is characterized as wide-spread renal fibrosis. The pathogenesis of renal fibrosis is a process of excessive accumulation and deposition of extracellular matrix components in vitro and in vivo studies, Liu and Yang de novo α-smooth muscle actin expression and actin reorganization, c) disruption of tubular basement membrane, and d) enhanced cell migration and invasion. Importantly, TGF-β1 was a key mediator during these steps. In our previous and current study, we provided the evidence that TGF-β1 could induce α-smooth muscle actin expression and decrease adhesive protein expression which could contribute to EMT in human renal tubular epithelia cells et alEMT has been firstly described in carcinogenesis in which the dedifferentiation of cells lose epithelial and get mesenchymal features In our study, we demonstrated that moesin, which is a member of ezrin/radixin/moesin (ERM) family, was involved in the process of renal fibrosis. It is reported that ERM proteins are highly conserved and high identity is observed in the FERM domain of different species Apart from the structural feature of moesin that is related to cytoskeleton regulation, post-transcriptional modification is also essential to its activation and function. Several studies have demonstrated that phosphorylation of threonine 558 is necessary for moesin activation In conclusion, our results showed that moesin might be involved during renal fibrosis. In view of the essential role of TGF-β in renal fibrosis and CKD, our findings offered novel insights into the mechanism underlying activation of TGF-β signaling and development of renal fibrosis. Our results suggested that moesin might be a novel target for attenuating renal fibrosis and could be served as possible strategy for treatment of CKD.