Alzheimer's disease (AD) is a major neurodegenerative disease and is one of the most common cause of dementia in older adults. Among several factors, neuroinflammation is known to play a critical role in the pathogenesis of chronic neurodegenerative diseases. In particular, studies of brains affected by AD show a clear involvement of several inflammatory pathways. Furthermore, depending on the brain regions affected by the disease, the nature and the effect of inflammation can vary. Here, in order to shed more light on distinct and common features of inflammation in different brain regions affected by AD, we employed a computational approach to analyze gene expression data of six site-specific neuronal populations from AD patients. Our network based computational approach is driven by the concept that a sustained inflammatory environment could result in neurotoxicity leading to the disease. Thus, our method aims to infer intracellular signaling pathways/networks that are likely to be constantly activated or inhibited due to persistent inflammatory conditions. The computational analysis identified several inflammatory mediators, such as tumor necrosis factor alpha (TNF-a)-associated pathway, as key upstream receptors/ligands that are likely to transmit sustained inflammatory signals. Further, the analysis revealed that several inflammatory mediators were mainly region specific with few commonalities across different brain regions. Taken together, our results show that our integrative approach aids identification of inflammation-related signaling pathways that could be responsible for the onset or the progression of AD and can be applied to study other neurodegenerative diseases. Furthermore, such computational approaches can enable the translation of clinical omics data toward the development of novel therapeutic strategies for neurodegenerative diseases. Alzheimer's disease (AD) is one of the most prevalent chronic neurodegenerative disease and is responsible for 60–70% of cases of dementia, thus laying important healthcare problems in countries with aging populations  and intracellular neurofibrillary tangles (NFTs) resulting from hyperphosphorylation of the microtubule-binding protein tau  that eventually cause a sustained activation of certain key intracellular signaling events in the cognate cells, possibly affecting their homeostatic states Figure . ConsequGSE5281. The samples consisted of 13 control subjects and 10 AD cases for entorhinal cortex, 13 control subjects and 10 AD cases for hippocampus, 12 control subjects and 16 AD cases for middle temporal gyrus, 13 control subjects and 9 AD cases for posterior cingulate, 11 control subjects and 23 AD cases for superior frontal gyrus, and 12 control subjects and 19 AD cases for primary visual cortex  directly from the original study  and sign (activation or inhibition) as an input for our method. For this, we used publicly available signaling interactions databases OmniPath and ReactomeFI . Since the exact mechanisms of chronic inflammation are not known, we represent them by introducing a dummy inflammation node in the raw signaling network. This dummy node is then connected to all upregulated receptors for each phenotype under consideration. Therefore, signal transduction from the inflammatory niche to DETFs must be propagated through at least one of the upregulated receptors. Such a representation of unknown external influence by a dummy node has been applied earlier  and the effect  that the receptor has on its downstream target TFs. For example, sign of a signaling path from a receptor to a TF that is up regulated or overexpressed must be positive (activation), while it must be negative (inhibition) for down regulated or under-expressed TF. We calculated the differential edge weight such that for a given phenotype (for example disease) it reflected the probability of the target gene of the specific interaction to be relatively more active when compared to the other phenotype  by considering the interaction sign and booleanized expression state of the interacting nodes. For example, considering an interaction A activating B with booleanized expression state of both the nodes being 1 (upregulated) for first phenotype and consequently −1 (downregulated) for the other phenotype. Here, since both nodes are upregulated in the first phenotype, the probability of B being differentially active in first phenotype will be higher in comparison to the second phenotype where both nodes are downregulated. However, when we consider an example of A inhibiting B, the probability of B being differentially active across the two phenotypes is low, and in such cases we consider equally low edge weights for the interaction. Since we worked with booleanized expression values, we considered the fixed probabilities, where an edge was assigned a probability of 0.9 if it was classified as high probability interaction and 0.1 when it was classified as low. Based on such differential edge-weighting scheme, we calculated differential edge weights for the two phenotypes under consideration by accounting for interaction sign and booleanized expression status of the interacting nodes.In this weighted raw signaling interactome, we aimed to identify signaling paths that are potentially affected by chronic inflammation and are responsible for the observed expression pattern of the DETFs. Our method considers that microenvironment maintains the cells in a stable state by a sustained effect on their TFs via constantly activated/inhibited intracellular signaling pathways compatible with the phenotype-specific TRN state. The fact that the cells exhibit differences in their phenotype  due to differential effect of the microenvironment suggests that the intracellular signaling events controlling the specific GRN for maintain the specific phenotype are also differentially active. For identifying such signaling sub-networks, we employed PCST formalism to infer sub-networks with the dummy inflammation node as the root or origin node and the DETFs as the terminal nodes employing a heuristic algorithm MSGSTEINER  or the entire pathway as a whole.We employed our computational method to identify signaling networks that are likely to be constantly perturbed in AD patients when compared to healthy elderly control individuals. Here, we discuss certain key receptors and their associated signaling components that were identified by our method. Although several of the identified receptors/ligands had a direct link with inflammatory immune responses, the method also inferred molecules not directly related to immune responses, but associated to the disease through other mechanisms. This could be possibly due to the fact that other processes that are responsible of AD progression can also exert sustained influence apart from inflammatory stimuli. Since the method attempts to infer sustained signaling components, irrespective of whether they are derived by inflammatory means or any other process linked to AD, such as deposition of amyloid-beta (Aβ) plaques or neuronal death, it can therefore gather components that are not associated to an inflammatory immune response, but linked to the disease itself. Further, it must be mentioned that we focused predominantly on characterizing the region specific inducers (receptors/ligands) of inflammation and not on all the downstream intermediates and target TFs that transmit the inflammatory signals based on the inferred signaling subnetworks. Table In the original study, the analysis was conducted comparing non-tangle-bearing neurons from AD patients with healthy neurons from control elderly subjects. In fact, the brain regions analyzed have been previously observed to show characteristic pathological differences in the brains of individuals afflicted with AD compared to healthy individuals. The entorhinal cortex and the hippocampus are two regions that have been found to be susceptible to early NFT formation  mediated signaling activity as a key upregulated component in neurons isolated from the hippocampus of AD patients Figure . FGFR1 sAmong the downregulated receptors, ADAM metallopeptidase domain 17 (ADAM17), a metalloprotease involved in the processing of tumor necrosis factor alpha (TNF) that has been described to counteract inflammation and further neuronal damage, was identified Figure .The precise function of the posterior cingulate cortex is not yet clearly established. However, it is known to be involved in cognitive tasks , a known pro-inflammatory signaling component, was a major player in the posterior cingulate cortex sub-network Figure . Excess Among the downregulated signaling networks, we identified sortilin 1 (SORT1), a pro-neurotrophin receptor which plays a major role in the clearance of apolipoprotein E (APOE)/Aβ complexes in neurons Figure . APOE seThis brain region is known to be involved in cognitive processes including, language and semantic memory processing, visual perception, and multimodal sensory integration  and ERB-B2 receptor tyrosine kinase 4 (ERRB4) mediated signaling were identified in our sub-networks to play key roles in the middle temporal gyrus area Table . ReductiIn the downregulated signaling networks, calreticulin (CALR) and its downstream network was identified by our method Figure . CalretiThe entorhinal cortex is thought to be a major input and output structure of the hippocampal formation, acting as the nodal point of cortico-hippocampal circuits -mediated signaling was identified as a key upregulated component of the entorhinal cortical neurons isolated from AD patients compared to healthy subjects Figure . AlthougLDL receptor related protein associated protein 1 (LRPAP1) and its downstream signaling network was found to be downregulated in this brain region Figure . InteresThe primary function of the early visual cortex is visual perception  and its downstream signaling to be specifically downregulated in the superior frontal gyrus area Figure . ImportaAfter identifying the signaling sub-networks likely to be constantly activated/inhibited in different neuronal subpopulations of AD affected brain, we identified the common and specific factors among different brain regions. As it could be seen from Figure In the case of inhibited signaling pathways Table , only SOTechnical advancements in sequencing allow the analysis of thousands of molecular profiles from clinical samples with high quality. These high-throughput techniques open-up opportunities for the development of computational analysis tools to infer meaningful patterns from big data. In this study, we have analyzed gene expression profiling data of specific neuronal populations collected by laser capture microdissection from postmortem samples of AD patients with the aim of identifying brain region specific signaling subnetworks affected by chronic inflammation. The network analysis we carried out enabled the identification of a fraction of receptor/ligands  as key inflammatory mediators from a large number of DERs and ligands. In fact, on average, there were about 100 DERs/ligand molecules in the datasets we analyzed, and our method was able to identify crucial inflammation mediators. This highlights the utility of network approaches to refine and extract accurate and relevant information from high-throughput datasets. Furthermore, computational analyses are amenable to consider more than one diseased region simultaneously and therefore can be used to evaluate the commonalities and distinct features of regulatory processes involved in different regions in an integrative manner. In addition, when data are available, approaches like ours can be employed to study differences and commonalities of a specific disease phenotype in the context of different regions or tissues that are affected due the disease.EGFR is one of the key molecule identified from our analysis to be common for four different regions namely, middle temporal gyrus, entorhinal cortex, primary visual cortex and superior frontal cortex of AD brain. Importantly, recent evidences suggest a crucial role for EGFR in AD pathogenesis, where potential interactions between Aβ oligomers and EGFR were found . This is because, although we considered inflammation to be one of the predominant cause of sustained signaling activities, our computational approach infers constantly activated/inhibited signaling pathways irrespective of whether these pathways are induced by inflammation or any other cellular processes. Although this aspect could represent a limitation of our approach, it rather demonstrates that it can be applied in cases where sustained signaling pathways need to be identified (Ravichandran et al., Overall, our analysis revealed mostly distinct and few common inflammatory signaling components across the different neuronal populations analyzed. However, despite the predominance of region specific factors, the analysis identified certain shared inflammatory factors, reflecting some environmental similarities across the affected brain regions, such as the presence of Aβ plaques. Further, we observed that not To our knowledge, this is one of the first study, which computationally analyzed the potential implication of chronic inflammation on AD to infer the induced signaling networks/pathways. In this regard, it must be mentioned that two recent studies have attempted to infer common and unique pathways/signatures in different neurodegenerative diseases (Li et al., In the context of neurodegenerative diseases, integrative approaches enable to obtain a holistic understanding of the processes and factors that initiate and sustain specific disease pathologies that act primarily at the cellular level. For instance, protective and pathogenic roles of glial cells, such as microglia and astrocytes, in addition to the activation of common inflammatory pathways in these cells in several neurodegenerative diseases, support the concept that glia-induced inflammation can possibly sustain the disease pathology (Burns and Iliffe, SR and AdS conceived the idea. SR performed the computational analysis. All authors contributed to the writing of the manuscript and approved the final version of the manuscript. AdS coordinated the overall project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JEE and handling Editor declared their shared affiliation.


Legionella pneumophila parasitizes environ mental amoebae and, upon inhalation, replicates in alveolar macrophages, thus causing a life-threatening pneumonia called “Legionnaires’ disease.” The opportunistic pathogen employs a bi-phasic life cycle, alternating between a replicative, non-virulent phase and a stationary, transmissive/virulent phase. L. pneumophila employs the Lqs (Legionella quorum sensing) system as a major regulator of the growth phase switch. The Lqs system comprises the autoinducer synthase LqsA, the homologous sensor kinases LqsS and LqsT, as well as a prototypic response regulator termed LqsR. These components produce, detect, and respond to the α-hydroxyketone signaling molecule LAI-1 . LAI-1-mediated signal transduction through the sensor kinases converges on LqsR, which dimerizes upon phosphorylation. The Lqs system regulates the bacterial growth phase switch, pathogen-host cell interactions, motility, natural competence, filament production, and expression of a chromosomal “fitness island.” Yet, LAI-1 not only mediates bacterial intra-species signaling, but also modulates the motility of eukaryotic cells through the small GTPase Cdc42 and thus promotes inter-kingdom signaling. Taken together, the low molecular weight compound LAI-1 produced by L. pneumophila and sensed by the bacteria as well as by eukaryotic cells plays a major role in pathogen-host cell interactions.The ubiquitous Gram-negative bacterium  N-acyl-homoserine lactone (HSL) autoinducers, diffusible signal factor (DSF), autoinducer-2 (AI-2) and its precursor 4,5-dihydroxy-2,3-pentanedione (DPD), 2-heptyl-3-hydroxy-4-quinolone (PQS), as well as dialkylresorcinol (DAR) and α-hydroxyketone (AHK) molecules  of the signaling molecule. Quorum sensing is mediated by various chemical classes of small molecules called “autoinducers” . Promineolecules .Vibrio species -HSL (HAI-1), autoinducer-2 , and (Z)-3-aminoundec-2-en-4-one . This process is termed inter-kingdom signaling . Promine elegans , and AHK elegans .Mycobacterium spp. , which are detected by a number of bacterial genera through the QseBC two-component system TCS; . Phagocyium spp. . In thisLegionella pneumophila is a ubiquitous Gram-negative bacterium that colonizes complex aquatic biofilm communities , wherein the bacteria naturally replicate  1 causes about 85% of all clinical cases in most parts of the world, while L. longbeachae accounts for about 30% of the reported cases in Australia and New Zealand , a conserved and essential global activator of replication and repressor of transmission traits . The system is encoded by genes arranged in a genomic cluster  and Vibrionaceae , but also in the Burkholderiaceae , Chlorobiaceae   . The hdefunction .32P]-ATP at their conserved histidine residues , located in the cytoplasmic C-terminal histidine kinase domain (108), the response regulator abrogated autophosphorylation of the sensor kinases by catalyzing the dephosphorylation of phospho-LqsS or phospho-LqsT. LqsR forms dimers upon phosphorylation at Asp108 by either acetyl-phosphate or phospho-LqsT. Upon heterologous production in Escherichia coli LqsT (but not LqsS) is autophosphorylated by ATP, and LqsR prevents phosphorylation of the sensor kinase under these conditions by catalyzing the dephosphorylation of phospho-LqsT. Taken together, phosphorylation signaling through the sensor histidine kinases LqsS and LqsT converges on LqsR, which forms dimers upon phosphorylation  promotes the phosphorylation of LqsS (but not LqsT). Moreover, synthetic LAI-1 promotes the motility of L. pneumophila in an LqsS/LqsT- and LqsR-dependent manner. Transcriptome analysis of L. pneumophila treated with LAI-1 revealed that the signaling molecule negatively regulates the RNA-binding global regulator crsA and positively regulates a number of genes, including the snRNAs rsmY and rsmZ. In summary, these findings indicate that LAI-1 regulates motility and the switch from the replicative to the transmissive growth phase of L. pneumophila by phosphorylation signaling through LqsS, LqsT and LqsR  were deleted from the L. pneumophila chromosome by double homologous recombination  shows a higher G+C content than the L. pneumophila core genome, is located adjacent to the tRNAThr gene lpg0972 and flanked by putative DNA-mobilizing genes such as integrases, transposases, and phage-like genes. Two regions can be discriminated: region I  harbors many (conserved) unknown genes, some of which encode putative pili components , and region II  encodes the subunits of a FoF1 ATP synthase and several metal ion resistance transporters. The region lpg1008-lpg1035 has been characterized previously as a 40 kb eﬄux pump genomic island, which is induced upon (but not required for) L. pneumophila infection of macrophages  is also severely defective for intracellular replication and outcompeted by the parental strain upon co-infection of amoebae   . The netas GacAS . Upon enrophages , as welltellanii . The ressor CsrA .L. pneumophila in protozoa and macrophages . In agreement with this notion, synthetic LAI-1 (as well as the Vibrio autoinducer CAI-1) dose-dependently inhibited cell migration . That is, LAI-1 reduced the forward migration index but not the velocity, suggesting that the directionality but not speed of the cells was affected.Small signaling molecules promote the communication not only between bacteria but also between prokaryotes and eukaryotes. To test the hypothesis that the Lqs system and LAI-1 affect eukaryotic cells, we used migration of eukaryotic cells as readout. t manner . Rather rne lqsA . InteresFigure 1), but not other modulators of Cdc42, or the small GTPases RhoA, Rac1 or Ran. Furthermore, upon treatment with LAI-1, IQGAP1 redistributed to the cell cortex, and Cdc42 was inactivated. Taken together, the results indicate that LAI-1 modulates migration of eukaryotic cells through a signaling pathway involving IQGAP1, Cdc42, and ARHGEF9. The eukaryotic receptor(s) of L. pneumophila LAI-1 is/are currently not known.In order to determine host cell factors implicated in LAI-1-dependent cell migration inhibition, a scratch wound healing assay with A549 lung epithelial cells was used, alongside RNA interference . The depSalmonella enterica serovar Typhimurium also modulates the actin cytoskeleton through Cdc42 and IQGAP1. The Salmonella type III-secreted GEF SopE directly activates Cdc42  O157:H7  and noradrenaline (norepinephrine) produced by eukaryotic cells . The adr O157:H7 . Accordi O157:H7 .Escherichia coli  had only a minor effect on growth inhibition by adrenergic antagonists, implying a different mode of action and/or the presence of another adrenergic sensing system. Yet, regardless of their bacterial target, adrenergic antagonists might represent potential lead compounds in screens for novel anti-infective compounds against Legionella or Mycobacterium spp. using phagocytes as host cells (The genes apparently encoding the QseBC homolog in ractions . Adrenalenhanced . Growth st cells .L. pneumophila, is an amoebae-resistant opportunistic pathogen, which employs the AHK molecule LAI-1 for intra-species as well as inter-kingdom communication. Since AHK molecules are wide-spread in nature, environmental bacteria likely employ this class of signals for inter-genera signaling. L. pneumophila produces, detects and responds to LAI-1 through the Lqs system, which comprises the autoinducer synthase LqsA, the homologous sensor kinases LqsS and LqsT, as well as the response regulator LqsR. As part of the stationary phase regulatory network, the Lqs system regulates the growth phase switch, pathogen-host cell interactions, bacterial motility, natural competence, filament production and expression of a chromosomal “fitness island.” The responses of L. pneumophila to LAI-1 might be exploited for anti-virulence drug development, as has been described for Vibrio cholerae using agonist and antagonist derivatives of the corresponding signaling molecule CAI-1 (L. pneumophila; however, the mere fact that the pathogen responds to adrenergic antagonists does not imply that this is the case. Future studies will address the question whether there are other classes of low molecular weight molecules produced and detected by Legionella spp. or by Legionella -infected host cells, and will explore the potential of small molecule signaling to interfere with Legionella virulence or eukaryote processes.The causative agent of Legionnaires’ disease, le CAI-1 . FurtherRH and HH wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The immunopathology of chronic obstructive pulmonary disease (COPD) is based on the innate and adaptive inflammatory immune responses to the chronic inhalation of cigarette smoking. In the last quarter of the century, the analysis of specimens obtained from the lower airways of COPD patients compared with those from a control group of age-matched smokers with normal lung function has provided novel insights on the potential pathogenetic role of the different cells of the innate and acquired immune responses and their pro/anti-inflammatory mediators and intracellular signaling pathways, contributing to a better knowledge of the immunopathology of COPD both during its stable phase and during its exacerbations. This also has provided a scientific rationale for new drugs discovery and targeting to the lower airways. This review summarises and discusses the immunopathology of COPD patients, of different severity, compared with control smokers with normal lung function.


High uric acid (UA) can act as a pro-oxidant in normal physiological conditions; however, emerging evidence is still debatable with regard to the association between high UA and poor outcomes among chronic hemodialysis (HD) patients. In the present study, 27,229 stable prevalent HD patients were enrolled and divided into four groups according to the quartiles of baseline UA concentration, and 5737 died during a median follow-up of 38 months. Multivariate Cox regression analysis showed that a UA level of <6.1 mg/dL was associated with a higher risk of all-cause mortality compared with a UA level of >8.1 mg/dL  adjusting for baseline demographic and biochemical parameters. Moreover, a UA level of <6.1 mg/dL was associated with greater risks of cardiovascular mortality  and stroke-related mortality , respectively. In vitro experiments further showed an increase in oxidative stress and an inhibition nitric oxide synthesis by indoxyl sulfate (IS) in human aortic endothelial cells, which were significantly attenuated by UA in a dose-dependent manner. We concluded that higher UA in serum was associated with lower risk of all-cause and cardiovascular mortality among HD patients probably through its antioxidant property in ameliorating the IS-related vascular toxicity. Uric acid (UA) is the end product of purine metabolism in humans. Diet and increased consumption of fructose and alcoholic beverages  affect tEmerging evidence is still controversial with regard to the relationship of high UA with poor outcomes among chronic hemodialysis (HD) patients. Three studies based on a relatively small sample size found a J-shaped relationship between uric acid and mortality in HD patients ,8,9. A mIncreased risk of CV morbidity and mortality is frequently encountered in HD patients. The Framingham risk equation does not capture the full extent of CV risk in HD patients . InsteadUA can initiate oxidative stress, but it also exhibits an antioxidant in plasma . TherefoBetween 2001 and 2006, a total of 60,196 incident HD patients were registered in the Taiwan Society of Nephrology (TSN) Dialysis Registry. After exclusion of patients for various reasons , 27,229 During a study period of 7 years , 5737 (21%) patients died. Overall, the main causes of death were CV-related (63%), malignant disease (11%), stroke (7%), and others (11%). In multivariate Cox proportional hazard model, the adjusted hazard ratios (aHRs) for all-cause mortality , CV mortality , and stroke mortality  were significantly higher in patients with UA below 6.1 mg/dL . HoweverWe then used an in vitro system to determine whether UA acts as an anti-oxidant in a uremic environment. We used the MTT assay to determine the effect of different IS and UA concentrations on the viability of human aorta endothelial cells (HACEs) . A high NO, produced by the endothelial NO synthase (eNOS), is fundamental to cardiovascular homeostasis, and an intact Akt-eNOS-NO signal pathway is critical for maintaining endothelial cell function ,20. ThusIn the current study, we used cell culture experiments to demonstrate that a high level of UA  attenuates endothelial dysfunction, and the inhibition of IS induced oxidative stress and the preservation of NO production by uric acid is one of the possible mechanisms. In concert with these in vitro experiments, we also found that a lower serum UA concentration is associated with higher risks for all-cause and CV mortality among HD patients. Our data highlight the potentially protective role of UA in vascular toxicity induced by IS in chronic HD patients .In the general population, there is abundant clinical and experimental evidence that UA contributes to the development and progression of heart and kidney disease because it increases hypertension, activity of the renin-angiotensin-aldosterone system (RAAS), and micro-inflammation, and decreases insulin sensitivity ,22,23. HThere is uncertainty regarding whether UA mediates disease or is only a surrogate of disease severity ,27. UA iIn the current study, UA reduced uremic toxin-induced oxidative stress in endothelial cells. Oxidative stress has emerged as a significant pathogenic feature of uremia. The presence of oxidative stress in ESRD is indicated by an overabundance of the oxidation products of lipids, carbohydrates, and proteins in the plasma and tissues of uremic patients. Uremia has long been interpreted as a sign of premature aging, along with DNA and mitochondrial damage, increased ROS generation, and accelerated vascular disease . Thus, UCompared to previous studies, the current study examined the largest number of HD patients and used in vitro experiments to examine the potential mechanism of the observed clinical effects. We conclude that UA ameliorated IS-induced endothelial damage, and that a high–normal level of UA is beneficial for HD patients. The results of the current study may also have implications for determining the target UA level in dialysis patients, and may influence the prescription of UA-lowering drugs in this population. However, several limitations should be noted. First, the TSN Dialysis Registry does not included data on C-reactive protein, asymmetric dimethylarginine, and NO, and these are important mediators or surrogate markers of endothelial function. Therefore, we cannot correlate serum UA and indoxyl sulfate level with endothelial function in our cohort study. Second, the TSN Dialysis Registry does not include non-fatal events, such as myocardial infarction, heart failure, or gout attack, so we could not examine the relationship of UA level with these events. Among HD patients, uricosuric drugs are not effective, and less than 5% of our HD patients received xanthine oxidase inhibitors. Although xanthine oxidase inhibitors also have anti-oxidant effects , we coulHigh UA exhibits various activities, from pro-oxidant activities in the general population to anti-oxidant activities in uremic patients. In chronic HD patients, high CV mortality is correlated with endothelial dysfunction partly related to uremic toxins, which exacerbate oxidative stress and reduce NO production. In the current study, we found that a high–normal serum UA concentration can preserve endothelial cell function in vitro and is associated with better survival among chronic HD patients. Therefore, a high–normal UA level in HD patients may be a compensatory mechanism that counteracts the oxidative damage and vascular toxicity from uremic toxins like IS.TSN Dialysis Registry maintained records of all ESRD patients requiring chronic dialysis in Taiwan . TSN DiaTo determine the role of UA on the long-term outcomes of HD patients, we reviewed the records of the TSN Dialysis Registry to identify all incident ESRD patients in Taiwan from 1 January 2001 to 31 December 2006. Patients treated by peritoneal dialysis, recipients of kidney transplantation, and those with incomplete biochemistry data were excluded. To avoid survival bias, we excluded patients younger than 40 years old and those who died within one year of initiation of dialysis [HAECs were cultured in endothelial growth medium  with 10% fetal bovine serum (FBS) as described previously . These cA commercially available indicator of general oxidative stress (CM-H2DCFDA) was used. The cell-permeable H2DCFDA  passively diffuses into cells and remains within the cell after cleavage by intracellular esterases. Upon oxidation, the non-fluorescent H2DCFDA was converted to the highly fluorescent CM-H2DCFDA, which was better retained within living cells than H2DCFDA. Thus, HACEs cultured in 24-well plates were incubated with 10 µM H2DCFDA for 30 min, washed with sterile PBS, and incubated with UA and IS at different concentrations for 3 h. To determine the effect of UA on IS-induced ROS production, HAECs were pre-treated with different concentrations of UA for 30 min, and then incubated with 200 µM IS for 3 h. The IS incubation time was determined according to previous studies ,35. FluoNO has an extremely short physiological half-life, so special methods are needed to measure the reaction products of NO biochemistry. Thus, we used a standard diaminonaphthalene-based assay to determine NO production according to manufacturer’s protocol . BrieflyProteins in cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membranes, and detected by Western blot analysis according to established protocols. The primary antibodies were: anti-phospho-eNOS and anti-eNOS , and anti-phospho-AKT and anti-AKT . Anti-β-actin was used as a loading control. Independent Western blotting experiments were performed four times, and protein expression was quantified by densitometry.p-values were two-sided, and the significance level was set at 0.05. All analyses were performed using commercially available software .All values were expressed as means and SDs unless otherwise specified. For the in vitro experiments, multiple groups were compared by analysis of variance (ANOVA), with Bonferroni’s post hoc correction. For clinical data, baseline characteristics were compared by ANOVA or chi-square tests. In a multivariable Cox regression model, the effect of UA was adjusted for age, gender, diabetes mellitus, comorbid diseases, cause of ESRD, type of vascular access, dialysis adequacy (Kt/V), normalized protein catabolic rate (nPCR), hematocrit, and serum biochemistry data. Results are expressed as Kaplan-Meier plots or hazard ratios (HRs). A constant HR over time  was evaluated by comparing estimated log-log survival curves for all time-independent covariates. All of these plots had two parallel lines, indicating no violation of this assumption. All


Uric acid (UA) is a kind of purine metabolism product and important in clinical diagnosis. In this work, we present a study of ZnS nanostructures-based electrochemical and photoelectrochemical biosensors for UA detection. Through a simple hydrothermal method and varying the ratio of reaction solvents, we obtained ZnS nanomaterials of one-dimensional to three-dimensional morphologies and they were characterized using field emission scanning electron microscopy (FESEM) and X-ray diffraction (XRD). To fabricate the UA biosensor and study the effect of material morphology on its performance, ZnS nanomaterials were deposited on indium tin oxide (ITO) conducting glass and then coated with uricase by physical absorption. Three kinds of working electrodes were characterized by cyclic voltammetry method. The effect of material morphology on performance of UA detection was investigated via amperometric response based electrochemical method based on enzymatic reaction. The ZnS urchin-like nanostructures electrode shows better sensitivity compared with those made of nanoparticles and nanoflakes because of its high surface-area-to-volume ratio. The photoelectrochemical method for detection of UA was also studied. The sensitivity was increased 5 times after irradiation of 300 nm UV light. These results indicate that ZnS nanostructures are good candidate materials for developing enzyme-based UA biosensors. Uric acid (UA) is a kind of purine metabolism product and important in clinical diagnosis. For a healthy adult male, the normal range of UA in serum is 149∼416 0∼300 μM  or other0∼300 μM ,11,12. M0∼300 μM  exhibit 0∼300 μM , electro0∼300 μM  and etc.0∼300 μM  and nanoIn this paper, we present a study of ZnS nanostructures-based electrochemical and photoelectrochemical biosensors for UA detection. A hydrothermal method was employed to synthesize ZnS nanomaterials with controllable morphology of one to three dimensions and a simple spin-coating method to fabricate UA biosensor without using any organic link molecule . The terAnhydrous zinc acetate  at a scanning rate of 0.1 V/s in the potential range of −0.2∼0.8 V. The CV curves can reflect the oxidization and reduction reaction that occur on the surface of the electrodes.It can be seen in The amperometric response for the UA of different biosensors were also measured. In y and x stand for current (A) and the concentration (mM) of UA separately. The biosensor made using ZnS urchin-like nanostructures shows a best sensitivity of 76.12 The current-to-concentration calibration curves of the UA biosensors fabricated with different ZnS nanostructures were shown in ZnS nanostructures possessed photoelectrochemical property for its potential application in photoelectric biosensors ,30. ElecThe photoelectrochemical UA detection was the same as electrochemical method with one exception of the 300 nm UV irradiation. The working electrode was tested while exposed to the illumination of a 200 W mercury lamp. The photocurrent also increased linearly with the addition of UA. The photocurrent response were about 5 times higher than that without irradiation when same amount of UA were added . The sen follows : ElectroThe storage stability of this UA biosensor was investigated. The sensors were stored in the fridge at 4 ZnS nanomaterials of different morphologies were synthesized through a simple hydrothermal method. With a spin-coating method, these nanomaterials were fabricated into Nafion/uricase/ZnS/ITO electrodes which have been successfully applied for electrochemical and photoelectrochemical UA detection in aqueous solution. The ZnS urchin-like nanostructures based electrochemical biosensor showed the highest sensitivity of 76.12


The Total Fertility Rate (TFR) is defined as the average number of births a woman would have if she were to live throughout the reproductive span and bear children at each age at the rates observed in a particular year or period. The current demographic explanation for decline in TFR is primarily attributed to an increase in postponement in pregnancy. Being cross-sectional, fertility measures can be confounded by changes in the timing of births across women’s lifetimes (tempo) and by changes in the numbers of children that they have by the time they end their childbearing (quantum). After a sharp fall in the last two decades, TFR of Bangladesh is now 2.3; whereas the TFR was greater than 3 in the last decade. However, mean age at childbearing showed decreasing trend in the last decade.This is a secondary analysis of data from the three consecutive Bangladesh Demographic Health Surveys; BDHS-2004, 2007 and 2011. The method of Bongaarts and Feeney has been applied to estimate the tempo of fertility. Life Table analyses were applied on birth intervals to explain the tempo effect.There was a sustained decline of the fertility quantum (the number of births per woman) as estimated by the conventional TFR; due to tempo effects during the last three BDHS surveys. Mean age at childbearing also showed decreasing trend in the last decade.The current study shows the presence of a significant tempo effect with variability of timing in having first or higher order births. If this trend continues, Bangladesh will be able to achieve below replacement level of fertility soon. Policy-makers have considered different strategies and interventions aimed at achieving a replacement level of fertility, while researchers search for factors and explanations affecting fertility. The most used indicator of fertility rate is Total Fertility Rate (TFR) which is conceptualized as the average number of children a woman will have over the course of her life if the prevailing age specific fertility rates (period fertility) are assumed to be constant. Demographers and policy makers rely profoundly on the TFR for examining trends in fertility for a number of conveniences . First, The age pattern of fertility may change for various reasons. Perturbations in the age pattern of childbearing are more subtle, and in recent years are facilitated by effective contraceptive methods. However, a postponement of births not only leads to a rising mean age at child bearing, but it also affects the observed fertility rates and thus the TFR , 4, 5. SBongaarts and Feeney estimated the effect of timing by decomposing age specific fertility rates applying the parity progression ratio in a calendar year. This method considered the shift in the mean age of childbearing for each birth (parity). Parity-specific analysis provides more detailed insights that cannot be observed using conventional age-specific analysis of fertility. Another important benefit of parity-specific analysis is its capability of providing few threshold measures that can be related more directly to behavioral responses than is the case with age specific fertility rates . Using tThe socio-economic and policy changes over the years regarding population and public health sectors in Bangladesh are associated with the fall in fertility. After a fall in the last two decades, TFR is now 2.3 in Bangladesh. If the present trend continues then it is expected that Bangladesh will be able to achieve its demographic goal as envisaged in the population policy. If we divide the country by east and west, BDHS 2011 demonstrates that the west has already achieved replacement fertility but eastern regions such Syllhet and Chittagong are lagging far behind. Health Nutrition Population Sector Development Program (HNPSDP) identified several factors such as utilization of mass media, women empowerment, increased enrolment of girls in education, participation of women in employment and migration. Although significant fertility transition occurred in Bangladesh between 1975 and 2011, Bangladesh has to pass many barriers including high rates of adolescent marriage, lower use of contraception by newly married couples, low education of women and low employment rate of women . DecliniBesides decline in the ASFRs, mean age of childbearing also declined since the mid 1970’s which obviously should have some effect on aggregate fertility level of the country. If the TFR changes in a particular year, how we can infer whether fertility rates of the cohorts who are bearing children in that year also changes? It is difficult to predict and interpret changes until relevant cohorts have completed child bearing. A timing change is also known as a tempo change and the tempo effect has a dramatic effect on period fertility rates, but probably has comparatively little effect on cohort rates. Influences on fertility which do affect cohort rates are called quantum effects. However, there have not been enough studies that provide better understanding of recent fertility transition (declining fertility rates from a higher level to replacement level) and its relationship with the change in population size and composition , 13. TheData for the analyses in this study come from Bangladesh Demographic and Health Surveys (BDHS). BDHS is a nationally representative survey that was conducted under the authority of the National Institute for Population, Research and Training (NIPORT) of the Ministry of Health and Welfare, Bangladesh and funded by USAID. Three consecutive surveys  have been included in the present study. Stratified Multi-stage Cluster Sampling design was used to collect data and BDHS 2004, BDHS 2007 and BDHS 2011 includes 11400, 10996 and 17749 ever-married women of child bearing age of all six divisions in the country respectively [*) for i-th birth order in a particular calendar year y is-The Bongaarts and Feeney method has been applied in this paper to estimate the tempo effect . The proi (y-1) denotes the mean age of child bearing of birth order ‘i’ (year 1). If we are estimating tempo effect for the year ‘y’ (year 2); than ‘y-1’ stands for year previous year of year ‘y’ and ‘y + 1’ mean the next year of the year ‘y’ (year 3), i.e. the immediate preceding year to the survey. The mean age of childbearing can be expressed as the weighted average with ASFRs as the weight:Here MAC5fx represents the age specific fertility rates for five year age group [Where, x = 15,20,25,35,40,45; (x + 2.5) represents the midpoint of each age interval and ge group . To compge group .TFR transition for the last three decades and ASFR for the last three BDHS is graphically presented below Figures  and 2. PThe decline in TFR over the period of 1975 to 2011 is remarkable for Bangladesh. Rapid decline was observed up to nineties; fertility level plateaued for about a decade before it began to fall again in approximately 2000 BDHS. Adolescent fertility is still high and its contribution is increasing the total specific fertility rate; it is now about one fourth of the total age specific fertility rate which may be explained due to high fertility in the past. ASFR peaked in the age group 20–24 for Bangladeshi women and later age parity progression diminished notably during these three consecutive BDHSs. The details of conventional and tempo adjusted TFR for BDHS 2004, 2007 and 2011 are presented in Table 1 suggest it is more than 19.7 years now indicating a shift in the age specific fertility rates. No change may be observed in first order birth of MAC1 in BDHS 2011. For birth order 2, 3 and 4 the MAC have an increasing trend for all three BDHSs. These results indicate the spread of spacing between births for all birth order up to 4th birth order. The conventional and tempo adjusted TFR for BDHS 2004, 2007 and 2011 are presented in Figure Application of the Bongaarts and Feeney (1998) method suggests the presence of a tempo effect in TFR of Bangladesh, and is significant. The tempo effect for BDHS 2004, 2007 and 2011 were 1.08, 1.12 and 0.51 respectively. From the comparison of the three BDHSs, the highest decline in fertility is observed in the first order birth. For each birth order mean age of child bearing (MAC) by birth order is estimated for the last three BDHSs. In the last three BDHSs , the MAC for first birth concentrated around 19 to 20 years though the linear trend of increasing MACThe case of birth order 5 or higher is different from parity progression from birth order 4. The trend of MAC is irregular for higher order births; in BDHS 2007 MAC5+ is lower than MAC5+ for BDHS 2004. This is due to rapid decline in higher order births as Bangladesh approaches a two child family. Tempo adjusted TFR obtained for three combined BDHS  was 0.6 ; while tComparing the tempo distortion in different parity progression, it is clear that highest timing effect is observed for first order birth. The median age at marriage was 14 years at BDHS 2004, while it increases to 15 years in next two BDHSs. Age at first birth centered around 17 years in all of three BDHSs . To examine the marriage to first birth interval, life table technique was applied in all the three BDHSs and the results indicate an increase of cumulative probability of having first birth having longer spacing  it is called ‘quintum’ [Here Bquintum’ . The proThe aim of the present paper is to understand to which extent the decline in Total Fertility Rate (TFR) can be attributed to the quantum effect and to what extent it is due to tempo effect. The period TFR is one of the most often used indices of fertility, but it is sensitive to variation in the parity distribution of women that results from shifting in the time of childbearing in successive cohorts. The Bongaarts-Feeney tempo adjusted TFR method is one of several attempts to produce better period measure  of the fertility quantum. Bangladesh, a country of Southeast Asia, has shown significant fertility transition in the last two decades. For Bangladesh, clearly, the impact of timing on the aggregate level of fertility is present. This tempo effect is the result of change in the birth intervals and mean age of childbearing for each birth order. The results indicate the importance of analysis of the timing effect on the aggregate level of fertility which has implications in policy consideration. The primary distortion was caused by an increase in the mean age at child bearing. The analysis shows that tempo adjusted TFR will be half a child higher than the estimates suggest in 2011 BDHS (2.81 as opposed to 2.30). However, the distortion effects of a changing mean age of child bearing by birth order are important in order to understand fertility behavior and its implications on future population growth.The tempo effect of TFR could be reduced by motivating mothers to widen spacing between consecutive births. Even if Bangladesh achieved replacement fertility by 2015 as envisaged in the population policy of Bangladesh, population growth will continue due to population momentum. The most feasible approaches to reduce the impact of population momentum should be taken into account as policy measures and include delayed age at marriage, delayed age at first birth and wider spacing of successive births.Despite this, there may be debate about the use of the period measure of TFR. If couples have their children earlier, the mean age at child bearing falls and the period rate will rise. On the other hand, if they have them later, it will fall. Using the Bongaarts and Feeney method one can estimate the effect of shifting in the MAC to the aggregate level fertility only for consecutive years (but not for long periods) , 17. HowLife table analysis shows that the highest tempo effect was obtained from age at first parity. The other important consideration from this analysis is the increase in variability in age at first birth and higher order births. Several explanations may be inferred from previous studies, such as lower family planning practices among the adolescents mothers, ‘catching-up’ effects of the educated mothers (having the first birth earlier to overcome the reproductive life span passed before marriage) and so on , 18. ThiThe current study shows the presence of a significant tempo effect. Decomposition of parity specific analysis suggests the highest tempo effect on first order birth which is strongly associated with age at marriage. Age at marriage is the most prominent and key-factor explaining fecundability for every sub-group of the study population. Findings from the current study suggest that new policy regarding the present child bearing situation will have important impact on the TFR and maternal and child heath. Further research using cohort measures may identify potentially relevant factors that impact the tempo effect in order to better understand the context.


Endothelial cells’ (EC) injury is a major step for the pathological progression of atherosclerosis. Recent study demonstrated that thymic stromal lymphopoietin (TSLP) exerts a protective role in atherosclerosis. However, the effect of TSLP and the exact molecular mechanism involved in EC remains unknown. In the present study, we found that long noncoding RNA (lncRNA) HOTAIR was much lower in EC from atherosclerotic plaque. Functional assays showed that HOTAIR facilitated cell proliferation and migration, and suppressed apoptosis in EC. Moreover, we demonstrated that TSLP functions upstream of HOTAIR. We found that serum level of TSLP was decreased in atherosclerosis patients and serum TSLP level positively correlated with HOTAIR expression in EC. Further investigation demonstrated that TSLP activated HOTAIR transcription through PI3K/AKT-IRF1 pathway and then regulates the EC proliferation and migration. TSLP-HOTAIR axis also plays a protective role in low-density lipoprotein (ox-LDL)-induced EC injury. Taken together, TSLP-HOTAIR may be a potential therapy for EC dysfunction in atherosclerosis. Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipids and fibrous elements in the arterial wall of large- and medium-sized arteries, and is the main cause of heart disease and stroke ,2. Endot−/−) mice treated with TSLP developed significantly fewer atherosclerotic plaques in the aortic root compared with controls, along with decreased inflammation in the aorta, indicating a protective role of TSLP in atherosclerosis [Thymic stromal lymphopoietin (TSLP), which belongs to an IL-7 like cytokine family, is mainly expressed by epithelial cells. Previous studies have reported that TSLP signal was activated through a heterodimeric receptor complex consisting of an IL-7 receptor-α chain and a unique TSLP receptor (TSLPR). TSLPs are often activated by infection related agents, allergens and cytokines including IL-4, IL-1 and TNF-α . After sclerosis . HoweverHOXC gene cluster present in chromosome 12 [Recently, increasing evidence demonstrated that noncoding RNAs (ncRNAs) play important roles in the regulation of gene expression via multiple mechanisms . Long noosome 12 . HOTAIR osome 12 –18. HOTAosome 12 ,20. A reosome 12 . HoweverThe complete details of the entire study design and procedures involved were in accordance with the Declaration of Helsinki. Informed written consent was obtained from all the subjects. The study process was approved by the Ethics Review Committee of the Tongji Medical College. A total of 40 atherosclerotic patients were included in the present study after angiographic documentation of coronary artery disease (CAD). Atherosclerotic plaques were collected from patients undergoing vascular surgery or at autopsy. Primary EC were isolated as previously described . The serSerum TSLP levels were determined by using an ELISA kit from eBioscience according to the manufacturer’s instructions.2 and in RPMI-1640 medium (Gibco) supplemented with 10% FBS (Gibco).Two EC cell lines, HUVEC and HAEC were obtained from Cell Bank of Chinese Academy of Sciences. Cells were cultured at 37°C in an atmosphere containing 5% COC−ΔΔtmethod was applied to calculate relative transcript levels. The primer sequence for HOTAIR was provided as follows: F-AGACCCTCAGGTCCCTAATATC, R-CCCTACTGCAGGCTTCTAAATC.Total RNA was isolated using TRIzol reagent  according to the manufacturer’s instructions. cDNA was synthesized by using PrimeScript First Strand cDNA Synthesis Kit (Takara). GAPDH served as an endogenous control. Real-time PCR was performed on the Applied Biosystems 7300 Real-Time PCR system using SYBR Green dye  as manufacturer’s instructions. All detections were performed in triplicate. The 2Cells were lysed in RIPA lysis buffer with protease inhibitor cocktail (Roche). Lysis was subjected to SDS/PAGE and transferred on to a PVDF membrane . The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h and then incubated with anti-IRF1 (Abcam), AKT , P-AKT  or GAPDH (Santa Cruz) antibodies for overnight at 4°C. p-Akt and total Akt were analyzed in different membranes. After washing, the membrane was reacted with HRP–conjugated secondary antibodies (Jackson) for 1 h at room temperature. Bands were visualized using the ECL detection system .HOTAIR was amplified from HUVEC and cloned into expression plasmid pCMV (Addgene). Cells were transfected with pCMV or pCMV-HOTAIR by using Turbofect (Thermo) following the manufacturer’s instructions. siRNAs targeting HOTAIR was purchased from Ribobio Company . The target sequence for HOTAIR was as follows: si1, CTGGGAGGCGCTAATTAAT; si2, AGGCAAATGTCAGAGGGTT. Cells transfected with scramble siRNA is taken as control group. siRNAs were transfected into cells by using using Hiperfect (Qiagen) following the manufacturer’s instructions. After 48 h of transfection, the following experiments were performed.A total of 48 h after transfection, the cells were treated with different concentrations of oxidized low-density lipoprotein  for 12 h or with 40 μg/ml ox-LDL for different time points. Cells were treated with recombinant human TSLP (rhTSLP) (R&D Systems) at different concentration for 24 h, with vehicle as control. We treated HUVEC with rhTSLP (10 pg/ml) and TSLP  or TSLPR antibody  or PI3K inhibitor LY294002  for 24 h. After drug treatment, the cells were used for flow cytometry or RNA extraction or cell viability detection.5 cells were seeded in serum-free RPMI-1640 in the upper chamber of an 8-μm pore 24-well transwell insert. The lower chamber was filled with RPMI-1640 containing 10% FBS. The 24-well plates were then incubated for 24 h in an atmosphere at 37°C. Cells that migrated through the membranes were fixed in 4% paraformaldehyde and then stained by Crystal Violet. Cells were counted in ten random fields under a microscope.A total of 1 × 10Cell proliferation and viability was determined by the CCK-8 (Dojindo) according to the manufacturer’s instructions. Cell proliferation rate was detected by measuring the absorbance at 450 nm with the microplate reader (Bio–Rad). The OD value of each group at day 1 was taken as control.The apoptosis was detected by using Apoptosis Detection Kit  according to the manufacturer’s instructions. Cells were stained with fluorescein isothiocyanate–conjugated Annexin V and 7-Aminoactinomycin D (7-AAD). Cells were then analyzed with a FACScan flow cytometer and the data were analyzed by Kaluza software. Annexin V labels externalized phosphatidylserine and 7-ADD labels DNA.ChIP assay was performed by using the EZ-Magna Chromatin Immunoprecipitation Kit (Millipore) according to the manufacturer’s protocol. Immunoprecipitate complexes were cultured with an anti-IRF1 antibody (Abcam) or negative control IgG antibody (Millipore) overnight at 4°C. The captured DNA was used for quantative-PCR (qPCR) analysis. The primers used for HOTAIR promoter sequence were provided as follows: F-CATGCTGTCTTAGCGGATCTT, R-ATGTCTTGCTGCTCGGATTAG.t test. For all tests, P<0.05 was taken as statistically significant.All statistical analysis was performed using SPSS 17.0 software. All results were expressed as mean ± S.D. and were statistically evaluated using a one-way ANOVA followed by the First, we examined the expression pattern of HOTAIR in EC isolated from atherosclerotic plaque in atherosclerosis patients. We detected HOTAIR expression in 40 pairs of EC isolated from atherosclerotic plaques and the corresponding vascular wall by qPCR. We found that HOTAIR expression was significantly lower in EC from atherosclerotic plaque compared with corresponding vascular wall A.Next, we determined the functional role of HOTAIR in EC by gain- and loss-of-function assays in two EC lines, HUVEC and HAEC. Cells were transfected with two different siRNAs against HOTAIR. Both the siRNAs significantly suppressed HOTAIR expression, and the target site one (siHOTAIR-1) is the most effective site that is chosen for the following experiments B. CCK-8 Migration of EC has also been implicated to be critical in the progression of atherosclerotic lesions ,24. We s2 =0.6702, P<0.0001), indicating that HOTAIR expression may be regulated by TSLP.Next, we determined the relationship between TSLP signal and HOTAIR. We detected the serum TSLP levels in 40 atherosclerotic patients and 40 healthy donors by ELISA assay. Serum TSLP levels were much lower in atherosclerosis patients than in healthy donors A. In addTo clarify the influence of TSLP on HOTAIR expression, we treated HUVEC with different concentrations of rhTSLP. The results showed that TSLP significantly increased HOTAIR expression in a dose-dependent manner C. We susHOTAIR promoter region. Inhibition of TSLP-TSLPR-PI3K/AKT-IRF1 pathway significantly abolished the rhTSLP-induced decrease in the IRF1 occupancy at HOTAIR promoter (Previous study demonstrated that osteopontin (OPN) regulated PI3K/AKT activation and IRF1 expression, subsequently influencing the expression of HOTAIR . Similarpromoter F. Taken We next determined the significance of HOTAIR in TSLP-mediated phenotypes of EC. We transfected HOTAIR siRNA into rhTSLP-treated HUVEC A. IntereIt has been reported that ox-LDL-induced oxidative injury is a critical step in EC injury and atherosclerotic plaque formation . We specIn the present study, HOTAIR expression was found to be much lower in EC from atherosclerotic plaques. Given that lncRNAs are critical for gene regulatory networks, our present study focused on the potential functional role regulated by HOTAIR. Our data demonstrated that HOTAIR facilitated cell proliferation and migration, and suppressed apoptosis in EC. Endothelial dysfunction is one of the initial steps in the development of atherosclerosis. EC showed a decreased proliferation and migration and increased apoptosis . Our res−/− mice. TSLP administration increased the levels of anti-ox-LDL IgM and IgG1, but decreased the levels of IgG2a in plasma, along with increased numbers of CD4+LAP+ regulatory T cells (Tregs) in the spleen and decreased inflammation in the aorta [In atherosclerosis, EC activation triggers platelet aggregation, immunocytes adhesion and vascular smooth muscle cells proliferation. The proliferation and migration of EC have been implicated to be a critical step in the progression of atherosclerotic lesions ,27. TSLPhe aorta . HoweverOx-LDL-induced EC injury, which mimics oxidative injury to EC, is taken as an initial and critical step in the atherosclerotic process . In the In conclusion, these results demonstrated that TSLP-PI3K/AKT-HOTAIR pathway positively regulates proliferation and migration of EC. These findings may reveal important insights into the role of lncRNAs in the pathogenesis and progression of atherosclerosis. TSLP and HOTAIR may serve as a novel therapy for atherosclerosis. However, numbers of specific lncRNAs might be involved in atherosclerotic disease. Thus, investigation of other lncRNAs and their functional association with atherosclerosis is necessary to overcome CADs.


The linear epitopes of gp46-I, gp46-II, gp21 and p19 are used in diagnosis of HTLV-I/-II infections. The aims of this study was to obtain high-level expression and purification of recombinant antigen (RA) containing these epitopes. Large-scale preparation of such antigen probably worths for diagnostic purpose.Escherichia coli BL21 (DE3) cells. Expression and distribution of the His-GST-RA protein were evaluated using SDS-PAGE. The soluble RA was purified utilizing Ni-NTA agarose beads under native conditions and was concentrated by ultra filtration. Using 20 sera specimens from HTLV infected patients, the antigenicity of the purified protein was confirmed in ELISA and western blotting analysis.The synthetic DNA encoding RA was synthesized and over-expressed as soluble in SDS-PAGE revealed that the purified protein was more than 90% pure. The final yield was approximately 25 mg per liter of culture medium. ELISA results showed that RA could specifically bind to anti-HTLV-I/-II antibodies in infected sera.RA could be a candidate for HTLV-I/-II screening and the strategy presented in this study could be used for easy production of this diagnostic protein. Human T-lymphotropic virus type I (HTLV-I) is a retrovirus known to be the etiological agent of adult T-cell leukemia/lymphoma (ATLL) and myelopathy/tropical spastic paraparesis (HAM/TSP) diseases . Human THTLV-I and -II infection is asymptomatic in more than 90% of cases, most of them are completely unaware of their infection. Screening of asymptomatic carriers is the main strategy to prevent infections with HTLV-I and -II and spreading . The mosWhole viral lysate, full length antigen and synthetic peptides are used as capture antigen for detection of HTLV antibodies. Viral lysate and full length antigens give some non-specific interaction with antibodies arises against other viral infections . TherefoIn previous study, Heydari-Zarnagh designed a recombinant protein (named SCA) for specific detection of HTLV-I antibodies incorporating linear epitopes of gp46, gp21 and p19 and flexible linker as spacer arm between epitopes  unless otherwise indicated. The oligonucleotides were synthesized by Biomatik Company . Restriction enzymes were purchased from Takara ; plasmid DNA extraction and gel purification kits were obtained from iNtRON ; T4 ligase, DNA marker, and IPTG were purchased from Fermentas ; protein marker was purchased from Cinaclone ; and Ni-NTA agarose resin was obtained from Qiagen .Serum samples were collected from 10 patients with HTLV-I (anti-HTLV-I positive) and 10 patients with HTLV-II (anti-HTLV-II positive) infection referred to Razavi hospital, Mashhad city, Iran. All samples were diagnosed as positive when tested with commercial ELISA kits . Samples from 20 healthy volunteers were used as controls and tested with ELISA.http://www.bioinformatics.org/sms2/rev_trans.html) and optimized according to E. coli codon usage by JAVA codon optimization tool (http://www.jcat.de/) and analyzed by rare codon analysis tool . The EcoRI and BamHI restriction sites were inserted into the ends of DNA sequence for cloning purpose.The amino acid sequence of immunodominant peptides derived from HTLV-I/-II diagnostic proteins  from both HTLV-I/II were connected together using flexible linker (amino acid sequence: EAAAK). The selected epitopes were investigated further using multiple sequence alignments tools. All sequence-profile alignments were performed using a standard pairwise alignment of Clustal Omega. The resulting chimeric protein was reversely translated  cells and the colonies were selected on a LB plate supplemented with 100 μg/ml ampicillin.The synthetic gene was sub-cloned into the E. coli BL21 (DE3) colony harboring pGS21a-RA expression vector was grown overnight at 37°C in 5 ml of LB medium with 100μg/ml ampicillin. This culture was inoculated into a baffled flask that contained 100ml of the LB medium with 100μg/ml ampicillin. The culture was incubated at 37°C until an OD600 of 0.6 was reached. Expression was induced by adding IPTG  and incubating the cells at 18°C for an additional 12h under constant shaking at 250 rpm. The cells were harvested by centrifugation at 5000 rpm for 10min at 4°C. The pellets were resuspended in PBS and mixed with 2× sample loading buffer heated at 100°C for 15 min. The samples were analyzed by SDS-PAGE in 12% polyacrylamide gels. The non-induced controls (as negative controls) were analyzed in parallel.An E. coli pellets obtained from 50 ml of culture following IPTG induction were re-suspended in phosphate buffered saline (PBS) buffer  and subjected to sonication  on ice. The sonication was done at a frequency of 20 kHz for 2 minutes, bacteria were pulsed for 6 × 10 s intervals. After sonication, the cell lysate was centrifuged at 12,000 rpm for 20 min at 4°C. The clear supernatant and pellets were analyzed by SDS-PAGE in 12% polyacrylamide gels.To check the solubility of His-GST-RA under conditions for expression, the +-NTA resin and protein was purified according to the manufacture instructions. A flow rate of 0.5 ml/min was used in all the chromatographic steps. The protein concentration was determined using the Bradford method  and then incubated overnight with serum containing HTLV antibodies at 4°C. After washing, the immunoreactive bands were visualized using horseradish peroxidase (HRP)-conjugated HTLV-I/-II chimeric antigen and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution.The ELISA plate wells were coated with RA (5–30 μg/ml) in sodium bicarbonate buffer  and incubated overnight at 4°C. The coated wells were washed twice with PBS and then blocked with blocking buffer (1% BSA in PBS with 1% Tween-20) for 2 h at room temperature. Then, 50 μl of the infected sera were added to the plates and incubated for 1 h at 37°C. The HRP-conjugated HTLV-I/-II-chimeric antigen was added to the plates and incubated for 1 h at 37°C. After washing for six times, TMB substrate solution was added and the absorbance was determined at 450 nm after 30 min. Bovine serum albumin (BSA) was used as a negative control under the same conditions.E. coli was improved by upgrading guanine-cytosine percent (GC%) and codon adaptation index (CAI) to 55.2% (GC% of E. coli is about 50) and 0.89 respectively. CAI of > 0.8 was regarded as good, in terms of high gene expression level  to generate pGS21a-RA. Accuracy of sub-cloning was confirmed by DNA sequencing.The synthetic DNA encoding RA was sub-cloned into E. coli BL21 (DE3) was analyzed by SDS-PAGE. The induction time was 12 h and the IPTG concentration was 0.4 mM. SDS-PAGE of the lysate of the induced pGS21a-RA/BL21 recombinant bacteria revealed a 63-kDa band, corresponding in size to the expected molecular weight of His-GST-RA , whereas no visible band was noted upon examining the pellet of the cell lysate .The His-GST-tagged RA was purified under native conditions using Ni-NTA agarose beads. The protein was visualized as a 63-kD band and its purity was around 90% .Using HTLV-I/-II infected sera, the binding characteristic of the His-GST-RA was confirmed by western blot analysis. As expected, the 63-kD band exhibited affinity to the antibodies .The binding activity of the His-GST-RA to the infected serum antibodies was determined by ELISA. As shown in HTLV-I and HTLV-II are major causes of neurologic disease and lymphocytic leukemia. Peptides derived from gp46-I, gp46-II, gp21 and p19 are important antigens that are widely used in the serological tests of HTLV infection . The preE. coli was selected as the host, owing to advantages such as low cost, ease of use, high level of productivity, and presence of an N-terminal His-GST-tag in the vector pGS21a, which allows easy purification  was constructed to be used in ELISA for screening the suspected cases for HTLV infection. Integration of the GST-fusion, low temperature induction and low IPTG concentration are excellent strategies for expressing this antigen as soluble form in


Functional neurological disorder (FND) is an elusive disorder characterized by unexplained neurological symptoms alongside aberrant cognitive processing and negative affect, often associated with amygdala reactivity.We examined the effect of negative conditioning on cognitive function and amygdala reactivity in 25 FND patients and 20 healthy volunteers (HV). Participants were first conditioned to stimuli paired with negative affective or neutral (CS +/CS −) information. During functional MRI, subjects then performed an instrumental associative learning task to avoid monetary losses in the context of the previously conditioned stimuli. We expected that FND patients would be better at learning to avoid losses when faced with negatively conditioned stimuli (increased harm avoidance). Multi-echo resting state fMRI was also collected from the same subjects and a robust denoising method was employed, important for removing motion and physiological artifacts.FND subjects were more sensitive to the negative CS + compared to HV, demonstrated by a reinforcement learning model. Contrary to expectation, FND patients were generally more impaired at learning to avoid losses under both contexts (CS +/CS −), persisting to choose the option that resulted in a negative outcome demonstrated by both behavioural and computational analyses. FND patients showed enhanced amygdala but reduced dorsolateral prefrontal cortex responses when they received negative feedback. Patients also had increased resting state functional connectivity between these two regions.FND patients had impaired instrumental avoidance learning, findings that parallel previous observations of impaired action-outcome binding. FND patients further show enhanced behavioural and neural sensitivity to negative information. However, this did not translate to improved avoidance learning. Put together, our findings do not support the theory of harm avoidance in FND. We highlight a potential mechanism by which negative contexts interfere with adaptive behaviours in this under-explored disorder. •Functional neurological disorder (FND) is a relatively common but elusive disorder characterized by unexplained neurological symptoms and negative affect.•One theory of FND symptomology is related to unconscious harm avoidance, in which symptom expression occurs to avoid a stressful situation.•We show that FND patients were more sensitive to negative information. However, this did not translate to increased harm avoidance as patients were impaired at goal-directed avoidance learning.•This study suggests that excessive negative conditioning in this group may engender deficient, rather than increased, goal-directed cognitive ability and loss avoidance. It has been proposed that excessive negative affect and anxiety can exacerbate a deficient top-down regulatory system, leading to psychogenic or ‘functional’ neurological symptoms . There iIn healthy populations, learning to associate and avoid external stimuli with probable negative outcomes is vital for the selection of appropriate behaviour and environmental adaptation. Several lines of evidence suggest that FND patients are more sensitive to negative conditioning, with heightened responses to both negative affective stimuli  and arouNeurally, negative emotional stimuli  and affeConnectivity between the dorsolateral PFC and amygdala is associated with the capacity to modulate negative emotional responses with cognitive strategies  thereby It is unclear how the enhancement in negative conditioning observed in FND impacts cognitive ability, and how this is expressed neurally. There is some evidence that FND patients are more likely to engage dissociative or avoidance-related strategies for coping with difficult life events, rather than planning and problem solving, or using the cognitive skills necessary to adapt to a dynamic world . Therefo22.1http://fndhope.org/). Diagnoses were made or confirmed by a neurologist from the FND clinic in Addenbrooke's Hospital, using the DSM-4 TR diagnostic criteria. All participants were screened by a psychiatrist for comorbidities and to record symptom severity. The FND patients were screened both clinically and using the Mini International Neuropsychiatric Inventory (MINI). Healthy controls were screened using the MINI. Symptom severity was rated for duration and severity . Exclusion criteria included subjects below the age of 18, any other major neurological, current major depression greater than moderate severity, psychotic or bipolar disorder and substance use disorder. Current mild major depression and elevated depression scores with no current major depression diagnosis were allowed in the FND group. Symptoms of pain, motor  and sensory  functions were assessed in clinical interview that included duration and severity. All subjects were able to remain still in the scanner. HV were recruited via community-based advertisements. All participants provided written informed consent and were reimbursed for their time. The study was approved by the University of Cambridge Research Ethics Committee.Data were collected from 25 FND patients and 20 healthy volunteers (HV). The FND subjects were recruited both from neurologists and psychiatrists at Addenbrookes Hospital and via the FND Hope website . Participants should thus learn to avoid the conditioned/familiar choice and instead choose the novel stimulus. The choice presentation phase (3000 ms) was followed by a response phase (1000 ms) in which participants made a left or right button press to indicate their response. Feedback was presented as either a £1 coin with a red cross overlaid (loss feedback) or as a grey square (no loss feedback) for 500 ms. This was followed by an inter-trial fixation cross interval varying between 1000 and 3000 ms. There were 30 trials per condition  that were randomly intermixed, resulting in 90 trials for the three conditions. This was repeated after a short break for the second set of conditioned stimuli, giving a total of 180 trials.Outcome measures were response accuracy and trials to acquisition . Response accuracy was also averaged across the 30 trials within conditions. Accuracy and trials to acquisition data were entered into repeated measures ANOVA testing for effects of condition (3) and group . Subject2.3Aversion learning task data was also assessed using a standard Q-learning model , a simplBetter or worse than expected outcomes increase or decrease the value, respectively, with a prediction error of:The amplitude of value change from one trial to the next is captured by α, the learning rate. The noisiness or choice randomness is captured by β, the temperature parameter.We also introduced a free parameter, γ, which was the estimated initial value of Q representing the CS +, CS − or familiar stimulus and was allowed to vary from − 1 to 0.2.43 voxel size, 0.75 mm gap, FA = 78°, 64 × 64 matrix size, 39 slices) and angled 30° to the AC-PC line to avoid susceptibility signal loss in the orbitofrontal regions. Anatomical images were acquired with a T1-weighted magnetization prepared rapid gradient echo (MPRAGE) sequence .Functional MRI data was collected during task performance at the Wolfson Brain Imaging Centre, University of Cambridge with a Siemens 3 T Tim Trio scanner and 32-channel head coil. Interleaved echo planar imaging (EPI) images were acquired . EPI data were realigned to the first image, unwarped and slice time corrected. Each individual's anatomical T1 image was co-registered with their EPI image and normalized to a standard MNI template. EPI data were smoothed using a 8 mm FWHM Gaussian kernel. Artifact Detection Tools (ART) repair toolbox for SPM . FastICA was used to first decompose the data. Components were then categorized as BOLD or non-BOLD based on their TE-dependence as measured by the pseudo-F-statistic kappa and rho, respectively (p < 0.05 was considered significant.Resting state fMRI (rsfMRI) data were acquired following the fMRI task for 10 min with eyes open. Data from 25 FND patients and 70 HV  were included in this analysis, including the HV that took part in the task-based fMRI study. We have previously reported reduced inferior parietal cortex (IPC) functional connectivity with frontal regions and increased IPC connectivity with premotor and supplementary motor cortices in this FND group . A multiectively . Non-BOLectively  for SPM83n = 17), pregabalin (n = 5), gabapentin (n = 1), lamotrigine (n = 2), topiramate (n = 1), and a synthetic opioid (n = 1).The participant characteristics have been extensively described . Symptom3.1 = 5.896, p = 0.019) of impaired accuracy in the FND group and a trend towards a main effect of condition  = 2.571, p = 0.088)  = 4.585, p = 0.038) with greater trials to acquisition in the FND group and no effect of condition. There were no group × condition interaction effects (p > 0.05).Repeated measures ANOVA for overall avoidance learning accuracy revealed a main effect of group (F= 0.088) . For tri3.2 = 4.658, p = 0.037) with impaired learning rate in the FND group, and no condition or interaction effects (p > 0.05)  = 5.480, p = 0.025) with greater choice noisiness in the FND group and a group × condition interaction effect  = 4.034, p = 0.026), and no main effect of condition (p > 0.05). We conducted post hoc pairwise comparisons which revealed a significant group difference in the CS + condition, , but not for the other two conditions (p > 0.05). For γ, there was a main effect of group  = 4.748, p = 0.036) of a lower initial value assigned to the stimuli and a trend towards a condition × group interaction  = 2.818, p = 0.073). We conducted post hoc pairwise comparisons, which revealed a significant group difference in the CS + condition  and a trend towards a group difference in the CS − condition .There was a main effect of group for α across the three conditions (F > 0.05) . For β, 3.3p = 0.048, Z = 2.83, xyz = − 22 − 6 − 14, p = 0.059, Z = 3.56, xyz = − 36 54 22, There were no between group differences in amygdala or dorsolateral PFC responses to the negative CS + during the choice presentation stage, when compared to CS − or familiar cues. When examining the feedback stage, FND subjects showed enhanced left amygdala responses to loss compared to HV  = 5.722, p = 0.024) in which FND subjects showed a negative relationship between amygdala reactivity and STAI and HV showed a positive relationship  = 0.006, Cluster = 181, Z = 4.07, xyz = 27 33 51, t = − 2.03, p = 0.059, Resting state functional connectivity of the bilateral amygdala and right dorsolateral PFC was increased in FND patients compared to HV  and responses and outcomes (R-O) and to use aversive goals to guide behaviours. These findings were confirmed by both behavioural and computational methods. We further show that FND patients had a more negative valuation of the aversive CS + and showed heightened amygdala but blunted dorsolateral PFC reactivity to negative outcomes.We show that FND patients have disrupted instrumental avoidance learning in the context of both affective information (CS +/CS − stimuli) and an affectively-neutral control condition (familiar stimuli). This observation parallels a previous report of decreased implicit action-outcome binding in FND relative to healthy controls suggesting an impaired sense of agency . The actPrevious studies have demonstrated that patients with FND show diminished cognitive processing such as working memory in situations of affective processing . AlthougFND patients also had enhanced amygdala and a trend towards reduced dorsolateral PFC responses to loss outcomes compared to HV. As discussed, the amygdala nuclei mediate the initial acquisition of fear via CS-US associations , regulatIndeed the neutral stimulus in this study might have gained ‘safe’ properties as neutral stimuli were intermixed with fearful CS + stimuli. A similar study in healthy individuals during fMRI has demonstrated reduced amygdala responses to neutral or safe/irrelevant stimuli compared to fearful stimuli . This coPatients with unipolar depression show a similar pattern of neural responses to affective information, with increased amygdala responses to affective information  that doeThe FND group also had elevated resting state functional connectivity between the amygdala and dorsolateral PFC. This was somewhat unexpected as increased resting connectivity between these regions might suggest an enhanced capacity for amygdala – dorsolateral PFC mediated affective and behavioural control. As mentioned, the dorsolateral PFC plays a regulatory role over the amygdala and voluntary emotional evaluation  and psycWe had anticipated that given the enhanced neural activity to negative affective stimuli and loss feedback, the greater salience of negative stimuli might be associated with greater harm avoidance and better avoidance learning in FND patients. However, the behavioural response to enhanced salience of negative stimuli was not adaptive but interfered with goal-directed learning with greater noise in behavioural choices with aversive contexts. These findings suggest that goal-directed harm avoidance is unlikely to underlie FND symptoms. There are several plausible mechanisms that might underlie the effect of the CS + on enhanced choice randomness in FND patients. The neural network implicated in fear/negative conditioning  overlaps with that of value representation of loss outcomes  but is not identical. Furthermore, the timing of the training and testing phases in this task differ which might induce conflict. The training phase involved a 1.5 s cue followed by the aversive stimulus lasting 2 s. The instrumental test phase involved a two-choice decision phase of 3 s followed by a 0.5 s choice and 0.5 s outcome phase. Thus, 1.5 s into the decision phase in the presence of a CS +, subjects were conditioned to expect an aversive outcome. The presence of the CS + in FND patients thus acted as a fearful context, possibly interfering with goal-directed learning and enhancing random noisy choices.We note that the sample size is small but we further emphasize the difficulties in recruiting FND subjects for studies. A current limitation is that multiple types of presentations of this disorder have currently been merged into a single group, assuming homogeneity in behavioural, cognitive and neural dysfunction. Since the group included both positive and negative motor symptoms, with about half experiencing non-epileptic seizures, it is likely that the disorder etiology differs between subjects. As there is currently a dearth of literature investigating this group, the current study is at least a step towards examining shared deficits. This dissociation between shared and unique aberrancies is certainly a crucial goal for future studies. The FND patients were also on different medications which may impact the findings. We further emphasize that our findings demonstrate an impairment in the capacity to acquire S-O or R-O representations relevant to goal-directed learning. However, the task was not designed to dissociate goal-directed and habitual behaviours. Furthermore, to learn 3 different stimuli-pairs and their associations is more difficult; greater cognitive demands required by this task may also contribute to the observed impairments. We emphasize that this current conditioning task uses robust aversive stimuli with unpleasant imagery of high intensity and paired with negative sounds such as nails scratching on a blackboard or high pitched screaming. Thus, the capacity to elicit effects as compared to more subtle aversive stimuli may differ.Together, we demonstrate that despite enhanced neural salience and computational value of negative stimuli in FND subjects, behavioural avoidance learning was not improved. There was no enhancement of goal-directed harm avoidance in FND patients, but rather, a generalized impairment in the capacity to use negative outcomes to guide goal-directed behaviours alongside increased amygdala reactivity to negative outcomes. Our findings suggest that learned negative or fearful environmental contexts  can impair goal-directed decision making in FND subjects. Pharmacological or behavioural interventions to enhance associative learning or to decrease the salience of negative stimuli or learned contexts may be important in FND.The following are the supplementary data related to this article.