NOD2-Nitric Oxide-responsive MicroRNA-146a Activates Sonic Hedgehog Signaling to Orchestrate Inflammatory Responses in Murine Model of Inflammatory Bowel Disease

Background: Genetic variants of NOD2 are linked to inflammatory bowel disease (IBD) etiology. Results: DSS model of colitis in wild-type and inducible nitric-oxide synthase (iNOS) null mice revealed that NOD2-iNOS/NO-responsive microRNA-146a targets NUMB gene facilitating Sonic hedgehog (SHH) signaling. Conclusion: miR-146a-mediated NOD2-SHH signaling regulates gut inflammation. Significance: Identification of novel regulators of IBD provides new insights into pathophysiology and development of new therapy concepts. Inflammatory bowel disease (IBD) is a debilitating chronic inflammatory disorder of the intestine. The interactions between enteric bacteria and genetic susceptibilities are major contributors of IBD etiology. Although genetic variants with loss or gain of NOD2 functions have been linked to IBD susceptibility, the mechanisms coordinating NOD2 downstream signaling, especially in macrophages, during IBD pathogenesis are not precisely identified. Here, studies utilizing the murine dextran sodium sulfate model of colitis revealed the crucial roles for inducible nitric-oxide synthase (iNOS) in regulating pathophysiology of IBDs. Importantly, stimulation of NOD2 failed to activate Sonic hedgehog (SHH) signaling in iNOS null macrophages, implicating NO mediated cross-talk between NOD2 and SHH signaling. NOD2 signaling up-regulated the expression of a NO-responsive microRNA, miR-146a, that targeted NUMB gene and alleviated the suppression of SHH signaling. In vivo and ex vivo studies confirmed the important roles for miR-146a in amplifying inflammatory responses. Collectively, we have identified new roles for miR-146a that established novel cross-talk between NOD2-SHH signaling during gut inflammation. Potential implications of these observations in therapeutics could increase the possibility of defining and developing better regimes to treat IBD pathophysiology.

Differential proteomic and genomic profiling of mouse striatal cell model of Huntington's disease and control; probable implications to the disease biology

Huntington's disease (HD) is an autosomal dominant disorder of central nervous system caused by expansion of CAG repeats in exon1 of the huntingtin gene (Htt). Among various dysfunctions originated from the mutation in Htt gene, transcriptional deregulation has been considered to be one of the most important abnormalities. Large numbers of investigations identified altered expressions of genes in brains of HD patients and many models of HD. In this study we employed 2D SDS-PAGE/MALDI-MS coupled with 2D-DIGE and real-time PCR experiments of an array of genes focused to HD pathway to determine altered protein and gene expressions in STHdh(Q111)/Hdh(Q111) cells, a cell model of HD and compared with STHdh(Q7)/Hdh(Q7) cells, its wild type counterpart. We annotated 76 proteins from these cells and observed differential expressions of 31 proteins (by 2D-DIGE) involved in processes like unfolded protein binding, negative regulation of neuron apoptosis, response to superoxides etc. Our PCR array experiments identified altered expressions of 47 genes. Altogether significant alteration of 77 genes/proteins could be identified in this HD cell line with potential relevance to HD biology. Biological significance: In this study we intended to find out differential proteomic and genomic profiles in HD condition. We used the STHdh cells, a cellular model for HD and control. These are mouse striatal neuronal cell lines harboring 7 and 111 knock -in CAG repeats in their two alleles. The 111Q containing cell line (STHdh(Q111)/Hdh(Q111)) mimics diseased condition, whereas the 7Q containing ones (STHdh(Q7)/Hdh(Q7)), serves as the proper control cell line. Proteomic experiments were performed earlier to obtain differential expressions of proteins in R6/2 mice models, Hdh(Q) knock -in mice and in plasma and CSF from HD patients. However, no earlier report on proteomic alterations in these two HD cell lines and control was available in literature. It was, therefore, an important objective to find out differential expressions of proteins in these two cell lines. In this study, we annotated 76 proteins from STHdh(Q7)/Hdh(Q7) and STHdh(Q111)/Hdh(Q111) cells using 2D-gel/mass spectrometry. Next, by performing 2D-DIGE, we observed differential expressions of 31 proteins (16 upregulated and 15 downregulated) between these two cell lines. We also performed customized qRT-PCR array focused to HD pathway and found differential expressions of 47 genes (8 gene exptessions increased and 39 genes were decreased significantly). A total of 77 genes/proteins (Htt downregulated in both the studies) were found to be significantly altered from both the experimental paradigms. We validated the differential expressions of Vim, Hypk, Ran, Dstn, Hspa5 and Sod2 either by qRT-PCR or Western blot analysis or both. Out of these 77, similar trends in alteration of 19 out of 31 and 38 out of 47 proteins/genes were reported in earlier studies. Thus our study confirmed earlier observations on differential gene/protein expressions in HD and are really useful. Additionally, we observed differential expression of some novel genes/proteins. One of this was Hypk, a Htt-interacting chaperone protein with the ability to solubilize mHtt aggregated structures in cell lines. We propose that downregulation of Hypk in STHdh-Qm (Q111)/Hdh(Q111) has a causal effect towards HD pathogenesis. Thus the novel findings from our study need further research and might be helpful to understand the molecular mechanism behind HD pathogenesis. (C) 2015 Elsevier B.V. All rights reserved.

A systems perspective of host-pathogen interactions: predicting disease outcome in tuberculosis

The complex web of interactions between the host immune system and the pathogen determines the outcome of any infection. A computational model of this interaction network, which encodes complex interplay among host and bacterial components, forms a useful basis for improving the understanding of pathogenesis, in filling knowledge gaps and consequently to identify strategies to counter the disease. We have built an extensive model of the Mycobacterium tuberculosis host-pathogen interactome, consisting of 75 nodes corresponding to host and pathogen molecules, cells, cellular states or processes. Vaccination effects, clearance efficiencies due to drugs and growth rates have also been encoded in the model. The system is modelled as a Boolean network. Virtual deletion experiments, multiple parameter scans and analysis of the system's response to perturbations, indicate that disabling processes such as phagocytosis and phagolysosome fusion or cytokines such as TNF-alpha and IFN-gamma, greatly impaired bacterial clearance, while removing cytokines such as IL-10 alongside bacterial defence proteins such as SapM greatly favour clearance. Simulations indicate a high propensity of the pathogen to persist under different conditions.

A systems perspective of host-pathogen interactions: predicting disease outcome in tuberculosis

The complex web of interactions between the host immune system and the pathogen determines the outcome of any infection. A computational model of this interaction network, which encodes complex interplay among host and bacterial components, forms a useful basis for improving the understanding of pathogenesis, in filling knowledge gaps and consequently to identify strategies to counter the disease. We have built an extensive model of the Mycobacterium tuberculosis host-pathogen interactome, consisting of 75 nodes corresponding to host and pathogen molecules, cells, cellular states or processes. Vaccination effects, clearance efficiencies due to drugs and growth rates have also been encoded in the model. The system is modelled as a Boolean network. Virtual deletion experiments, multiple parameter scans and analysis of the system's response to perturbations, indicate that disabling processes such as phagocytosis and phagolysosome fusion or cytokines such as TNF-alpha and IFN-gamma, greatly impaired bacterial clearance, while removing cytokines such as IL-10 alongside bacterial defence proteins such as SapM greatly favour clearance. Simulations indicate a high propensity of the pathogen to persist under different conditions.

Modelling studies on neurodegenerative disease causing triplet repeat sequences d(CGG/GCC) and d(CAG/CTG): Duplexes vs Quadruplexes

Model building and molecular mechanics studies have been carried out to examine the potential structures for d(GGC/GCC)5 and d(CAG/CTG)5 that might relate to their biological function and association with triplet repeat expansion diseases. Model building studies suggested that hairpin and quadruplex structures could be formed with these repeat sequences. Molecular mechanics studies have demonstrated that the hairpin and hairpin dimmer structures of triplet repeat sequences formed by looping out of the two strands are as favourable as the corresponding B-DNA type hetero duplex structures. Further, at high salt condition, Greek key type quadruplex structures are energetically comparable with hairpin dimer and B-DNA type duplex structures. All tetrads in the quadruplex structures are well stacked and provide favourable stacking energy values. Interestingly, in the energy minimized hairpin dimer and Greek key type quadruplex structures, all the bases even in the non-G tetrads are cyclically hydrogen bonded, even though the A, C and T-tetrads were not hydrogen bonded in the starting structures.

Enhanced J-protein interaction and compromised protein stability of mtHsp70 variants lead to mitochondrial dysfunction in Parkinsons disease

Parkinsons disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multifactorial in nature, a recent genetic screen involving PD patients identified two mitochondrial Hsp70 variants (P509S and R126W) that are suggested in PD pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD variants are centrally involved in PD progression is totally elusive. In this article, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD variants. Biochemically, the R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, the P509S variant exhibits significantly enhanced interaction with J-protein cochaperones involved in folding and import machinery, thus altering the overall regulation of chaperone-mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD mutations at the cellular level, we developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with omitochondrial dysfunction', including compromised growth, impairment in protein translocation, reduced functional mitochondrial mass, mitochondrial DNA loss, respiratory incompetency and increased susceptibility to oxidative stress. In addition to that, R103W protein is prone to aggregate in vivo due to reduced stability, whereas P486S showed enhanced interaction with J-proteins, thus remarkably recapitulating the cellular defects that are observed in human PD variants. Taken together, our findings provide evidence in favor of direct involvement of mtHsp70 as a susceptibility factor in PD.

Computational framework to integrate the effect of antigen recognition on disease epidemiology outcome: multi-scale approach

Human Leukocyte Antigen (HLA) plays an important role, in presenting foreign pathogens to our immune system, there by eliciting early immune responses. HLA genes are highly polymorphic, giving rise to diverse antigen presentation capability. An important factor contributing to enormous variations in individual responses to diseases is differences in their HLA profiles. The heterogeneity in allele specific disease responses decides the overall disease epidemiological outcome. Here we propose an agent based computational framework, capable of incorporating allele specific information, to analyze disease epidemiology. This framework assumes a SIR model to estimate average disease transmission and recovery rate. Using epitope prediction tool, it performs sequence based epitope detection for a given the pathogenic genome and derives an allele specific disease susceptibility index depending on the epitope detection efficiency. The allele specific disease transmission rate, that follows, is then fed to the agent based epidemiology model, to analyze the disease outcome. The methodology presented here has a potential use in understanding how a disease spreads and effective measures to control the disease.

A Generalist Predator Regulating Spread of a Wildlife Disease: Exploring Two Infection Transmission Scenarios

Ecoepidemiology is a well-developed branch of theoretical ecology, which explores interplay between the trophic interactions and the disease spread. In most ecoepidemiological models, however, the authors assume the predator to be a specialist, which consumes only a single prey species. In few existing papers, in which the predator was suggested to be a generalist, the alternative food supply was always considered to be constant. This is obviously a simplification of reality, since predators can often choose between a number of different prey. Consumption of these alternative prey can dramatically change their densities and strongly influence the model predictions. In this paper, we try to bridge the gap and explore a generic ecoepidemiological system with a generalist predator, where the densities of all prey are dynamical variables. The model consists of two prey species, one of which is subject to an infectious disease, and a predator, which consumes both prey species. We investigate two main scenarios of infection transmission mode: (i) the disease transmission rate is predator independent and (ii) the transmission rate is a function of predator density. For both scenarios we fulfil an extensive bifurcation analysis. We show that including a second dynamical prey in the system can drastically change the dynamics of the single prey case. In particular, the presence of a second prey impedes disease spread by decreasing the basic reproduction number and can result in a substantial drop of the disease prevalence. We demonstrate that with efficient consumption of the second prey species by the predator, the predator-dependent disease transmission can not destabilize interactions, as in the case with a specialist predator. Interestingly, even if the population of the second prey eventually vanishes and only one prey species finally remains, the system with two prey species may exhibit different properties to those of the single prey system.

An enzyme-linked immuno focus assay for rapid detection and enumeration, and a newborn mouse model for human non-polio enteroviruses associated with acute diarrhea

We have recently reported significant association of non-polio enteroviruses (NPEVs) with acute and persistent diarrhea (18-21% of total diarrheal cases), and non-diarrheal Increased Frequency of Bowel Movements (IFoBM-ND) (about 29% of the NPEV infections) in children and that the NPEV-associated diarrhea was as significant as rotavirus diarrhea. However, their diarrhea-causing potential is yet to be demonstrated in an animal model system. Since the determination of virus titers by the traditional plaque assay takes 4-7 days, there is a need for development of a rapid method for virus titer determination to facilitate active clinical research on enterovirus-associated diarrhea. The goal of this study is to develop a cell-based rapid detection and enumeration method and to demonstrate the diarrhea-inducing potential of purified and characterized non-polio enteroviruses, which were isolated from diarrheic children. Here we describe generation of monoclonal and polyclonal antibodies against purified strains belonging to different serotypes, and development of an enzyme-linked immuno focus assay (ELIFA) for detection and enumeration of live NPEV particles in clinical and purified virus samples, and a newborn mouse model for NPEV diarrhea. Plaque-purified NPVEs, belonging to different serotypes, isolated from children with diarrhea, were grown in cell culture and purified by isopycnic CsCl density gradient centrifugation. By ELIFA, NPEVs could be detected and enumerated within 12 h post-infection. Our results demonstrated that Coxsackievirus B1 (CVB1) and CVB5 strains, isolated from diarrheic children, induced severe diarrhea in orally-inoculated 9-12 day-old mouse pups, fulfilling Koch's postulates. The methods described here would facilitate studies on NPEV-associated gastrointestinal disease. (C) 2015 Elsevier B.V. All rights reserved.

MPTP activates ASK1-p38 MAPK signaling pathway through TNF-dependent Trx1 oxidation in parkinsonism mouse model

Activation of apoptosis signal regulating kinase 1 (ASK1)-p38 MAPK death signaling cascade is irn plicated in the death of dopaminergic neurons in substantia nigra in Parkinson's disease (PD). We investigated upstream activators of ASK1 using an MPTP mouse model of parkinsonism and assessed the temporal cascade of death signaling in ventral midbrain (VMB) and striatum (ST). MPTP selectively activated ASK1 and downstream 1)38 MAPK in a time dependent manner in VMB alone. This occurred through selective protein thiol oxidation of the redox-sensitive thiol disulfide oxidoreductase, thiorcdoxin (Trxl), resulting in release of its inhibitory association with ASK1, while glutathione-S-transferase ji 1 (GSTM1) remained in reduced form in association with ASK1. Levels of tumor necrosis factor (TNF), a known activator of ASK1, increased early after MPTP in VMB. Protein ovariation netvvork analysis (PCNA) using protein states as nodes revealed TNF to be an important node regulating the ASK1 signaling cascade. In confirmation, blocking MPTP-mecliated TNF signaling through intrathecal administration of TNFneutralizing antibody prevented Trxl oxidation and downstream ASK1-p38 MAPK activation. Averting an early increase in TNF, which leads to protein thiol oxidation resulting in activation of ASK1-p38 signaling, may be critical for neuroprotection in PD. Importantly, network analysis can help in understanding the cause/effect relationship within protein networks in complex disease states. (C) 2015 Published by Elsevier Inc.

Modelling staphylococcal pneumonia in a human 3D lung tissue model system delineates toxin-mediated pathology

Staphylococcus aureus necrotizing pneumonia is recognized as a toxin-mediated disease, yet the tissue-destructive events remain elusive, partly as a result of lack of mechanistic studies in human lung tissue. In this study, a three-dimensional (3D) tissue model composed of human lung epithelial cells and fibroblasts was used to delineate the role of specific staphylococcal exotoxins in tissue pathology associated with severe pneumonia. To this end, the models were exposed to the mixture of exotoxins produced by S. aureus strains isolated from patients with varying severity of lung infection, namely necrotizing pneumonia or lung empyema, or to purified toxins. The necrotizing pneumonia strains secreted high levels of alpha-toxin and Panton-Valentine leukocidin (PVL), and triggered high cytotoxicity, inflammation, necrosis and loss of E-cadherin from the lung epithelium. In contrast, the lung empyema strain produced moderate levels of PVL, but negligible amounts of alpha-toxin, and triggered limited tissue damage. alpha-toxin had a direct damaging effect on the epithelium, as verified using toxin-deficient mutants and pure alpha-toxin. Moreover, PVL contributed to pathology through the lysis of neutrophils. A combination of alpha-toxin and PVL resulted in the most severe epithelial injury. In addition, toxin-induced release of pro-inflammatory mediators from lung tissue models resulted in enhanced neutrophil migration. Using a collection of 31 strains from patients with staphylococcal pneumonia revealed that strains producing high levels of alpha-toxin and PVL were cytotoxic and associated with fatal outcome. Also, the strains that produced the highest toxin levels induced significantly greater epithelial disruption. Of importance, toxin-mediated lung epithelium destruction could be inhibited by polyspecific intravenous immunoglobulin containing antibodies against alpha-toxin and PVL. This study introduces a novel model system for study of staphylococcal pneumonia in a human setting. The results reveal that the combination and levels of alpha-toxin and PVL correlate with tissue pathology and clinical outcome associated with pneumonia.

Transgenic Drosophila model to study apolipoprotein E4-induced neurodegeneration

The epsilon 4 isoform of apolipoprotein E (ApoE4) that is involved in neuron-glial lipid metabolism has been demonstrated as the main genetic risk factor in late-onset of Alzheimer's disease. However, the mechanism underlying ApoE4-mediated neurodegeneration remains unclear. We created a transgenic model of neurodegenerative disorder by expressing epsilon 3 and epsilon 4 isoforms of human ApoE in the Drosophila melanogaster. The genetic models exhibited progressive neurodegeneration, shortened lifespan and memory impairment. Genetic interaction studies between amyloid precursor protein and ApoE in axon pathology of the disease revealed that over expression of hApoE in Appl-expressing neurons of Drosophila brain causes neurodegeneration. Moreover, acute oxidative damage in the hApoE transgenic flies triggered a neuroprotective response of hApoE3 while chronic induction of oxidative damage accelerated the rate of neurodegeneration. This Drosophila model may facilitate analysis of the molecular and cellular events implicated in hApoE4 neurotoxicity. (C) 2015 Elsevier B.V. All rights reserved.

Transgenic Drosophila model to study apolipoprotein E4-induced neurodegeneration

The epsilon 4 isoform of apolipoprotein E (ApoE4) that is involved in neuron-glial lipid metabolism has been demonstrated as the main genetic risk factor in late-onset of Alzheimer's disease. However, the mechanism underlying ApoE4-mediated neurodegeneration remains unclear. We created a transgenic model of neurodegenerative disorder by expressing epsilon 3 and epsilon 4 isoforms of human ApoE in the Drosophila melanogaster. The genetic models exhibited progressive neurodegeneration, shortened lifespan and memory impairment. Genetic interaction studies between amyloid precursor protein and ApoE in axon pathology of the disease revealed that over expression of hApoE in Appl-expressing neurons of Drosophila brain causes neurodegeneration. Moreover, acute oxidative damage in the hApoE transgenic flies triggered a neuroprotective response of hApoE3 while chronic induction of oxidative damage accelerated the rate of neurodegeneration. This Drosophila model may facilitate analysis of the molecular and cellular events implicated in hApoE4 neurotoxicity. (C) 2015 Elsevier B.V. All rights reserved.