Harish Changotra

Murine Norovirus 1 Infection Is Associated with Histopathological Changes in Immunocompetent Hosts, but Clinical Disease Is Prevented by STAT1-Dependent Interferon Responses

ABSTRACT Human noroviruses are the major cause of nonbacterial epidemic gastroenteritis worldwide. However, little is known regarding their pathogenesis or the immune responses that control them because until recently there has been no small animal model or cell culture system of norovirus infection. We recently reported the discovery of the first murine norovirus, murine norovirus 1 (MNV-1), and its cultivation in macrophages and dendritic cells in vitro. We further defined interferon receptors and the STAT-1 molecule as critical in both resistance to MNV-1-induced disease in vivo and control of virus growth in vitro. To date, neither histopathological changes upon infection nor viral replication in wild-type mice has been shown. Here we extend our studies to demonstrate that MNV-1 replicates and rapidly disseminates to various tissues in immunocompetent mice and that infection is restricted by STAT1-dependent interferon responses at the levels of viral replication and virus dissemination. Infection of wild-type mice is associated with histopathological alterations in the intestine (mild inflammation) and the spleen (red pulp hypertrophy and white pulp activation); viral dissemination to the spleen, liver, lung, and lymph nodes; and low-level persistent infection in the spleen. STAT-1 inhibits viral replication in the intestine, prevents virus-induced apoptosis of intestinal cells and splenocytes, and limits viral dissemination to peripheral tissues. These findings demonstrate that murine norovirus infection of wild-type mice is associated with initial enteric seeding and subsequent extraintestinal spread, and they provide mechanistic evidence of the role of STAT-1 in controlling clinical norovirus-induced disease.

TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle-wasting cytokine

TWEAK cytokine has been implicated in several biological responses including inflammation, angiogenesis, and osteoclastogenesis. We have investigated the role of TWEAK in regulating skeletal muscle mass. Addition of soluble TWEAK protein to cultured myotubes reduced the mean myotube diameter and enhanced the degradation of specific muscle proteins such as CK and MyHCf. The effect of TWEAK on degradation of MyHCf was stronger than its structural homologue, TNF‐α. TWEAK increased the ubiquitination of MyHCf and the transcript levels of atrogin‐1 and MuRF1 ubiquitin ligases. TWEAK inhibited phosphor‐ylation of Akt kinase and its downstream targets GSK‐3β, FOXO1, mTOR, and p70S6K Furthermore, TWEAK increased the activation of NF‐κB transcription factor in myotubes. Adenoviral‐mediated overexpression of IκBαΔN (a degradation‐resistant mutant of NF‐κB inhibitory protein ΙκBα) in myotubes blocked the TWEAK‐induced degradation of MyHCf. Chronic administration of TWEAK in mice resulted in reduced body and skeletal muscle weight with an associated increase in the activity of ubiquitin‐proteasome system and NF‐κB. Finally, muscle‐specific transgenic overexpression of TWEAK decreased the body and skeletal muscle weight in mice. Collectively, our data suggest that TWEAK induces skeletal muscle atrophy through inhibition of the PI3K/Akt signaling pathway and activation of the ubiquitin‐proteasome and NF‐kB systems.—Dogra, C., Changotra, H., Wedhas, N., Qin, X., Wergedal, J. E., Kumar, A. TNF‐related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle‐wasting cytokine. FASEB J. 21, 1857–1869 (2007)

Tumor Necrosis Factor-like Weak Inducer of Apoptosis Inhibits Skeletal Myogenesis through Sustained Activation of Nuclear Factor-κB and Degradation of MyoD Protein

In this study we have investigated the effect and the mechanisms by which tumor necrosis factor-like weak inducer of apoptosis (TWEAK) modulates myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK inhibited their differentiation evident by a decrease in the expression of creatine kinase, myosin heavy chain-fast twitch, myogenin, and the formation of multinucleated myotubes. TWEAK also inhibited the differentiation of mouse primary myoblasts. Conversely, the proliferation of C2C12 myoblasts and the expression of a cell-cycle regulator cyclin D1 were increased in response to TWEAK treatment. Inhibition of cellular proliferation using hydroxyurea only partially reversed the inhibitory effect of TWEAK on myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK resulted in the activation of nuclear factor-κB (NF-κB), the (IkappaB) IκB kinase (IKK) complex, and the phosphorylation and degradation of IκBα protein. Inhibition of NF-κB activity by overexpression of a dominant negative mutant of IκBα (IκBαΔN) significantly increased the myogenic differentiation in TWEAK-treated C2C12 cultures. Furthermore, overexpression of a dominant negative mutant of IKKβ (IKKβK44A) but not IKKα (IKKαK44M) reversed the inhibitory effect of TWEAK on myogenesis. TWEAK inhibited the expression of myogenic regulatory factors MyoD and myogenin and also induced the degradation of MyoD protein. Finally, inhibition of NF-κB activation through overexpression of IKKβK44A prevented the degradation of MyoD protein. Overall, our data suggest that TWEAK inhibits myogenesis through the activation of NF-κB signaling pathway and degradation of MyoD protein.

Regulation of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappa B signaling pathways in dystrophin-deficient skeletal muscle in response to mechanical stretch

Phosphatidylinositol 3‐kinase (PI3K)/Akt and nuclear factor‐kappa B (NF‐κB) signaling pathways play a critical role in mediating survival signals. In this study we have investigated how loss of dystrophin (the primary cause of Duchenne muscular dystrophy) modulates the activation of PI3K/Akt and NF‐κB signaling pathways in skeletal muscle in response to mechanical stimulation. Activation of Akt was significantly higher in diaphragm muscle from dystrophin‐deficient mdx mice compared to normal mice at both prenecrotic and necrotic states. Higher activation of Akt was also observed in cultured dystrophin‐deficient primary myotubes differentiated in vitro. Application of passive mechanical stretch ex vivo synergistically increased the activation of Akt in diaphragm of mdx mice. Stretch‐induced activation of PDK‐1 and PI3K were also higher in diaphragm of mdx mice compared to normal mice. Pretreatment of diaphragm with PI3K inhibitor LY294002 blocked the activation of Akt in normal and mdx mice. Higher activation of Akt was associated with increased phosphorylation of its downstream targets glycogen synthase kinase 3β (GSK3β), FKHR, and mammalian target of rapamycin (mTOR). Treatment of diaphragm muscle with LY294002 inhibited the stretch‐induced activation of IkappaB (IκB) kinase (IKK) and NF‐κB transcription factor in normal and mdx mice. Mechanical stretch also reduced the interaction of HDAC1 with RelA subunit of NF‐κB in diaphragm muscle. Finally, cellular levels of Bcl‐2, cIAP1, and integrin β1 and activation of integrin linked kinase were higher in diaphragm muscle of mdx mice compared to normal mice. Taken together, our data suggest that loss of dystrophin and/or mechanical stretch results in the up‐regulation of PI3K/Akt and NF‐κB signaling pathways in skeletal muscle. J. Cell. Physiol. 208: 575–585, 2006.

Type I and Type II Interferons Inhibit the Translation of Murine Norovirus Proteins

ABSTRACT Human noroviruses are responsible for more than 95% of nonbacterial epidemic gastroenteritis worldwide. Both onset and resolution of disease symptoms are rapid, suggesting that components of the innate immune response are critical in norovirus control. While the study of the human noroviruses has been hampered by the lack of small animal and tissue culture systems, our recent discovery of a murine norovirus (MNV) and its in vitro propagation have allowed us to begin addressing norovirus replication strategies and immune responses to norovirus infection. We have previously demonstrated that interferon responses are critical to control MNV-1 infection in vivo and to directly inhibit viral replication in vitro. We now extend these studies to define the molecular basis for interferon-mediated inhibition. Viral replication intermediates were not detected in permissive cells pretreated with type I interferon after either infection or transfection of virion-associated RNA, demonstrating a very early block to virion production that is after virus entry and uncoating. A similar absence of viral replication intermediates was observed in infected primary macrophages and dendritic cells pretreated with type I IFN. This was not due to degradation of incoming genomes in interferon-pretreated cells since similar levels of genomes were present in untreated and pretreated cells through 6 h of infection, and these genomes retained their integrity. Surprisingly, this block to the translation of viral proteins was not dependent on the well-characterized interferon-induced antiviral molecule PKR. Similar results were observed in cells pretreated with type II interferon, except that the inhibition of viral translation was dependent on PKR. Thus, both type I and type II interferon signaling inhibit norovirus translation in permissive myeloid cells, but they display distinct dependence on PKR for this inhibition.

Depression mediates impaired glucose tolerance and cognitive dysfunction: A neuromodulatory role of rosiglitazone

Comorbidity of depression and diabetes is a serious risk factor worsening the complications such as cognitive function and locomotion. Treatment under this condition becomes extremely complicated. Insulin signaling and autophagy pathways are involved in modulation of learning and memory. Rosiglitazone (ROSI) ameliorate cognitive deficit associated with depression and insulin resistance. In the present study, we investigated the effect of ROSI against chronic unpredictable stress (CUS) induced depression as a risk factor for diabetes and behavioral dysfunctions. Adult male Swiss albino mice were exposed to CUS alongside ROSI (5mg/kg/day) treatment for 21days. Thereafter, animals were subjected to different behavioral studies to assess depressive like behavior, cognition and locomotion. The effect of ROSI on insulin signaling, autophagy and apoptosis were evaluated in the hippocampus. CUS resulted in depressive like behavior, cognitive impairment and hypolocomotion associated with oxidative stress, impaired glucose tolerance and hypercorticosteronemia. CUS significantly impaired hippocampal insulin signaling, membrane translocation of glucose transporter type 4 (GLUT4) as well as decreased the expression of autophagy5, autophagy7, B-cell lymphoma 2 and apoptosis inhibitory protein 2. ROSI significantly reduced depressive like behavior, postprandial blood glucose, hypercorticosteronemia, oxidative and inflammatory stress, and apoptosis in stressed mice. Moreover, ROSI treatment effectively improved hippocampal insulin signaling, GLUT4 membrane translocation and cognitive performance in depressed mice. ROSI administration might prove to be effective for neurological disorders associated with depressive like behavior and impaired glucose tolerance.

Rotaviruses: Is their surveillance needed?

Rotaviruses, a major cause of gastroenteritis in children worldwide accounts for around 0.5 million deaths annually. Owing to their segmented genome and frequently evolving capability, these display a wide variation in their genotypes. In addition to commonly circulating genotypes (G1, G2, G3, G4, G9, P[4] and P[8]), a number of infrequent genotypes are being continuously reported to infect humans. These viral strains exhibit variation from one geographical setting to another in their distribution. Though the introduction of vaccines (RotaTeq and Rotarix) proved to be very effective in declining rotavirus associated morbidity and mortality, the number of infections remained same. Unusual genotypes significantly contribute to the rotavirus associated diarrhoeal burden, may reduce the efficacy of the vaccines in use and hence vaccinated individuals may not be benefited. Vaccine introduction may bring about a notable impact on the distribution and prevalence of these viruses due to selection pressure. Moreover, there is a sudden emergence of G2 and G3 in Brazil and United States, respectively, during the years 2006-2008 post-vaccination introduction; G9 and G12 became predominant during the years 1986 through 1998 before the vaccine introduction and now are commonly prevalent strains; and disparity in the predominance of strains after introduction of vaccines and their natural fluctuations poses a vital question on the impact of vaccines on rotavirus strain circulation. This interplay between vaccines and rotavirus strains is yet to be explored, but it certainly enforces the need to continuously monitor these changes in strains prevalence in a particular region. Furthermore, these fluctuations should be considered while administration or development of a vaccine, if rotavirus associated mortality is ever to be controlled.

Unc-51 like kinase 1 (ULK1) in silico analysis for biomarker identification: a vital component of autophagy.

Autophagy is a degradation pathway involving lysosomal machinery for degradation of damaged organelles like the endoplasmic reticulum and mitochondria into their building blocks to maintain homeostasis within the cell. ULK1, a serine/threonine kinase, is conserved across species, from yeasts to mammals, and plays a central role in autophagy pathway. It receives signals from upstream modulators such as TIP60, mTOR and AMPK and relays them to its downstream substrates like Ambra1 and ZIP kinase. The activity of this complex is regulated through protein-protein interactions and post-translational modifications. Applying in silico analysis we identified (i) conserved patterns of ULK1 that showed its evolutionary relationship between the species which were closely related in a family compared to others. (ii) A total of 23 TFBS distributed throughout ULK1 and nuclear factor (erythroid-derived) 2 (NFE2) is of utmost significance because of its high importance rate. NEF2 has already been shown experimentally to play a role in the autophagy pathway. Most of these were of zinc coordinating class and we suggest that this information could be utilized to modulate this pathway by modifying interactions of these TFs with ULK1. (iii) CATTT haplotype was prominently found with frequency 0.774 in the studied population and nsSNPs which could have harmful effect on ULK1 protein and these could further be tested. (iv) A total of 83 phosphorylation sites were identified; 26 are already known and 57 are new that include one at tyrosine residue which could further be studied for its involvement in ULK1 regulation and hence autophagy. Furthermore, 4 palmitoylation sites at positions 426, 927, 1003 and 1049 were also found which could further be studied for protein-protein interactions as well as in trafficking.

Predominance of unusual rotavirus G1P[6] strain in North India: An evidence from hospitalized children and adult diarrheal patients

Group A Rotavirus remains the leading cause of gastroenteritis in children and accounts for 0.2 million fatalities each year; out of which, approximately 47,100 deaths occur in India. In adults also, rotavirus is reported to be responsible for diarrhea severe enough to require hospitalizations. India has recently introduced rotavirus vaccine in the Universal Immunization Programme and Himachal Pradesh became the first Indian state to implement this project. This study is an attempt to provide the pre-vaccination data on rotavirus gastroenteritis burden and circulating genotypes in Himachal Pradesh, India. A total of 607 faecal specimens (247 children ≤5years, 50 older children and 310 adults) from hospitalized diarrheal patients from Himachal Pradesh, India were screened for rotavirus using ELISA and RT-PCR. The positive samples were further G/P genotyped using semi-nested PCR. Rotavirus was detected in 25.2% and 28.3% of samples with ELISA and RT-PCR, respectively. In children, rotavirus frequency was significantly high with positivity in 49.0% cases whereas 14.0% adult samples have rotavirus in them. Genotyping of the positive samples revealed predominance of G1 (66.0%) and P[6] (66.7%) genotypes. The most common G and P combination was G1P[6] (62.8%) followed by G1P[8] (16.5%), G9P[6] (7.4%) and G12P[6] (5.0%). Molecular analysis reveals the belonging of P[6] strains in Lineage 1a. This pre-vaccination data on rotavirus prevalence and diversity would be helpful for assessing the affect of vaccination on the disease burden and its comparison with post-vaccination data of circulating genotypes would help in studying the effect on diversity of rotavirus strains possibly due to vaccine selection pressure.

Investigating regulatory signatures of human autophagy related gene 5 (ATG5) through functional in silico analysis

Autophagy is an essential, homeostatic process which removes damaged cellular proteins and organelles for cellular renewal. ATG5, a part of E3 ubiquitin ligase-like complex (Atg12-Atg5/Atg16L1), is a key regulator involved in autophagosome formation - a crucial phase of autophagy. In this study, we used different in silico methods for comprehensive analysis of ATG5 to investigate its less explored regulatory activity. We have predicted various physico-chemical parameters and two possible transmembrane models that helped in exposing its functional regions. Twenty four PTM sites and 44 TFBS were identified which could be targeted to modulate the autophagy pathway. Furthermore, LD analysis identified 3 blocks of genotyped SNPs and 2 deleterious nsSNPs that may have damaging impact on protein function and thus could be employed for carrying genome-wide association studies. In conclusion, the information obtained in this study could be helpful for better understanding of regulatory roles of ATG5 and provides a base for its implication in population-based studies.