Search

Hepatic dysfunction is a feature of dengue virus (DENV) infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show apoptosis, which relates to the pathogenesis of DENV infection. However, how DENV induced liver injury is not fully understood. In this study, we aim to identify the factors that influence cell death by employing an apoptosis-related siRNA library screening. Our results show the effect of 558 gene silencing on caspase 3-mediated apoptosis in DENV-infected Huh7 cells. The majority of genes that contributed to apoptosis were the apoptosis-related kinase enzymes. Tumor necrosis factor superfamily member 12 (TNFSF12), and sphingosine kinase 2 (SPHK2), were selected as the candidate genes to further validate their influences on DENV-induced apoptosis. Transfection of siRNA targeting SPHK2 but not TNFSF12 genes reduced apoptosis determined by Annexin V/PI staining. Knockdown of SPHK2 did not reduce caspase 8 activity; however, did significantly reduce caspase 9 activity, suggesting its involvement of SPHK2 in the intrinsic pathway of apoptosis. Treatment of ABC294649, an inhibitor of SPHK2, reduced the caspase 3 activity, suggesting the involvement of its kinase activity in apoptosis. Knockdown of SPHK2 significantly reduced caspase 3 activity not only in DENV-infected Huh7 cells but also in DENV-infected HepG2 cells. Our results were consistent across all of the four serotypes of DENV infection, which supports the pro-apoptotic role of SPHK2 in DENV-infected liver cells.

Embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells are attractive in vitro models of vascular development, therapeutic angiogenesis, and tissue engineering. However, distinct ESC and iPS cell lines respond differentially to the same microenvironmental factors. Developing improved/optimized differentiation methodologies tailored/applicable in a number of distinct iPS and ESC lines remains a challenge in the field. Currently published methods for deriving endothelial cells (EC) robustly generate high numbers of endothlelial progenitor cells (EPC) within a week, but their maturation to definitive EC is much more difficult, taking up to 2 months and requiring additional purification. Therefore, we set out to examine combinations/levels of putative EC induction factors-utilizing our stage-specific chemically-defined derivation methodology in 4 ESC lines including: kinetics, cell seeding density, matrix signaling, as well as medium treatment with vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The results indicate that temporal development in both early and late stages is the most significant factor generating the desired cells. The generation of early Flk-1+/KDR+ vascular progenitor cells (VPC) from pluripotent ESC is directed predominantly by high cell seeding density and matrix signaling from fibronectin, while VEGF supplementation was NOT statistically significant in more than one cell line, especially with fibronectin matrix which sequesters autocrine VEGF production by the differentiating stem cells. Although some groups have shown that the GSK3-kinase inhibitor (CHIR) can facilitate EPC fate, it hindered the generation of KDR+ cells in our preoptimized medium formulations. The methods summarized here significantly increased the production of mature vascular endothelial (VE)-cadherin+ EC, with up to 93% and 57% purity from mouse and human ESC, respectively, before VE-cadherin+ EC purification.

This paper depicts the first report from an Indian population on the association between the variant Arg399Gln of XRCC1 locus in the DNA repair system and schizophrenia, the debilitating disease that affects 1% of the world population. Genotypic analysis of a total of 523 subjects (260 patients and 263 controls) revealed an overwhelming presence of Gln399Gln in the case subjects against the controls (P < 0.0068), indicating significant level of association of this nsSNP with schizophrenia; the Gln399 allele frequency was also perceptibly more in cases than in controls (p < 0.003; OR = 1.448). The results of the genotypic studies were further validated using pathogenicity and stability prediction analysis employing computational tools [I-Mutant Suite, iStable, PolyPhen2, SNAP, and PROVEAN], with a view toassess the magnitude of deleteriousness of the mutation. The pathogenicity analysis reveals that the nsSNP could be deleterious inasmuch as it could affect the functionality of the gene, and interfere with protein function. Molecular dynamics simulation of 60ns was performed using GROMACS to analyse structural change due to a mutation (Arg399Gln) that was never examined before. RMSD, RMSF, hydrogen bonds, radius of gyration and SASA analysis showedthe existence of asignificant difference between the native and the mutant protein. The present study gives astrong indication that the XRCC1 locus deserves serious attention, as it could be a potential candidatecontributing to the etio-pathogenesis of the disease.

Dengue virus (DENV) infection causes organ injuries, and the liver is one of the most important sites of DENV infection, where viral replication generates a high viral load. The molecular mechanism of DENV-induced liver injury is still under investigation. The mitogen activated protein kinases (MAPKs), including p38 MAPK, have roles in the hepatic cell apoptosis induced by DENV. However, the in vivo role of p38 MAPK in DENV-induced liver injury is not fully understood. In this study, we investigated the role of SB203580, a p38 MAPK inhibitor, in a mouse model of DENV infection. Both the hematological parameters, leucopenia and thrombocytopenia, were improved by SB203580 treatment and liver transaminases and histopathology were also improved. We used a real-time PCR microarray to profile the expression of apoptosis-related genes. Tumor necrosis factor α, caspase 9, caspase 8, and caspase 3 proteins were significantly lower in the SB203580-treated DENV-infected mice than that in the infected control mice. Increased expressions of cytokines including TNF-α, IL-6 and IL-10, and chemokines including RANTES and IP-10 in DENV infection were reduced by SB203580 treatment. DENV infection induced the phosphorylation of p38MAPK, and its downstream signals including MAPKAPK2, HSP27 and ATF-2. SB203580 treatment did not decrease the phosphorylation of p38 MAPK, but it significantly reduced the phosphorylation of MAPKAPK2, HSP27, and ATF2. Therefore, SB203580 modulates the downstream signals to p38 MAPK and reduces DENV-induced liver injury.

N. meningitidis induces extensive gene expression changes in human monocytes, suggesting that complex networks of signaling pathways are activated during meningococcal sepsis. These effects are modulated by the anti-inflammatory cytokine interleukin-10 (IL-10). To further study changes in signal transduction suggested by mRNA data, we used kinase substrate arrays to identify composite kinase activities induced by lysates from a primary human monocyte model system. Cell lysates were prepared from monocytes treated with the following experimental conditions: 106 N. meningitidis/mL, 25 ng/mL IL-10, 106 N. meningitidis/mL in combination with 25 ng/mL IL-10, and vehicle. Lysates were subjected to kinase activity profiling with Tyrosine Kinase PamChip® arrays containing 144 kinase peptide substrates. In our experimental model, we were not able to detect a statistically significant large-scale change in ex vivo array peptide phosphorylation by lysates from monocytes treated for 15 minutes. Targets of the IL-10 anti-inflammatory response were not identified. A profound inhibition of array peptide phosphorylation by monocytes treated for 60 minutes was identified, suggesting low activity of a large number of kinases associated with different signaling pathways and immune cell functions, including STAT3 activity, Nf-κB and VEGF signaling, and PTEN signaling activity. The peptide representing ZBTB16, which was reduced in phosphorylation by lysates from all three experimental conditions, was in Ingenuity Pathway Analysis identified to be linked to reduced cytokine release and mRNA levels of tumor necrosis factor (TNF), IL-6, and CXCL10. Further studies should investigate changes in tyrosine kinase-mediated signal transduction in human immune cells, in order to evaluate the potential clinical application of kinome profiling in the study of systemic inflammatory responses to pathogens.

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1−/− CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

Herbal remedies are increasingly being recognised in recent years as alternative medicine for a number of diseases including cancer. Curcuma longa L., commonly known as turmeric is used as a culinary spice in India and in many Asian countries has been attributed to lower incidences of gastrointestinal cancers. Curcumin, a secondary metabolite isolated from the rhizomes of this plant has been shown to have significant anticancer properties, in addition to antimalarial and antioxidant effects. We sequenced the transcriptome of the rhizome of the 3 varieties of Curcuma longa L. using Illumina reversible dye terminator sequencing followed by de novo transcriptome assembly. Multiple databases were used to obtain a comprehensive annotation and the transcripts were functionally classified using GO, KOG and PlantCyc. Special emphasis was given for annotating the secondary metabolite pathways and terpenoid biosynthesis pathways. We report for the first time, the presence of transcripts related to biosynthetic pathways of several anti-cancer compounds like taxol, curcumin, and vinblastine in addition to anti-malarial compounds like artemisinin and acridone alkaloids, emphasizing turmeric's importance as a highly potent phytochemical. Our data not only provides molecular signatures for several terpenoids but also a comprehensive molecular resource for facilitating deeper insights into the transcriptome of C. longa.

Bioinformatics of disordered proteins is especially challenging given high mutation rates for homologous proteins and that functionality may not be strongly related to sequence. Here we have performed a novel bioinformatic analysis, based on the spatial clustering of physically relevant features such as binding motifs and charges within disordered proteins, on thousands of Nuclear Pore Complex (NPC) FG motif containing proteins (FG nups). The biophysical mechanism by which FG nups regulate nucleocytoplasmic transport has remained elusive. Our analysis revealed a set of highly conserved spatial features in the sequence structure of individual FG nups, such as the separation, localization, and ordering of FG motifs and charged residues along the protein chain. These functionally conserved features provide insight into the particular biophysical mechanisms responsible for regulation of nucleocytoplasmic traffic in the NPC, strongly constraining current models. Additionally this method allows us to identify potentially functionally analogous disordered proteins across distantly related species.

The present paper describes a detailed study on the distribution of radionuclides along Chavara - Neendakara placer deposit, a high background radiation area (HBRA) along the Southwest coast of India (Kerala). Judged from our studies using HPGe gamma spectrometric detector, it becomes evident that Uranium ((238)U), Thorium ((232)Th) and Potassium ((40)K) are the major sources for radioactivity prevailing in the area. Our statistical analyses reveal the existence of a high positive correlation between (238)U and (232)Th, implicating that the levels of these elements are interdependent. Our SEM-EDAX analyses reveal that titanium (Ti) and zircon (Zr) are the major trace elements in the sand samples, followed by aluminum, copper, iron, ruthenium, magnesium, calcium, sulphur and lead. This is first of its kind report on the radiation hazard indices on this placer deposit. The average absorbed dose rates (9795 nGy h(-1)) computed from the present study is comparable with the top-ranking HBRAs in the world, thus offering the Chavara-Neendakara placer the second position, after Brazil; pertinently, this value is much higher than the World average. The perceptibly high absorbed gamma dose rates, entrained with the high annual external effective dose rates (AEED) and average annual gonadal dose equivalent (AGDE) values existing in this HBRA, encourage us to suggest for a candid assessment of the impact of the background radiation, if any, on the organisms that inhabit along this placer deposit. Future research could effectively address the issue of the possible impact of natural radiation on the biota inhabiting this HBRA.

Although targeting of cancer cells using drug-delivering nanocarriers holds promise for improving therapeutic agent specificity, the strategy of maximizing ligand affinity for receptors overexpressed on cancer cells is suboptimal. To determine design principles that maximize nanocarrier specificity for cancer cells, we studied a generalized kinetics-based theoretical model of nanocarriers with one or more ligands that specifically bind these overexpressed receptors. We show that kinetics inherent to the system play an important role in determining specificity and can in fact be exploited to attain orders of magnitude improvement in specificity. In contrast to the current trend of therapeutic design, we show that these specificity increases can generally be achieved by a combination of low rates of endocytosis and nanocarriers with multiple low-affinity ligands. These results are broadly robust across endocytosis mechanisms and drug-delivery protocols, suggesting the need for a paradigm shift in receptor-targeted drug-delivery design.

Here we show that bortezomib induces effective proteasome inhibition and accumulation of poly-ubiquitinated proteins in diffuse large B-cell lymphoma (DLBCL) cells. This leads to induction of endoplasmic reticulum (ER) stress as demonstrated by accumulation of the protein CHOP, as well as autophagy, as demonstrated by accumulation of LC3-II proteins. Our data suggest that recruitment of both ubiquitinated proteins and LC3-II by p62 directs ubiquitinated proteins, including I-κBα, to the autophagosome. Degradation of I-κBα results in increased NF-κB nuclear translocation and transcription activity. Since bortezomib treatment promoted I-κBα phosphorylation, ubiquitination and degradation, this suggests that the route of I-κBα degradation was not via the ubiquitin-proteasome degradation system. The autophagy inhibitor chloroquine (CQ) significantly inhibited bortezomib-induced I-κBα degradation, increased complex formation with NF-κB and reduced NF-κB nuclear translocation and DNA binding activity. Importantly, the combination of proteasome and autophagy inhibitors showed synergy in killing DLBCL cells. In summary, bortezomib-induced autophagy confers relative DLBCL cell drug resistance by eliminating I-κBα. Inhibition of both autophagy and the proteasome has great potential to kill apoptosis-resistant lymphoma cells.