A Comprehensive Proteomic Analysis of the Type III Secretome of Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease. Central to their pathogenesis is a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). The T3SS is used to inject both LEE- and non-LEE-encoded effector proteins into the host cell, where these effectors modulate host signaling pathways and immune responses. Identifying the effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Here we analyzed the type III secretome of C. rodentium using the highly sensitive and quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometry. This approach not only confirmed nearly all known secreted proteins and effectors previously identified by conventional biochemical and proteomic techniques, but also identified several new secreted proteins. The T3SS-dependent secretion of these new proteins was validated, and five of them were translocated into cultured cells, representing new or additional effectors. Deletion mutants for genes encoding these effectors were generated in C. rodentium and tested in a murine infection model. This study comprehensively characterizes the type III secretome of C. rodentium, expands the repertoire of type III secreted proteins and effectors for the A/E pathogens, and demonstrates the simplicity and sensitivity of using SILAC-based quantitative proteomics as a tool for identifying substrates for protein secretion systems.     

Synthesis, biological evaluation and molecular docking of aryl hydrazines and hydrazides for anticancer activity

Aryl hydrazine and hydrazide analogues were synthesized based on p-tolyl hydrazine, isolated as a breakdown product of a secondary metabolite from the mushroom, Agaricus bisporus, and tested to be highly active molecule than 5-fluorouracil in in vitro anticancer studies. The synthesized analogues were tested for anticancer activity using NCI protocol. Anolgues 12 and 15 emerged as molecules with significant in vitro anticancer activity. Molecular docking study revealed the binding orientations of aryl hydrazines and hydrazides analogues in the active sites of thymidylate synthase.     

Structure–toxicity relationship of phenolic analogs as anti-melanoma agents: An enzyme directed prodrug approach

The aim of this study was to identify a phenolic prodrug compound that is minimally metabolized by rat liver microsomes, but yet could form quinone reactive intermediates in melanoma cells as a result of its bioactivation by tyrosinase. In current work, we investigated 24 phenolic compounds for their metabolism by tyrosinase, rat liver microsomes and their toxicity towards murine B16-F0 and human SK-MEL-28 melanoma cells. A linear correlation was found between toxicities of phenolic analogs towards SK-MEL-28 and B16-F0 melanoma cells, suggesting similar mechanisms of toxicity in both cell lines. 4-HEB was identified as the lead compound. 4-HEB (IC₅₀ 48 h, 75 μM) showed selective toxicity towards five melanocytic melanoma cell lines SK-MEL-28, SK-MEL-5, MeWo, B16-F0 and B16-F10, which express functional tyrosinase, compared to four non-melanoma cells lines SW-620, Saos-2, PC3 and BJ cells and two amelanotic SK-MEL-24, C32 cells, which do not express functional tyrosinase. 4-HEB caused significant intracellular GSH depletion, ROS formation, and showed significantly less toxicity to tyrosinase specific shRNA transfected SK-MEL-28 cells. Our findings suggest that presence of a phenolic group in 4-HEB is critical for its selective toxicity towards melanoma cells.     

The dazzling array of basal branches in the mtDNA macrohaplogroup M from India as inferred from complete genomes

Many efforts based on complete mitochondrial DNA (mtDNA) genomeshave been made to depict the global mtDNA landscape, but thephylogeny of Indian macrohaplogroup M has not yet been resolvedin detail. To fill this lacuna, we took the same strategy asin our recent analysis of Indian mtDNA macrohaplogroup N andselected 56 mtDNAs from over 1,200 samples across India forcomplete sequencing, with the intention to cover all Indianautochthonous M lineages. As a result, the phylogenetic statusof previously identified haplogroups based on control-regionand/or partial coding-region information, such as M2, M3, M4,M5, M6, M30, and M33, was solidified or redefined here. Moreover,seven novel basal M haplogroups (viz., M34–M40) were identified,and yet another five singular branches of the M phylogeny werediscovered in the present study. The comparison of matrilinealcomponents among India, East Asia, Southeast Asia, and Oceaniaat the deepest level yielded a star-like and nonoverlappingpattern, reflecting a rapid mode of modern human dispersal alongthe Asian coast after the initial "out-of-Africa" event.     

Differential effects of insulin-like growth factor-1 and atheroma-associated cytokines on cell proliferation and apoptosis in plaque smooth muscle cells of symptomatic and asymptomatic patients with carotid stenosis

Morbidity and mortality from atherosclerosis are associated with complicated atherosclerotic lesions due to plaque rupture, which is regulated by a balance between proliferation and apoptosis of vascular smooth muscle cells (VSMC). We examined insulin-like growth factor-1 (IGF-1)-induced survival of plaque VSMC from carotid endarterectomy specimens and investigated the underlying cellular mechanisms in the presence and absence of IL-12 and IFN-gamma. Both IL-12 and IFN-gamma were strongly expressed in symptomatic atherosclerotic plaques as compared with asymptomatic plaques. In asymptomatic plaque VSMC, IGF-1 induced the survival and proliferation of VSMC and accelerated VSMC into S-phase. IL-12 or IFN-gamma inhibited proliferation and VSMC were arrested in the G0-G1 phase. IGF-1 markedly inhibited the expression of p27(kip) and p21(cip) and significantly induced cyclin E and cyclin D. Both cytokines by themselves increased the expression of p27(kip) and p21(cip) and inhibited cyclin E and cyclin D. On the contrary, in symptomatic VSMC there was already increased apoptosis of VSMC and there was no significant effect of IGF-1 or inflammatory cytokines on proliferation, apoptosis or the expression of p27(kip) and p21(cip) and cyclin D and E. These data suggest that IGF-1 is more potent in inducing the survival of VSMC from the endarterectomy specimens of asymptomatic patients as compared to that of symptomatic subjects and cytokines associated with atheroma lesions decrease the activity of IGF-1-induced survival in the VSMC of asymptomatic plaques. The different expression and activity of cell cycle regulatory proteins could be responsible for apoptosis of VSMC and destabilization of atherosclerotic plaques.     

Immunobiology of Toll-like receptors: emerging trends

Toll-like receptors (TLR), a family of evolutionarily conserved pathogen recognition receptors, play pivotal role as primary sensors of invading pathogens. TLR identify molecular motifs of infectious agents (pathogen associated molecular patterns) and elicit an effective defensive response against them. Mammalian TLR derive their name from the Drosophila Toll protein, with which they share sequence similarity. So far, expression of 10 TLR is known in humans. The adaptor proteins, myeloid differentiation factor 88 and Toll IL-1 receptor (TIR) domain containing adaptor inducing IFN-beta (TRIF) are the key players in the TLR signalling cascade leading to the activation of nuclear factor (NF)-kappaB and interferon regulatory factor-3, respectively. Targeted manipulation of the TLR signalling pathway has immense therapeutic potential and may eventually prove to be a boon in the development of innovative treatments for diverse disease conditions. There is accumulating evidence that TLR agonists have tremendous potential as novel therapeutic targets. In this review, we have discussed the immunobiology of TLR and emphasize significant advances made within the ever-expanding field of TLR that provide intriguing insights efficacious in unravelling the complexities associated with TLR.