Molecular mechanism of methyl jasmonate induced apoptosis in human cancer cells

This study investigated the cytotoxic activity of four plant derived compounds including perillic acid (PA), Perillyl alcohol (POH), cis jasmone (CJ) and methyl jasmonate (MJ) on a variety of human cancer cell lines: Breast (MDA-MB-435, MDA-MB-231, DC4, DB46, T47D, SKBR3 and ZR75-1), Prostate (PC-3 and DU-145) and Lung (A549, H520 and H2170). It was demonstrated that these compounds induce cell cycle arrest and apoptosis by themselves or in combination with other agents. Specifically, MJ decreases membrane fluidity resulting in an activation of tumor necrosis factor receptor 1 (TNFR1) which signals apoptosis via caspase 8 and 2. Combination studies demonstrated that POH, MJ and cisplatin decreased viability, induced cell cycle arrest and activated TNFR1. These results indicate that plant compounds show promise as an alternate treatment option and the combination of these compounds with chemotherapeutic drugs may decrease toxicity and improve efficacy over single agent therapy

Dicer regulates activation of the NLRP3 inflammasome

Inflammation plays a critical role in initiation of adaptive immunity, pathogen clearance and tissue repair. Interleukin (IL)-1β is a potent pro-inflammatory cytokine and therefore its production is tightly regulated: its secretion requires the assembly of a macromolecular protein complex, termed the inflammasome. Aberrant activation of the inflammasome has been linked to debilitating human diseases including chronic inflammatory and autoimmune diseases. Thus, there is a great interest in understanding how inflammasomes are regulated. Here we show that Dicer, an enzyme necessary for the production of mature micro-RNAs (miRNAs), is required for optimal activation of NLRP3 inflammasomes in bone marrow macrophages. Our data indicate that miRNAs may play an important role in promoting inflammasome activation

MicroRNAs Modulate Pathogenesis Resulting from Chlamydial Infection in Mice

Not all women infected with chlamydiae develop upper genital tract disease, but the reason(s) for this remains undefined. Host genetics and hormonal changes associated with the menstrual cycle are possible explanations for variable infection outcomes. It is also possible that disease severity depends on the virulence of the chlamydial inoculum. It is likely that the inoculum contains multiple genetic variants, differing in virulence. If the virulent variants dominate, then the individual is more likely to develop severe disease. Based on our previous studies, we hypothesized that the relative degree of virulence of a chlamydial population dictates the microRNA (miRNA) expression profile of the host, which, in turn, through regulation of the host inflammatory response, determines disease severity. Thus, we infected C57BL/6 mice with two populations of Chlamydia muridarum, each comprised of multiple genetic variants and differing in virulence: an attenuated strain (NiggA) and a virulent strain (NiggV). NiggA and NiggV elicited upper tract pathology in 54% and 91% of mice, respectively. miRNA expression analysis in NiggV-infected mice showed significant downregulation of miRNAs involved in dampening fibrosis (miR-200b, miR-200b-5p, and 200b-3p miR-200a-3p) and in transcriptional regulation of cytokine responses (miR-148a-3p, miR-152-3p, miR-132, and miR-212) and upregulation of profibrotic miRNAs (miR-142, and miR-147). Downregulated miRNAs were associated with increased expression of interleukin 8 (IL-8), CXCL2, IL-1β, tumor necrosis factor alpha (TNF-α), and IL-6. Infection with NiggV but not NiggA led to decreased expression of Dicer and Ago 2, suggesting that NiggV interaction with host cells inhibits expression of the miRNA biogenesis machinery, leading to increased cytokine expression and pathology

Investigation of translocation, DNA unwinding, and protein displacement by NS3h, the helicase domain from the Hepatitis C virus helicase

Helicases are motor proteins that are involved in DNA and RNA metabolism, replication, recombination, transcription and repair. The motors are powered by ATP binding and hydrolysis. Hepatitis C virus encodes a helicase called non-structural protein (NS3). NS3 possesses protease and helicase activities on its N-terminal and C-terminal domains respectively. The helicase domain of NS3 protein is referred as NS3h. In vitro, NS3h catalyzes RNA and DNA unwinding in a 3’ to -5’ direction. The directionality for unwinding is thought to arise in part from the enzyme's ability to translocate along DNA, but translocation has not been shown explicitly. We examined the DNA translocase activity of NS3h by using single-stranded oligonucleotide substrates containing a fluorescent probe on the 5’ end. NS3h can bind to the ssDNA and in the presence of ATP, move towards the 5’-end. When the enzyme encounters the fluorescent probe, a fluorescence change is observed that allows translocation to be characterized. Under conditions that favor binding of one NS3h per DNA substrate (100 nM NS3h, 200 nM oligonucleotide) we find that NS3h translocates on ssDNA at a rate of 46 ± 5 nt s−1 and that it can move for 230 ± 60 nt before dissociating from the DNA. The translocase activity of some helicases is responsible for displacing proteins that are bound to DNA. We studied protein displacement by using a ssDNA oligonucleotide covalently linked to biotin on the 5’-end. Upon addition of streptavidin, a ‘protein-block’ was placed in the pathway of the helicase. Interestingly, NS3h was unable to displace streptavidin from the end of the oligonucleotide, despite its ability to translocate along the DNA. The DNA unwinding activity of NS3h was examined using a 22 bp duplex DNA substrate under conditions that were identical to those used to study translocation. NS3h exhibited little or no DNA unwinding under single cycle conditions, supporting the conclusion that NS3h is a relatively poor helicase in its monomeric form, as has been reported. In summary, NS3h translocates on ssDNA as a monomer, but the translocase activity does not correspond to comparable DNA unwinding activity or protein-displacement activity under identical conditions

The DNA Sensor, Cyclic GMP–AMP Synthase, Is Essential for Induction of IFN-β during Chlamydia trachomatis Infection

IFNβ has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFNβ expression during chlamydial infection. We determined that TREX-1, a host 3’to 5’ exonuclease, reduced IFNβ expression significantly during chlamydial infection using siRNA and gene knock out fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cGAS has been shown to bind cytosolic DNA to generate cGAMP, which binds to the signaling adaptor STING to induce IFNβ expression. We determined that cGAS is required for IFNβ expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFNβ, co-culture of cells depleted for either STING or cGAS rescued IFNβ expression. These data demonstrate that cGAMP produced in infected cGAS+STING− cells can migrate into adjacent cells via gap junctions to function in trans in cGAS−STING+ cells. Further, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated-DNA sensing directs IFNβ expression during C.trachomatis infection and suggests that effectors from infected cells can directly upregulate IFNβ expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis

Endocervical miRNA Expression Profiles in Women Positive for Chlamydia trachomatis with Clinical Signs and/or Symptoms Are Distinct from Those in Women Positive for Chlamydia trachomatis without Signs and Symptoms

Chlamydia trachomatis is the leading cause of sexually transmitted infections that may progress to pelvic inflammatory disease and infertility. No effective vaccine exists for Chlamydia, nor are there biomarkers available that readily predict disease progression. In this cross-sectional pilot study, we recruited symptomatic and asymptomatic women with C. trachomatis (CT) infection and asymptomatic, uninfected control women from an urban sexually transmitted disease clinic to determine if there were differences in microRNA (miRNA) expression. Infected women with signs and/or symptoms (CTSS) have distinct miRNA profiles compared to asymptomatic infected women (CTNS). In the CTSS group, miR-142 and -147 showed 2.2- to 6.9-fold increases in expression. In the CTNS group, miR-449c, -6779, -519d, -449a, and -2467 showed 3.9- to 9.0-fold increases in expression. In the CTNS group, cyclins and cell cycle regulation and IL-17 pathways were likely downregulated, while the same signaling pathways were upregulated in the CTSS group. In addition, in the CTSS group, additional inflammatory pathways associated with TNFR1 and IL-8 appear to be upregulated. The miRNA expression patterns differ between CT-infected symptomatic and asymptomatic women, and these differences may warrant further study