Project description:Primary objectives: The primary objective is to investigate circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
Primary endpoints: circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
Project description:The natural maintenance cycles of many mosquito-borne pathogens require establishment of persistent non-lethal infections in the invertebrate host. The mechanism by which this occurs is not well understood, but we have previously shown that an antiviral response directed by small interfering RNAs (siRNAs) is important in modulating the pathogenesis of alphavirus infections in the mosquito. However, we report here that infection of mosquitoes with an alphavirus also triggers the production of another class of virus-derived small RNAs that exhibit many similarities to ping-pong-dependent piwi-interacting RNAs (piRNAs). However, unlike ping-pong-dependent piRNAs that have been described previously from repetitive elements or piRNA clusters, our work suggests production in the soma and a role for dsRNA in biogenesis. We also present evidence that suggests virus-derived piwi-like RNAs are capable of modulating the pathogenesis of alphavirus infections in dicer-2 null mutant mosquito cell lines defective in viral siRNA production. Overall, our results suggest that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication.
Project description:The natural maintenance cycles of many mosquito-borne pathogens require establishment of persistent non-lethal infections in the invertebrate host. The mechanism by which this occurs is not well understood, but we have previously shown that an antiviral response directed by small interfering RNAs (siRNAs) is important in modulating the pathogenesis of alphavirus infections in the mosquito. However, we report here that infection of mosquitoes with an alphavirus also triggers the production of another class of virus-derived small RNAs that exhibit many similarities to ping-pong-dependent piwi-interacting RNAs (piRNAs). However, unlike ping-pong-dependent piRNAs that have been described previously from repetitive elements or piRNA clusters, our work suggests production in the soma and a role for dsRNA in biogenesis. We also present evidence that suggests virus-derived piwi-like RNAs are capable of modulating the pathogenesis of alphavirus infections in dicer-2 null mutant mosquito cell lines defective in viral siRNA production. Overall, our results suggest that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication. Comparison of virus derived small RNAs in several mosquito cell types
Project description:Several alphaviruses bypass the blood-brain barrier (BBB), causing debilitating or fatal encephalitis. Sindbis virus (SINV) has been extensively studied in vivo to understand alphavirus neuropathogenesis; yet the molecular details of neuroinvasion at the BBB remain poorly understood. We investigated alphavirus-BBB interactions by pairing a physiologically-relevant, human pluripotent stem cell-derived model of brain microvascular endothelial cells (BMECs) with our model neuroinvasive SINV strains. Our system demonstrates that SINV neuroinvasion correlates with robust infection of the BBB. Specifically, SINV genetic determinants of neuroinvasion enhance viral entry into BMECs. We also identify solute carrier family 2 member 3 (SLC2A3, also named GLUT3) as a potential BMEC-specific entry factor exploited for neuroinvasion. Strikingly, efficient BBB infection is a conserved phenotype that correlates with the neuroinvasive capacity of several Old World alphaviruses, including chikungunya virus. Here, we reveal BBB infection as a shared pathway for alphavirus neuroinvasion that can be targeted for preventing alphavirus-induced encephalitis.
Project description:While viremia in the vertebrate host is a critical determinant of arboviral reservoir competency, transmission efficiency, and disease severity, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, MARCO-expressing Kupffer cells in the liver remove circulating alphavirus particles, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the lymph node and liver has important implications as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease outcomes are more severe. These findings uncover a previously unrecognized pathogen scavenging role for LECs and improve our mechanistic understanding of viremia control during arboviral infections.
Project description:A time course of infection of the alphavirus Sindbis virus (SINV) was used to investigate the presence of viral specific vsRNA and the changes in miRNAs profiles in human embryonic kidney 293 cells (HEK293) by high throughput DNA sequencing. Deep sequencing of small RNAs early in SINV infection (4 and 6 hpi) showed low abundance (0.8%) of viral specific RNAs (vsRNAs) , with a random uniform distribution not typical of Dicer products, suggesting they arise from non-specific degradation. Sequencing showed little variation of cellular microRNAs (miRNAs) at 4 and 6 hpi compared to uninfected cells. Twelve miRNAs exhibiting some minor differential expression by sequencing, showed insignificant modulation by Northern blot analysis.
Project description:The study is intended to collect specimens to support the application of genome analysis technologies, including large-scale genome sequencing. This study will ultimately provide cancer researchers with specimens that they can use to develop comprehensive catalogs of genomic information on at least 50 types of human cancer. The study will create a resource available to the worldwide research community that could be used to identify and accelerate the development of new diagnostic and prognostic markers, new targets for pharmaceutical interventions, and new cancer prevention and treatment strategies. This study will be a competitive enrollment study conducted at multiple institutions.
Project description:Although oral antibiotics can predispose to joint inflammation, this phenomenon remains poorly understood. Here, we leverage mouse models of alphavirus arthritis to investigate the gut commensals, metabolites, and host mechanisms that promote musculoskeletal inflammation. Mice treated with a short course of oral antibiotics exhibited worsened arthritis after chikungunya virus (CHIKV) infection. This phenotype was associated with loss of short chain fatty acids (SCFA) and greater intestinal permeability, and required TLR4 signaling, MyD88 expression, monocytes, antigen-specific and bystander CD4+ T cells, and pro-inflammatory cytokines. Administration of exogenous SCFA or colonization of mice with bacterial species that generate SCFA mitigated CHIKV-induced joint inflammation. Single cell RNA sequencing revealed that gut-derived SCFA restrain the inflammatory phenotype of synovial CD4+ T cells and limit activation of monocytes and osteoclast-like cells. Thus, antibiotic-triggered gut dysbiosis exacerbates alphavirus arthritis by shaping the inflammatory profile of both infiltrating and resident immune cells in joint tissues.