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

Distribution and Habitat Associations of Juvenile Common Snook in the Lower Rio Grande, Texas

Common Snook Centropomus undecimalis were once abundant off the Texas coast, but these populations are now characterized by low abundance and erratic recruitment. Most research concerning Common Snook in North America has been conducted in Florida and very little is known about the specific biology and habitat needs of Common Snook in Texas. The primary objective of this study was to describe the habitat use patterns of juvenile Common Snook and their role in the fish assemblage in the lower portion of the Rio Grande, Texas. Secondarily, we documented the relationship between age and juvenile reproductive development. Fish were collected during January–March 2006 from the lower 51.5 km of the Rio Grande using a bottom trawl and boat-mounted electrofisher. Measurements of water quality and other habitat traits were recorded at each sampling site. We captured 225 Common Snook exclusively in freshwater habitats above river kilometer 12.9. The distribution of juvenile Common Snook was not random, but influenced primarily by turbidity and dissolved oxygen. Sex differentiation and gonadal development based on histological examination of gonads established that age-1 and age-2 Common Snook were juvenile, prepubertal males. There was no difference between the age groups in their overall distribution in the river. However, age-2 Common Snook were associated with deeper areas with faster currents, higher conductivity, and steeper banks. Overall, Common Snook in the lower Rio Grande show substantial differences in habitat use than their counterparts in other parts of the range of the species, but it is unclear whether this is due to differences in habitat availability, behavioral plasticity, or some combination thereof

Genomic heterogeneity and clinical characterization of SARS-CoV-2 in Oregon

The first reported case of COVID-19 in the State of Oregon occurred in late February 2020, with subsequent outbreaks occurring in the populous Portland metro area but also with significant outbreaks in less-populous and rural areas. Here we report viral sequences from 188 patients across the hospitals and associated clinics in the Providence Health System in the State of Oregon dating back to the early days of the outbreak. We show a significant shift in dominant clade lineages over time in Oregon, with the rapid emergence and dominance of Spike D614G-positive variants. We also highlight significant diversity in SARS-CoV-2 sequences in Oregon, including a large number of rare mutations, indicative that these genomes could be utilized for outbreak tracing. Lastly, we show that SARS-CoV-2 genomic information may offer additional utility in combination with clinical covariates in the prediction of acute disease phenotypes

Probabilistic modeling of personalized drug combinations from integrated chemical screen and molecular data in sarcoma.

BackgroundCancer patients with advanced disease routinely exhaust available clinical regimens and lack actionable genomic medicine results, leaving a large patient population without effective treatments options when their disease inevitably progresses. To address the unmet clinical need for evidence-based therapy assignment when standard clinical approaches have failed, we have developed a probabilistic computational modeling approach which integrates molecular sequencing data with functional assay data to develop patient-specific combination cancer treatments.MethodsTissue taken from a murine model of alveolar rhabdomyosarcoma was used to perform single agent drug screening and DNA/RNA sequencing experiments; results integrated via our computational modeling approach identified a synergistic personalized two-drug combination. Cells derived from the primary murine tumor were allografted into mouse models and used to validate the personalized two-drug combination. Computational modeling of single agent drug screening and RNA sequencing of multiple heterogenous sites from a single patient's epithelioid sarcoma identified a personalized two-drug combination effective across all tumor regions. The heterogeneity-consensus combination was validated in a xenograft model derived from the patient's primary tumor. Cell cultures derived from human and canine undifferentiated pleomorphic sarcoma were assayed by drug screen; computational modeling identified a resistance-abrogating two-drug combination common to both cell cultures. This combination was validated in vitro via a cell regrowth assay.ResultsOur computational modeling approach addresses three major challenges in personalized cancer therapy: synergistic drug combination predictions (validated in vitro and in vivo in a genetically engineered murine cancer model), identification of unifying therapeutic targets to overcome intra-tumor heterogeneity (validated in vivo in a human cancer xenograft), and mitigation of cancer cell resistance and rewiring mechanisms (validated in vitro in a human and canine cancer model).ConclusionsThese proof-of-concept studies support the use of an integrative functional approach to personalized combination therapy prediction for the population of high-risk cancer patients lacking viable clinical options and without actionable DNA sequencing-based therapy