The oncoprotein H-RasV12 increases mitochondrial metabolism

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Virus detection and identification using random multiplex (RT)-PCR with 3'-locked random primers

<p>Abstract</p> <p>Background</p> <p>PCR-based detection and identification of viruses assumes a known, relatively stable genome. Unfortunately, high mutation rates may lead to extensive changes in viral nucleic acid sequences making dedicated PCR primer use problematic. Furthermore, in bioterrorism, viral consensus sequences can be genetically modified as a countermeasure to RT-PCR and DNA chip detection. Accordingly, there is a great need for the development of rapid and universal virus detection and identification technologies.</p> <p>Results</p> <p>We report herein that viral genomic DNA or RNA can be separated from host nucleic acids in plasma by filtration and nuclease digestion, and randomly amplified in a single PCR using a mixture of primers designed to be resistant to primer-dimer amplification (5'-VVVVVVVVAA-3', V = A, G or C; 3⁸or 6561 primers). We have termed this novel PCR method Random Multiplex (RT)-PCR since hundreds of overlapping PCR amplifications occur simultaneously. Using this method, we have successfullydetected and partially sequenced 3 separate viruses in human plasma without using virus-specific reagents (<it>i.e., </it>Adenovirus Type 17, Coxsackievirus A7, and Respiratory Syncytial Virus B). The method is sensitive to ~1000 genome equivalents/ml and may represent the fastest means of detection of unknown viruses.</p> <p>Conclusion</p> <p>These studies suggest that the further development of random multiplex (RT)-PCR may lead to a diagnostic assay that can universally detect viruses in donated blood products as well as in patients suffering with idiopathic disease states of possible viral etiology.</p

VCU-UNITE: Identifying Recognition Mechanisms for University-Community Engagement

The aims of the project were to determine what process would be appropriate to recognize significant contributions of university and community partners to joint community engagement projects. Our view was that several different award ceremonies exist in VCU, none of which are well attended and suggesting that this form of recognition may not be a high priority for participants. Thus, we sought to determine the awareness and extent of involvement of VCU faculty, staff and students in community engagement projects, and to find out what format of recognition process would be appealing to both university and community participants. We gathered this information through surveys and focus groups, analyzed the data and determined that university and community partners had differing views on what types of recognition would be desirous. Next, we designed a web portal through which nominations could be made by members of the university and by the community. We propose a streamlined nomination and online review process, leading to a quarterly award consisting of a letter of thanks from the Division of Community Engagement, and a photograph and narrative placed on the VCU website. Recipients of quarterly awards would automatically compete for an annual award, such as P&T credit or additional community leave time (university), or a scholarship, a plaque, or recognition at a VCU event (community)

Fructose-2,6-Bisphosphate Synthesis by 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase 4 (PFKFB4) Is Required for the Glycolytic Response to Hypoxia and Tumor Growth

Fructose-2,6-bisphosphate (F2,6BP) is a shunt product of glycolysis that allosterically activates 6-phosphofructo-1-kinase (PFK-1) resulting in increased glucose uptake and glycolytic flux to lactate. The F2,6BP concentration is dictated by four bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) with distinct kinase:phosphatase activities. PFKFB4 is over-expressed in human cancers, induced by hypoxia and required for survival and growth of several cancer cell lines. Although PFKFB4 appears to be a rational target for anti-neoplastic drug development, it is not clear whether its kinase or phosphatase activity is required for cancer cell survival. In this study, we demonstrate that recombinant human PFKFB4 kinase activity is 4.3-fold greater than its phosphatase activity, siRNA and genomic deletion of PFKFB4 decrease F2,6BP, PFKFB4 over-expression increases F2,6BP and selective PFKFB4 inhibition in vivo markedly reduces F2,6BP, glucose uptake and ATP. Last, we find that PFKFB4 is required for cancer cell survival during the metabolic response to hypoxia, presumably to enable glycolytic production of ATP when the electron transport chain is not fully operational. Taken together, our data indicate that the PFKFB4 expressed in multiple transformed cells and tumors functions to synthesize F2,6BP. We predict that pharmacological disruption of the PFKFB4 kinase domain may have clinical utility for the treatment of human cancers

Randomized, Double-Blind, Placebo-Controlled, Global Phase III Trial of Talimogene Laherparepvec Combined With Pembrolizumab for Advanced Melanoma

Purpose: The combination of talimogene laherparepvec (T-VEC) and pembrolizumab previously demonstrated an acceptable safety profile and an encouraging complete response rate (CRR) in patients with advanced melanoma in a phase Ib study. We report the efficacy and safety from a phase III, randomized, double-blind, multicenter, international study of T-VEC plus pembrolizumab (T-VEC-pembrolizumab) versus placebo plus pembrolizumab (placebo-pembrolizumab) in patients with advanced melanoma. Methods: Patients with stage IIIB-IVM1c unresectable melanoma, naïve to antiprogrammed cell death protein-1, were randomly assigned 1:1 to T-VEC-pembrolizumab or placebo-pembrolizumab. T-VEC was administered at ≤ 4 × 106 plaque-forming unit (PFU) followed by ≤ 4 × 108 PFU 3 weeks later and once every 2 weeks until dose 5 and once every 3 weeks thereafter. Pembrolizumab was administered intravenously 200 mg once every 3 weeks. The dual primary end points were progression-free survival (PFS) per modified RECIST 1.1 by blinded independent central review and overall survival (OS). Secondary end points included objective response rate per mRECIST, CRR, and safety. Here, we report the primary analysis for PFS, the second preplanned interim analysis for OS, and the final analysis. Results: Overall, 692 patients were randomly assigned (346 T-VEC-pembrolizumab and 346 placebo-pembrolizumab). T-VEC-pembrolizumab did not significantly improve PFS (hazard ratio, 0.86; 95% CI, 0.71 to 1.04; P = .13) or OS (hazard ratio, 0.96; 95% CI, 0.76 to 1.22; P = .74) compared with placebo-pembrolizumab. The objective response rate was 48.6% for T-VEC-pembrolizumab (CRR 17.9%) and 41.3% for placebo-pembrolizumab (CRR 11.6%); the durable response rate was 42.2% and 34.1% for the arms, respectively. Grade ≥ 3 treatment-related adverse events occurred in 20.7% of patients in the T-VEC-pembrolizumab arm and in 19.5% of patients in the placebo-pembrolizumab arm. Conclusion: T-VEC-pembrolizumab did not significantly improve PFS or OS compared with placebo-pembrolizumab. Safety results of the T-VEC-pembrolizumab combination were consistent with the safety profiles of each agent alone

Safety profile and pharmacokinetic analyses of the anti-CTLA4 antibody tremelimumab administered as a one hour infusion

BACKGROUND: CTLA4 blocking monoclonal antibodies provide a low frequency but durable tumor responses in patients with metastatic melanoma, which led to the regulatory approval of ipilimumab based on two randomized clinical trials with overall survival advantage. The similarly fully human anti-CTLA4 antibody tremelimumab had been developed in the clinic at a fixed rate infusion, resulting in very prolonged infusion times. A new formulation of tremelimumab allowed testing a shorter infusion time. METHODS: A phase 1 multi-center study to establish the safety and tolerability of administering tremelimumab as a 1-hour infusion to patients with metastatic melanoma. Secondary endpoints included pharmacokinetic and clinical effects of tremelimumab. RESULTS: No grade 3 or greater infusion-related adverse events or other adverse events preventing the administration of the full tremelimumab dose were noted in 44 treated patients. The overall side effect profile was consistent with prior experiences with anti-CTLA4 antibodies. Objective tumor responses were noted in 11% of evaluable patients with metastatic melanoma, which is also consistent with the prior experience with CTLA4 antagonistic antibodies. CONCLUSIONS: This study did not identify any safety concerns when tremelimumab was administered as a 1-hour infusion. These data support further clinical testing of the 1-hour infusion of tremelimumab. (Clinical trial registration number NCT00585000)

DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle

Physical mapping of DNA can be used to detect structural variants and for whole-genome haplotype assembly. Here, the authors use CRISPR-Cas9 and high-speed atomic force microscopy to ‘nanomap’ single molecules of DNA