RAD51 and other members of the RecA family of strand exchange proteins assemble on ssDNA to form presynaptic filaments, which carry out the central steps of homologous recombination. A microplate-based assay was developed for high-throughput measurement of hRAD51 filament formation on ssDNA. With this method, a 10,000 compound library was screened, leading to the identification of a small molecule (RS-1) that enhances hRAD51 binding in a wide range of biochemical conditions. Salt titration experiments showed that RS-1 can enhance filament stability. Ultrastructural analysis of filaments formed on ssDNA showed that RS-1 can increase both protein-DNA complex lengths and the pitch of helical filament turns. RS-1 stimulated hRAD51-mediated homologous strand assimilation (D-loop) activity by at least 5-to 11-fold, depending on the condition. This D-loop stimulation occurred even in the presence of Ca 2؉ or adenylyl-imidodiphosphate, indicating that the mechanism of stimulation was distinct from that conferred by Ca 2؉ and/or inhibition of ATPase. No D-loop activity was observed in the absence of a nucleotide triphosphate cofactor, indicating that the compound does not substitute for this requirement. These results indicate that RS-1 enhances the homologous recombination activity of hRAD51 by promoting the formation of active presynaptic filaments. Cell survival assays in normal neonatal human dermal fibroblasts demonstrated that RS-1 promotes a dose-dependent resistance to the cross-linking chemotherapeutic drug cisplatin. Given that RAD51-dependent recombination is a major determinant of cisplatin resistance, RS-1 seems to function in vivo to stimulate homologous recombination repair proficiency. RS-1 has many potential applications in both research and medical settings.cross-linking chemotherapy ͉ DNA repair ͉ high-throughput screen ͉ recombinase ͉ strand exchange H omologous recombination (HR) has multiple roles in DNA repair, including the repair of double strand breaks (DSBs) and recovery from the replication-blocking lesions formed by DNA cross-linking agents. HR repairs DSBs by locating a homologous stretch of DNA and replicating the missing genetic information from this homologous template. In contrast to DSB repair by nonhomologous end joining, HR repair generally occurs without mutations. Because of this, HR repair is critically important in the maintenance of genomic stability (reviewed in ref. 1). The proposed mechanism for this pathway begins with 5Ј to 3Ј nuclease activity at the DSB, resulting in a 3Ј singlestranded tail. The tail is coated by replication protein A, which is subsequently replaced by a helical filament of RAD51 protein. This displacement of replication protein A by RAD51 seems to be controlled by a number of mediator proteins, which include BRCA2, RAD52, and RAD51 paralogue complexes (2-5). The RAD51-coated 3Ј tail then locates and invades a homologous template of dsDNA. After invasion, templated DNA synthesis initiated at 3Ј ends leads to formation of branched DNA intermediates, which ...

Objectives Opioid pain reliever (OPR) prescribing at Emergency Department (ED) discharge has increased in the past decade but specific prescription details are lacking. Prior ED OPR prescribing estimates relied on national survey extrapolation or prescription databases. The main goal of this study was to utilize a research consortium to analyze the characteristics of patients and opioid prescriptions using a national sample of ED patients. We also aimed to examine the indications for OPR prescribing, characteristics of opioids prescribed both in the ED and at the time of discharge, and characteristics of patients who received OPRs compared with those who did not. Methods This observational, multi-centered, retrospective cohort study assessed OPR prescribing to consecutive patients presenting to the consortium EDs during 1 week in October 2012. The consortium study sites consisted of 19 EDs representing 1.4 million annual visits, varied geographically, and were predominantly academic centers. Medical records of all patients aged 18-90 years discharged with an OPR (excluding tramadol) were individually abstracted via standardized chart review by investigators for detailed analysis. Descriptive statistics were generated. Results During the study week, 27,516 patient visits were evaluated in the consortium EDs. 19,321 (70.2%) were discharged and 3,284 patients (11.9% of all patients and 17.0% of discharged patients) received an OPR prescription. For those prescribed an OPR, mean age was 41.1 (SD 14.7) years and 1,694 (51.6%) were female. Mean initial pain score was 7.7 (SD 2.4). The most common diagnoses associated with OPR prescribing were back pain (10.2%), abdominal pain (10.1%), and extremity fracture (7.1%) or sprain (6.5%). The most common OPRs prescribed were oxycodone (52.3%), hydrocodone (40.9%) and codeine (4.8%). >99% were immediate release, 90.0% were combination preparations, and the mean and median number of pills was 16.6 (SD 7.6) and 15 (IQR=12-20) respectively. Conclusion In a study of ED patients treated over a single week across the country, 17% of discharged patients were prescribed OPRs. The majority of the prescriptions had small pill counts and almost exclusively immediate release formulations.

Radiation therapy and chemotherapy are commonly used treatments for head and neck cancer. RAD51 is a highly conserved DNA repair protein that serves a central function in the homologous recombination pathway. High levels of RAD51 protein expression have been reported in number of human cancer cell lines, and studies suggest that RAD51 overexpression can increase cellular resistance to radiation and some chemotherapeutic drugs. In this study, RAD51 protein levels were quantified by immunohistochemistry in tumor samples from twelve head and neck cancer patients who received identical treatment with induction chemotherapy (paclitaxel and carboplatinum) followed by radiation therapy given concurrently with additional chemotherapy (paclitaxel, fluorouracil, hydroxyurea). Patients with high RAD51 protein levels in their pre-treatment tumor biopsies demonstrated poorer cancer-specific survival rates than patients with lower RAD51 levels (33.3% vs. 88.9% at 2 years; p=0.025). In addition, within a subgroup of patients with normal tumor cell p53 expression, there was a non-significant trend toward better induction chemotherapy response rates observed in the tumors with lower RAD51 protein levels. These results suggest that tumor cell RAD51 expression levels may influence the outcome of patients with head and neck cancer treated with chemotherapy and radiotherapy.

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