The recA protein of E. coli : regulation and function in recombination and repair : UV induction, autoregulatory model, DNA binding protein, DNA renaturation, strand assimilation

In Escherichia coli, the recA function is required for general recombination, repair of DNA damage and a diverse group of functions which are coordinately expressed following DNA damage or arrest of DNA synthesis (SOS functions ). These latter functions include UV mutagenesis, W- reactivation of damaged phage DNA, prophage induction and cell filamentation. In this paper we summarize experiments which have elucidated regulatory and functional aspects of the recA gene product. Biochemical and genetic data suggest a model for control of recA gene expression in which the recA protein auto regulates its own synthesis following UV irradiation or other forms of DNA damage. The features of this autoregulatory mechanism are different from those proposed for gene 32 protein of phage T4. Biochemical experiments with homogeneous wild type and mutant forms of RecA protein reveal that this unique DNA binding protein catalyzes DNA renaturation and single strand assimilation reactions which are coupled to ATP hydrolysis. These results indicate that RecA protein catalyzes strand transfer during the initiation of genetic recombination and during post-replication repair of damaged DNA in vivo.KEVIN MCENTEE AND GEORGE M. WEINSTOCK, Department of Biochemistry, Stanford University School of Medicine, Stanford, California

Outage Detection via Real-time Social Stream Analysis: Leveraging the Power of Online Complaints

Over the past couple of years, Netflix has significantly expanded its online streaming offerings, which now encompass multiple delivery platforms and thousands of titles available for instant view. This paper documents the design and development of an outage detection system for the online services provided by Netflix. Unlike other internal quality control measures used at Netflix, this system uses only publicly available information: the tweets, or Twitter posts, that mention the word “Netflix,” and has been developed and deployed externally, on servers independent of the Netflix infrastructure. This paper discussed the system and provides assessment of the accuracy of its real-time detection and alert mechanisms

BRAF- and MEK-targeted small molecule inhibitors exert enhanced antimelanoma effects in combination with oncolytic reovirus through ER stress

Reovirus type 3 (Dearing) (RT3D) infection is selective for cells harboring a mutated/activated RAS pathway. Therefore, in a panel of melanoma cell lines (including RAS mutant, BRAF mutant and RAS/BRAF wild-type), we assessed therapeutic combinations that enhance/suppress ERK1/2 signaling through use of BRAF/MEK inhibitors. In RAS mutant cells, the combination of RT3D with the BRAF inhibitor PLX4720 (paradoxically increasing ERK1/2 signaling in this context) did not enhance reoviral cytotoxicity. Instead, and somewhat surprisingly, RT3D and BRAF inhibition led to enhanced cell kill in BRAF mutated cell lines. Likewise, ERK1/2 inhibition, using the MEK inhibitor PD184352, in combination with RT3D resulted in enhanced cell kill in the entire panel. Interestingly, TCID 50 assays showed that BRAF and MEK inhibitors did not affect viral replication. Instead, enhanced efficacy was mediated through ER stress-induced apoptosis, induced by the combination of ERK1/2 inhibition and reovirus infection. In vivo, combined treatments of RT3D and PLX4720 showed significantly increased activity in BRAF mutant tumors in both immune-deficient and immune-competent models. These data provide a strong rationale for clinical translation of strategies in which RT3D is combined with BRAF inhibitors (in BRAF mutant melanoma) and/or MEK inhibitors (in BRAF and RAS mutant melanoma)

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

[2] Purification and properties of DNA polymerase II from Escherichia coli

This chapter describes a simple rapid procedure to obtain highly purified enzymes from wild-type pol B cells and from an exonucleasedeficient pol B mutant strain (D155A/E157A), which are suitable for obtaining crystals for analysis by X-ray diffraction. Three DNA polymerases have been isolated and purified from Escherichia coli. DNA polymerase I (Pol I) has been shown to be involved in a variety of DNA repair pathways and is responsible for removing the RNA primer portion of Okazaki fragments. The structural gene for Polymerase II (Pol II) is the damage-inducible pol B gene. Its expression is regulated by the Lex A repressor as a part of the SOS response to DNA damage in E. coli, and the enzyme has been classified as an α-type polymerase. Pol II incorporates nucleotides opposite abasic template sites 6 and incorporates chain terminating dideoxy- and arabinonucleotides. It contains 3'-exonuclease activity, and its high exonuclease to polymerase ratio is similar in magnitude to wildtype bacteriophage T4 polymerase. The wild-type Pol II and exonuclease-deficient Pol II mutant behave similarly during purification

Purification and Properties of Wild-type and Exonuclease-deficient DNA Polymerase II from Escherichia coli(∗)

Wild-type DNA polymerase II (pol II) and an exonuclease-deficient pol II mutant (D155A/E157A) have been overexpressed and purified in high yield from Escherichia coli. Wild-type pol II exhibits a high proofreading 3′-exonuclease to polymerase ratio, similar in magnitude to that observed for bacteriophage T4 DNA polymerase. While copying a 250-nucleotide region of the lacZα gene, the fidelity of wild-type pol II is high, with error rates for single-base substitution and frameshift errors being ≤10-6. In contrast, the pol II exonuclease-deficient mutant generated a variety of base substitution and single base frameshift errors, as well as deletions between both perfect and imperfect directly repeated sequences separated by a few to hundreds of nucleotides. Error rates for the pol II exonuclease-deficient mutant were from ≥13- to ≥240-fold higher than for wild-type pol II, depending on the type of error considered. These data suggest that from 90 to >99% of base substitutions, frameshifts, and large deletions are efficiently proofread by the enzyme. The results of these experiments together with recent in vivo studies suggest an important role for pol II in the fidelity of DNA synthesis in cells