Two distinct modes of RecA action are required for DNA polymerase V-catalyzed translesion synthesis

SOS mutagenesis in Escherichia coli requires DNA polymerase V (pol V) and RecA protein to copy damaged DNA templates. Here we show that two distinct biochemical modes for RecA protein are necessary for pol V-catalyzed translesion synthesis. One RecA mode is characterized by a strong stimulation in nucleotide incorporation either directly opposite a lesion or at undamaged template sites, but by the absence of lesion bypass. A separate RecA mode is necessary for translesion synthesis. The RecA1730 mutant protein, which was identified on the basis of its inability to promote pol V (UmuD'(2)C)-dependent UV-mutagenesis, appears proficient for the first mode of RecA action but is deficient in the second mode. Data are presented suggesting that the two RecA modes are "nonfilamentous". That is, contrary to current models for SOS mutagenesis, formation of a RecA nucleoprotein filament may not be required for copying damaged DNA templates. Instead, SOS mutagenesis occurs when pol V interacts with two RecA molecules, first at a 3' primer end, upstream of a template lesion, where RecA mode 1 stimulates pol V activity, and subsequently at a site immediately downstream of the lesion, where RecA mode 2 cocatalyzes lesion bypass. We posit that in vivo assembly of a RecA nucleoprotein filament may be required principally to target pol V to a site of DNA damage and to stabilize the pol V-RecA interaction at the lesion. However, it is only a RecA molecule located at the 3' filament tip, proximal to a damaged template base, that is directly responsible for translesion synthesis.     

Mitral valve repair with aortic valve replacement in rheumatic heart disease

From 1992 to 2001, 609 patients with rheumatic heart disease underwent aortic valve replacement with either mitral valve repair (n = 201) or mitral valve replacement (n = 408). Follow-up extended to 10 years. Thirty-day mortality was 1.4% for mitral valve repair and 0.7% for mitral valve replacement (p = 0.4). Survival at 9 years was 96.5 +/- 1.4% after mitral valve repair and 89.7 +/- 7.8% after mitral valve replacement (p = 0.73). Freedom from major bleeding at 9 years was 94.8 +/- 2.4% after mitral valve repair and 81 +/- 7.2% after mitral valve replacement (p = 0.03). Freedom from other valve-related complications and from mitral valve re-operation was similar for the two groups. This study showed that in patients with rheumatic heart disease the results of mitral valve repair with aortic valve replacement were comparable to those of double valve replacement. Major bleeding was less frequent after mitral valve repair with aortic valve replacement. Therefore, whenever feasible, mitral valve repair should be attempted in patients with rheumatic heart disease who need concomitant aortic valve replacement.     

A sensitive retroviral pseudotype assay for influenza H5N1‐neutralizing antibodies

Background The World Health Organisation (WHO) recommended the development of simple, safe, sensitive and specific neutralization assays for avian influenza antibodies. We have used retroviral pseudotypes bearing influenza H5 hemagglutinin (HA) as safe, surrogate viruses for influenza neutralization assays which can be carried out at Biosafety Level 2. Results Using our assay, sera from patients who had recovered from infection with influenza H5N1, and sera from animals experimentally immunized or infected with H5 tested positive for the presence of neutralizing antibodies to H5N1. Pseudotype neutralizing antibody titers were compared with titers obtained by hemagglutinin inhibition (HI) assays and microneutralization (MN) assays using live virus, and showed a high degree of correlation, sensitivity and specificity. Conclusions The pseudotype neutralization assay is as sensitive as horse erythrocyte HI and MN for the detection of antibodies to H5N1. It is safer, and can be applied in a high‐throughput format for human and animal surveillance and for the evaluation of vaccines.     

The Role of Intracoronary Plaque Imaging with Intravascular Ultrasound,Optical Coherence Tomography,and Near-Infrared Spectroscopy in Patients with Coronary Artery Disease

The development of multiple diagnostic intracoronary imaging modalities has increased our understanding of coronary atherosclerotic disease. These imaging modalities, intravascular ultrasound (IVUS), optical coherence tomography (OCT), and near-infrared spectroscopy (NIRS), have provided a method to study plaques and introduced the concept of plaque vulnerability. They are being increasingly used for percutaneous coronary intervention (PCI) optimization and are invaluable tools in research studying the pathophysiology of acute coronary syndrome (ACS), in-stent thrombosis and in-stent restenosis. IVUS has the ability to visualize the intracoronary lumen and the vessel wall and can be used to detect early atherosclerotic disease even in the setting of positive arterial remodeling. Studies supporting the use of IVUS to optimize stent deployment and apposition have shown a significant reduction in cardiovascular events. OCT provides even higher resolution imaging and near microscopic detail of plaques, restenoses, and thromboses; thus, it can identify the etiology of ACS. Ongoing trials are evaluating the role of OCT in PCI and using OCT to study stent endothelialization and neointimal proliferation. NIRS is a modality capable of localizing and quantifying lipid core burden. It is usually combined with IVUS and is used to characterize plaque composition. The benefits of NIRS in the setting of ACS have been limited to case reports and series. The utilization of all these intracoronary imaging modalities will continue to expand as their indications for clinical use and research grow. Studies to support their use for PCI optimization resulting in improved outcomes with potential to prevent downstream events are ongoing.     

Moniliform deformation of retinal ganglion cells by formaldehyde‐based fixatives

Protocols for characterizing cellular phenotypes commonly use chemical fixatives to preserve anatomical features, mechanically stabilize tissue, and stop physiological responses. Formaldehyde, diluted in either phosphate‐buffered saline or phosphate buffer, has been widely used in studies of neurons, especially in conjunction with dyes and antibodies. However, previous studies have found that these fixatives induce the formation of bead‐like varicosities in the dendrites and axons of brain and spinal cord neurons. We report here that these formaldehyde formulations can induce bead formation in the dendrites and axons of adult rat and rabbit retinal ganglion cells, and that retinal ganglion cells differ from hippocampal, cortical, cerebellar, and spinal cord neurons in that bead formation is not blocked by glutamate receptor antagonists, a voltage‐gated Na⁺ channel toxin, extracellular Ca²⁺ ion exclusion, or temperature shifts. Moreover, we describe a modification of formaldehyde‐based fixatives that prevents bead formation in retinal ganglion cells visualized by green fluorescent protein expression and by immunohistochemistry. J. Comp. Neurol. 523:545–564, 2015. © 2014 Wiley Periodicals, Inc.     

Second-Line HIV Therapy Outcomes and Determinants of Mortality at the Largest HIV Referral Center in Southern Vietnam

The growing numbers of HIV-infected patients requiring second-line antiretroviral therapy (ART) in Vietnam make essential the evaluation of treatment efficacy to guide treatment strategies.We evaluated all patients aged ≥15 years who initiated second-line ART after documented failure of first-line therapy at the Hospital for Tropical Diseases in Ho Chi Minh City. The primary outcome was time from second-line ART initiation to death, or to a new or reoccurrence of a WHO-defined immunological or clinical failure event, whichever occurred first. Risks of treatment failure and death were evaluated using Cox proportional hazards modeling.Data from 326 of 373 patients initiating second-line ART between November 2006 and August 2011 were included in this analysis. The median age was 32 years (IQR: 28–36). Eighty one percent were men. The median CD4 count was 44 cells/μL (IQR: 16–84). During a median follow-up of 29 months (IQR: 15–44), 60 (18.4%) patients experienced treatment failure, including 12 immunological failures, 4 WHO stage IV AIDS events, and 44 deaths (13.5%). Sixty percent of deaths occurred during the first 6–12 months. The Kaplan–Meier estimates of treatment failure after 1, 2, 3, and 4 years were 13.1% (95% CI: 9.2–16.8), 18.6% (95% CI: 14.0–23.1), 20.4% (95% CI: 15.4–25.1), and 22.8% (95% CI: 17.2–28.1), respectively. Older age, history of injection drug use, lower CD4 count, medication adherence <95%, and previous protease inhibitor use independently predicted treatment failure.While treatment efficacy was similar to that reported from other resource-limited settings, mortality was higher. Early deaths may be averted by prioritizing second-line therapy for those with lower CD4 counts and by improving treatment adherence support.     

Improved Screening Mammogram Workflow by Maximizing PACS Streamlining Capabilities in an Academic Breast Center

The aim of this study was to perform an operational improvement project targeted at the breast imaging reading workflow of mammography examinations at an academic medical center with its associated breast centers and satellite sites. Through careful analysis of the current workflow, two major issues were identified: stockpiling of paperwork and multiple worklists. Both issues were considered to cause significant delays to the start of interpreting screening mammograms. Four workflow changes were suggested (scanning of paperwork, worklist consolidation, use of chat functionality, and tracking of case distribution among trainees) and implemented in July 2015. Timestamp data was collected 2 months before (May–Jun) and after (Aug–Sep) the implemented changes. Generalized linear models were used to analyze the data. The results showed significant improvements for the interpretation of screening mammograms. The average time elapsed for time to open a case reduced from 70 to 28 min (60 % decrease, p?<?0.001), report turn-around time with preliminary signature decreased from 151 to 107 min (29 % decrease, p?<?0.001), and report turn-around time final signature from 153 to 139 min (9 % decrease, p?=?0.002). These improvements were achieved while keeping the efficiency of the workflow for diagnostic mammograms at large unaltered even with increased volume of mammography examinations (31 % increase of 4344 examinations for May–Jun to 5678 examinations for Aug–Sep). In conclusion, targeted efforts to improve the breast imaging reading workflow for screening mammograms in a teaching environment provided significant performance improvements without affecting the workflow of diagnostic mammograms.     

Fluocinolone Acetonide Is a Potent Synergistic Factor of TGF‐β3–Associated Chondrogenesis of Bone Marrow–Derived Mesenchymal Stem Cells for Articular Surface Regeneration

Articular cartilage repair remains a challenging problem. Based on a high‐throughput screening and functional analysis, we found that fluocinolone acetonide (FA) in combination with transforming growth factor beta 3 (TGF‐β3) strongly potentiated chondrogenic differentiation of human bone marrow–derived mesenchymal stem cells (hBMSCs). In an in vivo cartilage defect model in knee joints of immunocompromised mice, transplantation of FA/TGF‐β3–treated hBMSCs could completely repair the articular surface. Analysis of the intracellular pathways revealed that FA enhanced TGF‐β3–induced phosphorylation of Smad2 and Smad3. Additionally, we performed a pathway array and found that FA activates the mTORC1/AKT pathway. Chemical inhibition of mTORC1 with rapamycin substantially suppressed FA effect, and inhibition of AKT completely repressed chondrogenesis of hBMSCs. Inhibition of glucocorticoid receptor with mifepristone also suppressed FA effect, suggesting that FA involves binding to the glucocorticoid receptor. Comparative analysis with other glucocorticoids (triamcinolone acetonide [TA] and dexamethasone [DEX]) revealed the unique ability of FA to repair articular cartilage surgical defects. Analysis of intracellular pathways showed that the mTORC1/AKT pathway and the glucocorticoid receptor was highly activated with FA and TA, but to a lesser extent with DEX. Collectively, these results show a unique ability of FA to enhance TGF‐β3–associated chondrogenesis, and suggest that the FA/TGF‐β3 combination may be used as major inducer of chondrogenesis in vitro. Additionally, FA/TGF‐β3 could be potentially applied in a clinical setting to increase the efficiency of regenerative approaches based on chondrogenic differentiation of stem cells. © 2015 American Society for Bone and Mineral Research.