Role of RPA Phosphorylation in the ATR-Dependent G2 Cell Cycle Checkpoint.

Cells respond to DNA double-strand breaks by initiating DSB repair and ensuring a cell cycle checkpoint. The primary responder to DSB repair is non-homologous end joining, which is an error-prone repair pathway. However, when DSBs are generated after DNA replication in the G2 phase of the cell cycle, a second DSB repair pathway, homologous recombination, can come into action. Both ATM and ATR are important for DSB-induced DSB repair and checkpoint responses. One method of ATM and ATR working together is through the DNA end resection of DSBs. As a readout and marker of DNA end resection, RPA is phosphorylated at Ser4/Ser8 of the N-terminus of RPA32 in response to DSBs. Here, the significance of RPA32 Ser4/Ser8 phosphorylation in response to DNA damage, specifically in the S phase to G2 phase of the cell cycle, is examined. RPA32 Ser4/Ser8 phosphorylation in G2 synchronized cells is necessary for increases in TopBP1 and Rad9 accumulation on chromatin and full activation of the ATR-dependent G2 checkpoint. In addition, our data suggest that RPA Ser4/Ser8 phosphorylation modulates ATM-dependent KAP-1 phosphorylation and Rad51 chromatin loading in G2 cells. Through the phosphorylation of RPA Ser4/Ser8, ATM acts as a partner with ATR in the G2 phase checkpoint response, regulating key downstream events including Rad9, TopBP1 phosphorylation and KAP-1 phosphorylation/activation via the targeting of RPA32 Ser4/Ser8.

Prediabetes and the risk of type 2 diabetes: Investigating the roles of depressive and anxiety symptoms in the Lifelines cohort study.

AIMS: Depression and anxiety may increase the risk of progressing from prediabetes to type 2 diabetes. The present study examined the interactions between prediabetes status and elevated depressive and anxiety symptoms with the risk of type 2 diabetes. METHODS: Participants (N = 72,428) were adults aged 40 years and above without diabetes at baseline from the Lifelines Cohort Study (58% female; mean age = 51.4 years). The Mini-International Neuropsychiatric Interview screened for elevated symptoms of major depressive disorder and generalized anxiety disorder. Glycated haemoglobin A1c (HbA1c ) levels determined prediabetes status at baseline (2007-2013), and HbA1c and self-reported diabetes diagnoses determined diabetes status at follow-up (2014-2017). Groups were formed for elevated depressive and anxiety symptoms, respectively, and prediabetes status at baseline (elevated depressive/anxiety symptoms with prediabetes, elevated depressive/anxiety symptoms alone, and prediabetes alone), and compared to a reference group (no prediabetes or anxiety/depression) on the likelihood of developing diabetes during the follow-up period. RESULTS: N = 1300 (1.8%) participants developed diabetes. While prediabetes alone was associated with incident diabetes (OR = 5.94; 95% CI = 5.10-6.90, p < 0.001), the group with combined prediabetes and depressive symptoms had the highest likelihood of developing diabetes over follow-up (OR = 8.29; 95% CI = 5.58-12.32, p < 0.001). Similar results were found for prediabetes and anxiety symptoms (OR = 6.57; 95% CI = 4.62-9.33, p < 0.001), compared to prediabetes alone (OR = 6.09; 95% CI = 5.23-7.11, p < 0.001), though with a smaller effect. The interaction between depressive symptoms and prediabetes was synergistic in age-and-sex adjusted analyses. CONCLUSIONS: Individuals with elevated depressive, and to some extent anxiety, symptoms in combination with prediabetes may represent a high-risk subgroup for type 2 diabetes.

Carbon uptake in Eurasian boreal forests dominates the high-latitude net ecosystem carbon budget.

Arctic-boreal landscapes are experiencing profound warming, along with changes in ecosystem moisture status and disturbance from fire. This region is of global importance in terms of carbon feedbacks to climate, yet the sign (sink or source) and magnitude of the Arctic-boreal carbon budget within recent years remains highly uncertain. Here, we provide new estimates of recent (2003-2015) vegetation gross primary productivity (GPP), ecosystem respiration (Reco ), net ecosystem CO2 exchange (NEE; Reco - GPP), and terrestrial methane (CH4 ) emissions for the Arctic-boreal zone using a satellite data-driven process-model for northern ecosystems (TCFM-Arctic), calibrated and evaluated using measurements from >60 tower eddy covariance (EC) sites. We used TCFM-Arctic to obtain daily 1-km2 flux estimates and annual carbon budgets for the pan-Arctic-boreal region. Across the domain, the model indicated an overall average NEE sink of -850 Tg CO2 -C year-1 . Eurasian boreal zones, especially those in Siberia, contributed to a majority of the net sink. In contrast, the tundra biome was relatively carbon neutral (ranging from small sink to source). Regional CH4 emissions from tundra and boreal wetlands (not accounting for aquatic CH4 ) were estimated at 35 Tg CH4 -C year-1 . Accounting for additional emissions from open water aquatic bodies and from fire, using available estimates from the literature, reduced the total regional NEE sink by 21% and shifted many far northern tundra landscapes, and some boreal forests, to a net carbon source. This assessment, based on in situ observations and models, improves our understanding of the high-latitude carbon status and also indicates a continued need for integrated site-to-regional assessments to monitor the vulnerability of these ecosystems to climate change.

The effects of foam rolling on ankle dorsiflexion range of motion in healthy adults: A systematic literature review.

BACKGROUND: Reduced ankle dorsiflexion is associated with lower limb injury and dysfunction, with static stretching mostly used to increase ankle range of motion. Foam rolling is an alternative intervention, shown to immediately increase ankle range of motion, while the long-term application has conflicting evidence. AIMS: To assess the effects of single and multiple foam rolling interventions on ankle dorsiflexion range of motion in healthy adults and appraise the methodological quality of the included studies. DESIGN: Systematic literature review. METHODS: Five electronic databases were systematically searched to identify randomised controlled trials reporting the effects of foam rolling on ankle dorsiflexion. Data was extracted from studies that met the inclusion criteria and independently appraised by each reviewer using the PEDro scale. RESULTS: Thirty-two articles were identified; six studies included foam rolling compared to other interventions on ankle dorsiflexion range of motion. Five of the six studies reported a significant increase (p < 0.05) in ankle dorsiflexion within groups compared to baseline measurements, after a single foam rolling intervention. One study found a significant within group increase in long-term effects after foam rolling on ankle dorsiflexion over seven weeks. The mean PEDro score for all studies was 6/10 indicating a high-quality level of evidence. CONCLUSION: There is strong evidence suggesting that foam rolling may be effective in increasing range of motion in a healthy adult population in the short term up to 30 min; however, definitive conclusions on long-term effects cannot be drawn due to a lack of evidence, with further research recommended.

COVID-19 Outbreak in A Forward-Deployed Expeditionary Military Command: A Retrospective Review on Preventative Measures and Outbreak Characteristics.

BACKGROUND: An outbreak of coronavirus disease 2019 (COVID-19) occured within a land based 2,000-member cohort stationed on a remote air base in the Middle East from June to August 2020. We retrospectively reviewed base characteristics and mitigation measures instituted during the outbreak. We also reviewed documentation on the individuals that were either quarantined or placed in isolation and provide data on demographics, real-time reverse transcriptase polymerase chain reaction (rRT-PCR) results, occupation, and workdays lost. RESULTS: During the reporting period, 46 individuals or 3.84% of the population had a positive COVID-19 rRT-PCR test. Aviation personnel represented 50% of the COVID-19 positive tests. Sixteen percent of health care personnel were tested positive. Overall, 10% of personnel were placed in isolation or quarantine, resulting in the loss of 1,552 workdays. CONCLUSIONS: The data show a disproportionate impact on healthcare workers and personnel involved in aviation operations. The purpose of this study is 2-fold: to describe the characteristics of the outbreak and to highlight the effectiveness of mitigation measures implemented to control it during military operations. This study may serve to inform medical professionals and military leaders in the management of a similar outbreak in a congregate living setting.

Comment on "Recent global decline of CO2 fertilization effects on vegetation photosynthesis".

Wang et al. (Research Articles, 11 December 2020, p. 1295), using photosynthesis proxies from long-term satellite records, report a significant decline in CO2 fertilization effects. We find that small systematic biases in Advanced Very High Resolution Radiometer (AVHRR) data affect their analysis to the degree that the key finding is not robust. Caution is recommended when using AVHRR to detect changes in near-infrared reflectance of vegetation (NIRv) trends and vegetation indices.

Retrieval of Additional Epinephrine from Adrenaclick-Style Epinephrine Autoinjectors.

Anaphylaxis is a life-threatening allergic reaction involving multiple organ systems that can result in significant morbidity and mortality if left untreated. Epinephrine is the mainstay of treatment. Most episodes of anaphylaxis resolve after a single dose of epinephrine, but biphasic and protracted courses of anaphylaxis are well described. The need for additional doses of epinephrine poses a significant challenge in the wilderness setting, because patients and providers may only carry a single autoinjector. Prior work has demonstrated successful disassembly of various brands of epinephrine autoinjectors to retrieve additional drug product for repeat dosing. We describe 2 techniques to retrieve additional doses of epinephrine from Adrenaclick-style epinephrine autoinjectors. The techniques described are off-label and are not approved by the manufacturer or the Food and Drug Administration. Wilderness providers should familiarize themselves with techniques for retrieval of additional epinephrine from various autoinjectors in light of significant differences in product design.

Replication protein A, the laxative that keeps DNA regular: The importance of RPA phosphorylation in maintaining genome stability.

The eukaryotic ssDNA-binding protein, Replication protein A (RPA), was first discovered almost three decades ago. Since then, much progress has been made to elucidate the critical roles for RPA in DNA metabolic pathways that help promote genomic stability. The canonical RPA heterotrimer (RPA1-3) is an essential coordinator of DNA metabolism that interacts with ssDNA and numerous protein partners to coordinate its roles in DNA replication, repair, recombination and telomere maintenance. An alternative form of RPA, termed aRPA, is formed by a complex of RPA4 with RPA1 and RPA3. aRPA is expressed differentially in cells compared to canonical RPA and has been shown to inhibit canonical RPA function while allowing for regular maintenance of cell viability. Interestingly, while aRPA is defective in DNA replication and cell cycle progression, it was shown to play a supporting role in nucleotide excision repair and recombination. The binding domains of canonical RPA interact with a growing number of partners involved in numerous genome maintenance processes. The protein interactions of the RPA-ssDNA complex are not only governed by competition between the binding proteins but also by post-translation modifications such as phosphorylation. Phosphorylation of RPA2 is an important post-translational modification of the RPA complex, and is essential for directing context-specific functions of the RPA complex in the DNA damage response. Due to the importance of RPA in cellular metabolism, it was identified as an appealing target for chemotherapeutic drug development that could be used in future cancer treatment regimens.

Enteric microbiome profiles during a randomized Phase 2 clinical trial of surotomycin versus vancomycin for the treatment of Clostridium difficile infection.

OBJECTIVES: The effects of surotomycin (CB-183,315, MK-4261), a bactericidal cyclic lipopeptide, and vancomycin, the current standard-of-care for Clostridium difficile infection (CDI), on intestinal pathogens and microbiota were evaluated parallel to a Phase 2 randomized, double-blind clinical trial. METHODS: The single-centre cohort included 26 patients receiving surotomycin [125 or 250 mg twice daily (n = 9 each)] or oral vancomycin [125 mg four times daily (n = 8)] for 10 days. Faecal samples were collected at days 0-42 to quantify both C. difficile by conventional culture and the major components of the microbiome by quantitative PCR. RESULTS: Surotomycin 250 mg twice daily or vancomycin 125 mg four times daily reduced faecal C. difficile counts from ∼105-107 log10 cfu/g at baseline to ≤ 102 cfu/g by days 4-10 of treatment. Day 10 counts of C. difficile in 3/9 patients receiving surotomycin 125 mg twice daily remained detectable, including one patient who failed to achieve clinical cure. Bacteroidetes and Prevotella mean counts increased 0.7 log10 or remained unchanged with surotomycin 125 and 250 mg twice daily, respectively, whereas vancomycin reduced counts by 2.5-3.2 log10 (P < 0.02). Vancomycin reduced Firmicutes counts by 2.5-2.8 log10; surotomycin moderately suppressed these microbes in a dose-dependent manner. CONCLUSIONS: In this Phase 2 trial substudy, compared with vancomycin 125 mg four times daily, surotomycin 250 mg twice daily is as active in vivo against C. difficile, but was more sparing of microbiota. Surotomycin is no longer in development due to failed Phase 3 efficacy results.

Should serum calcitonin be routinely measured in patients presenting with thyroid nodule?

BACKGROUND: The European Thyroid Association recommends serum calcitonin measurement in thyroid nodule cases. In contrast, the American Thyroid Association is ambivalent. In this institution, thyroid nodules cases are subject to a multidisciplinary evaluation of the clinical history and examination, ultrasound and scintigraphy, CT scan and sometimes MRI scan, biochemistry and histopathology of biopsies. We report on the current use of plasma calcitonin measurements in the context of changing practice which has not included screening of all thyroid nodules. METHODS: Laboratory records were searched from the beginning of January 2010 to the end of April 2016 for all serum calcitonin measurements. RESULTS: There were 44 patients (30 females, age range 31 to 87 years with median 57.5) and 14 males, age range 20 to 85 years with median 53.4 years) who had a serum calcitonin measured. Of these 33 patients did not have a detectable serum calcitonin. There were 3 patients who had an initial elevated serum calcitonin which became undetectable over time. Over the same time period, a total of 2070 patients presented with thyroid nodules. Medullary thyroid cancer (MTC) was found in 7 cases. Thus assuming all MTC cases had calcitonin measured, MTC is 7 of 341 (2.05%) of the total thyroid cancer burden at the hospital and 7 of 2070 (0.338%) of all thyroid nodules. Our practice is not to routine screen all nodules for MTC. CONCLUSIONS: Because patients with a nodule are subjected to ultrasound scanning and biopsy, when the nodule size is greater than 5 cm or when there is a modifying ultrasound or clinical characteristic, the consensus at the multidisciplinary conference on thyroids rather than universal calcitonin screening of all nodules is the better option in our judgement.

An Analysis of the Top-cited Articles in Emergency Medicine Education Literature.

INTRODUCTION: Dissemination of educational research is critical to improving medical education, promotion of faculty and ultimately patient care. The objective of this study was to identify the top 25 cited education articles in the emergency medicine (EM) literature and the top 25 cited EM education articles in all journals, as well as report on the characteristics of the articles. METHODS: Two searches were conducted in the Web of Science in June 2016 using a list of education-related search terms. We searched 19 EM journals for education articles as well as all other literature for EM education-related articles. Articles identified were reviewed for citation count, article type, journal, authors, and publication year. RESULTS: With regards to EM journals, the greatest number of articles were classified as articles/reviews, followed by research articles on topics such as deliberate practice (cited 266 times) and cognitive errors (cited 201 times). In contrast in the non-EM journals, research articles were predominant. Both searches found several simulation and ultrasound articles to be included. The most common EM journal was Academic Emergency Medicine (n = 18), and Academic Medicine was the most common non-EM journal (n=5). A reasonable number of articles included external funding sources (6 EM articles and 13 non-EM articles.). CONCLUSION: This study identified the most frequently cited medical education articles in the field of EM education, published in EM journals as well as all other journals indexed in Web of Science. The results identify impactful articles to medical education, providing a resource to educators while identifying trends that may be used to guide EM educational research and publishing efforts.

S4S8-RPA phosphorylation as an indicator of cancer progression in oral squamous cell carcinomas.

Oral cancers are easily accessible compared to many other cancers. Nevertheless, oral cancer is often diagnosed late, resulting in a poor prognosis. Most oral cancers are squamous cell carcinomas that predominantly develop from cell hyperplasias and dysplasias. DNA damage is induced in these tissues directly or indirectly in response to oncogene-induced deregulation of cellular proliferation. Consequently, a DNA Damage response (DDR) and a cell cycle checkpoint is activated. As dysplasia transitions to cancer, proteins involved in DNA damage and checkpoint signaling are mutated or silenced decreasing cell death while increasing genomic instability and allowing continued tumor progression. Hyperphosphorylation of Replication Protein A (RPA), including phosphorylation of Ser4 and Ser8 of RPA2, is a well-known indicator of DNA damage and checkpoint activation. In this study, we utilize S4S8-RPA phosphorylation as a marker for cancer development and progression in oral squamous cell carcinomas (OSCC). S4S8-RPA phosphorylation was observed to be low in normal cells, high in dysplasias, moderate in early grade tumors, and low in late stage tumors, essentially supporting the model of the DDR as an early barrier to tumorigenesis in certain types of cancers. In contrast, overall RPA expression was not correlative to DDR activation or tumor progression. Utilizing S4S8-RPA phosphorylation to indicate competent DDR activation in the future may have clinical significance in OSCC treatment decisions, by predicting the susceptibility of cancer cells to first-line platinum-based therapies for locally advanced, metastatic and recurrent OSCC.

In silico and in vitro methods to identify ebola virus VP35-dsRNA inhibitors.

Ebola virus continues to be problematic as sporadic outbreaks in Africa continue to arise, and as terrorist organizations have considered the virus for bioterrorism use. Several proteins within the virus have been targeted for antiviral chemotherapy, including VP35, a dsRNA binding protein that promotes viral replication, protects dsRNA from degradation, and prevents detection of the viral genome by immune complexes. To augment the scope of our antiviral research, we have now employed molecular modeling techniques to enrich the population of compounds for further testing in vitro. In the initial docking of a static VP35 structure with an 80,000 compound library, 40 compounds were selected, of which four compounds inhibited VP35 with IC50 <200µM, with the best compounds having an IC50 of 20µM. By superimposing 26 VP35 structures, we determined four aspartic acid residues were highly flexible and the docking was repeated under flexible parameters. Of 14 compounds chosen for testing, five compounds inhibited VP35 with IC50 <200µM and one compound with an IC50 of 4µM. These studies demonstrate the value of docking in silico for enriching compounds for testing in vitro, and specifically using multiple structures as a guide for detecting flexibility and provide a foundation for further development of small molecule inhibitors directed towards VP35.

Identification of inhibitors for single-stranded DNA-binding proteins in eubacteria.

OBJECTIVES: The increasing threat of drug-resistant bacteria establishes a continuing need for the development of new strategies to fight infection. We examine the inhibition of the essential single-stranded DNA-binding proteins (SSBs) SSBA and SSBB as a potential antimicrobial therapy due to their importance in DNA replication, activating the SOS response and promoting competence-based mechanisms of resistance by incorporating new DNA. METHODS: Purified recombinant SSBs from Gram-positive (Staphylococcus aureus and Bacillus anthracis) and Gram-negative (Escherichia coli and Francisella tularensis) bacteria were assessed in a high-throughput screen for inhibition of duplex DNA unwinding by small molecule inhibitors. Secondary electrophoretic mobility shift assays further validated the top hits that were then tested for MICs using in vitro assays. RESULTS: We have identified compounds that show cross-reactivity in vitro, as well as inhibition of both F. tularensis and B. anthracis SSBA. Five compounds were moderately toxic to at least two of the four bacterial strains in vivo, including two compounds that were selectively non-toxic to human cells, 9-hydroxyphenylfluoron and purpurogallin. Three of the SSBA inhibitors also inhibited S. aureus SSBB in Gram-positive bacteria. CONCLUSIONS: Results from our study support the potential for SSB inhibitors as broad-spectrum antibacterial agents, with dual targeting capabilities against Gram-positive bacteria.

Physiological and health characteristics of ex-jockeys.

OBJECTIVES: The long-term health implications resulting from extreme and chronic weight cycling and the associated energy restricted lifestyle of a professional jockey remain unknown. The aim of the present study was to describe and evaluate the physiological and health characteristics of retired jockeys. DESIGN: Cross-sectional. METHODS: Retired male jockeys (n=28; age 59±6yr; height 1.65±0.03m; body mass 73.7±10.9kg; BMI 26.9±3.8kgm(-2)) participated. Body composition (by dual energy X-ray absorptiometry; DXA), resting metabolic rate (RMR), glucose metabolism (by oral glucose tolerance test; OGTT), lipids and thyroid function were assessed. All data were interpreted in relation to established age specific reference ranges for the particular assessment undertaken. RESULTS: Forty three percent of the retired jockeys were classified as overweight, while 21% were obese. RMR was calculated as 1447±201kcalday(-1). Mean total cholesterol was 5.93±1.5mmolL(-1) for those not on cholesterol lowering medication and 5.09±1.1mmolL(-1) for those currently taking medication. No abnormalities in glucose metabolism and thyroid function were found. CONCLUSIONS: Chronic weight cycling during a career as a professional jockey did not induce any health consequences in the retired jockeys assessed in this study, however enhanced weight gain and dyslipidaemia were evident. Results suggest jockeys should be tracked longitudinally throughout their racing career and beyond to further establish long-term physical health implications associated with the current lifestyle of a professional jockey.

Differences of the Fecal Microflora With Clostridium difficile Therapies.

BACKGROUND: During treatment of Clostridium difficile infection (CDI), patterns of pathogen reduction in relationship to changes in components of the normal microbiota are hypothesized to be predictive of response to treatment and subsequent sustained cure. METHODS: At a single center, subjects enrolled into phase 2 and 3 C. difficile treatment clinical trials (2003-2008) provided fecal samples to assess killing of C. difficile and changes to components of the microbiome. Quantitative bacterial cultures, measurement of C. difficile toxin titers, quantitative polymerase chain reaction of fecal samples for Bacteroidetes, Clostridium clusters XIVa and IV, and C. difficile were performed. RESULTS: Quantitative bacterial cultures showed a mean log10 C. difficile count (colony-forming units [CFU]) of 6.7 ± 2.0 at study entry; vancomycin treatment consistently reduced C. difficile counts to the limit of detection (2.0 log10 CFU/g), whereas metronidazole was associated with mean C. difficile counts 1.5-2 log10 higher at 10 days of treatment. In patients receiving tolevamer, C. difficile persisted in high counts during treatment; response to treatment was correlated with neutralization of toxin along with persistence of normal microbiota components. However, this was achieved in approximately half of subjects. Both vancomycin and metronidazole further suppressed microbiome components during treatment of CDI. Lactobacilli were observed to be a microbiome component that persisted during treatment of CDI. CONCLUSIONS: Differences of pathogen clearance and microbiome perturbation during treatment of CDI appear to explain treatment outcomes. The hypothesis that probiotic microbes could help prevent onset of CDI is supported by the observation of persistence of lactobacilli during and after treatment of CDI.