A New Role for Translation Initiation Factor 2 in Maintaining Genome Integrity

Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA–damaging agent is present

Barriers and facilitators to using NHS Direct: a qualitative study of ‘users’ and ‘non-users’

Background NHS Direct, introduced in 1998, has provided 24/7 telephone-based healthcare advice and information to the public in England and Wales. National studies have suggested variation in the uptake of this service amongst the UK¿s diverse population. This study provides the first exploration of the barriers and facilitators that impact upon the uptake of this service from the perspectives of both `users¿ and `non- users¿. Methods Focus groups were held with NHS Direct `users¿ (N?=?2) from Bedfordshire alongside `non-users¿ from Manchester (N?=?3) and Mendip, Somerset (N?=?4). Each focus group had between five to eight participants. A total of eighty one people aged between 21 and 94 years old (M: 58.90, SD: 22.70) took part in this research. Each focus group discussion lasted approximately 90 minutes and was audiotape-recorded with participants¿ permission. The recordings were transcribed verbatim. A framework approach was used to analyse the transcripts. Results The findings from this research uncovered a range of barriers and facilitators that impact upon the uptake of NHS Direct. `Non-users¿ were unaware of the range of services that NHS Direct provided. Furthermore, `non-users¿ highlighted a preference for face-to face communication, identifying a lack of confidence in discussing healthcare over the telephone. This was particularly evident among older people with cognitive difficulties. The cost to telephone a `0845¿ number from a mobile was also viewed to be a barrier to access NHS Direct, expressed more often by `non-users¿ from deprived communities. NHS Direct `users¿ identified that awareness, ease of use and convenience were facilitators which influenced their decision to use the service. Conclusions An understanding of the barriers and facilitators which impact on the access and uptake of telephone-based healthcare is essential to move patients towards the self-care model. This research has highlighted the need for telephone-based healthcare services to increase public awareness; through the delivery of more targeted advertising to promote the service provision availabl

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Multidimensional signals and analytic flexibility: Estimating degrees of freedom in human speech analyses

Recent empirical studies have highlighted the large degree of analytic flexibility in data analysis which can lead to substantially different conclusions based on the same data set. Thus, researchers have expressed their concerns that these researcher degrees of freedom might facilitate bias and can lead to claims that do not stand the test of time. Even greater flexibility is to be expected in fields in which the primary data lend themselves to a variety of possible operationalizations. The multidimensional, temporally extended nature of speech constitutes an ideal testing ground for assessing the variability in analytic approaches, which derives not only from aspects of statistical modeling, but also from decisions regarding the quantification of the measured behavior. In the present study, we gave the same speech production data set to 46 teams of researchers and asked them to answer the same research question, resulting insubstantial variability in reported effect sizes and their interpretation. Using Bayesian meta-analytic tools, we further find little to no evidence that the observed variability can be explained by analysts’ prior beliefs, expertise or the perceived quality of their analyses. In light of this idiosyncratic variability, we recommend that researchers more transparently share details of their analysis, strengthen the link between theoretical construct and quantitative system and calibrate their (un)certainty in their conclusions

Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo

Meeting Abstracts: Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo Clearwater Beach, FL, USA. 9-11 June 201

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Role of IF2 isoforms in the major replication restart pathways.

<p>A) The major restart pathways and the influence of IF2 isoforms. Genetic analysis indicates that IF2-1 influences the restart pathways dependent on PriA helicase, including not only PriA-PriC pathway but also part of the PriA-PriB pathway. IF2-2/3 may play a prominent role in the pathways that do not require PriA helicase. The diagram should not be interpreted to indicate that IF2-2/3 cannot participate in reactions involving PriA helicase or that IF2-1 cannot participate in reactions where PriA's helicase is inactive. However, under these circumstances restart pathways do not function optimally to maintain maximal cell viability. The minor, less robust pathways such as the Rep-PriC pathway are not shown here. These PriA-independent pathways require suppressor mutations in <i>dnaC</i> to support a significant level of cell viability <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002648#pgen.1002648-Sandler1" target="_blank">[18]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002648#pgen.1002648-Sandler3" target="_blank">[38]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002648#pgen.1002648-Sandler4" target="_blank">[39]</a>. B) Model for the IF2-2/PriA helicase pathway. i) The starting point is a stalled replication fork with bound IF2-1. ii) Binding of restart proteins. The key protein to bind at this stage is PriA, which is poised to displace IF2-1 upon activation of its helicase activity. iii) Replisome assembly. PriA helicase action disassembles IF2-1 from the template, making way for initiation of DNA replication. It is hypothesized that removal of IF2-1 by PriA helicase can be regulated, extensive damage to the DNA template being able to inhibit this process and prevent replication restart. Removal of IF2-2/3 by PriA helicase bypasses this regulation, and IF2-2/3 may also be removed from the DNA by other mechanisms.</p

Binding of S-IF2 to Mu ends upon induction of phage replication by transposition.

<p>Mu development in GTN373 (<i>his::</i>Mu<i>cts62 priA300 del(gpt-lac)5</i>) and GTN622 (<i>his::</i>Mu<i>cts62 priA300 clpX::kan del(gpt-lac)5</i>) transformed with pBAD24-S-IF2-1, pBAD24-S-IF2-2, or pBAD24-IF2-2 were induced by incubation at 42°C, and immunoprecipitation with anti-S-tag monoclonal antibody was conducted with samples from 0 and 35 min postinduction. The presence of host and Mu DNA sequences in immunocomplexes were detected by amplifying 200–400 bp segments of template DNA (2 µl of indicated DNA dilutions in 10-µl reactions) in a 26-cycle PCR, using primers for amplifying <i>thrA</i>, <i>pyrD</i>, <i>serA</i>, Mu left end (L), Mu center (C), and Mu right end (R). A) Pull-down of Mu sequences by anti-S-tag antibody during Mu development. 1:20 dilutions of the immunoprecipitation (IP) and the no antibody control and 1∶2500 dilutions of total DNA were used. B) Comparison of IP with S-tagged and untagged IF2-2. GTN373 transformed with the indicated plasmids were used. C) Binding of S-IF2-2 concentrated at or near Mu ends. Analysis was conducted with induced pBAD24-S-IF2-2/GTN373, using one-fifth the standard amount of antibody.</p