DNA binding proteins of the phage λ ninR region

Bacteriophages are known to disable host functions in order to impose their own enzymes to hijack the bacterium for maximal replication of the virus. One well-characterised example of this is the Red genetic recombination system of phage λ which blocks host RecBCD and replaces it with an exonuclease and synaptase to promote genomic rearrangements. Other substitute enzymes from λ include Orf, which can replace the RecA-loading activity of RecFOR, and Rap, which can substitute for the RuvC Holliday junction resolvase. This study has investigated the DNA binding activities of three gene products from the ninR region of phage λ that are linked to genetic recombination. These are Orf (NinB), Rap (NinG) and NinH, the latter an uncharacterised protein of 68 residues. DNA binding analysis with selected substitution mutants of the Orf protein established that the central channel and adjacent regions are critical for binding to single-stranded DNA. The predicted HNH catalytic site of the Rap DNA structure-specific endonuclease was also investigated by site-directed mutagenesis confirming that the HNH domain is essential for Holliday junction cleavage and may also participate in branched DNA binding. Finally, the NinH protein which resembles the bacterial nucleoid associated protein (NAP) Fis, was also analysed for its ability to bind double-stranded DNA. The in vitro data showed that NinH binds DNA and preferentially favours bent duplex substrates, consistent with its similarity to Fis. NinH could not compensate for the loss of various NAPs from E. coli in deletion strains, including a fis mutant, and generally conferred a negative effect on the growth of these strains. Thus NinH may play a role in perturbing normal bacterial replication to promote phage production. The results offer several new insights into the properties of these three DNA binding proteins and their possible roles in phage DNA replication and recombination

Preliminary evaluation of a rapid lateral flow calprotectin test for the diagnosis of prosthetic joint infection

Aims This pilot study tested the performance of a rapid assay for diagnosing prosthetic joint infection (PJI), which measures synovial fluid calprotectin from total hip and knee revision patients. Methods A convenience series of 69 synovial fluid samples from revision patients at the Norfolk and Norwich University Hospital were collected intraoperatively (52 hips, 17 knees) and frozen. Synovial fluid calprotectin was measured retrospectively using a new commercially available lateral flow assay for PJI diagnosis (Lyfstone AS) and compared to International Consensus Meeting (ICM) 2018 criteria and clinical case review (ICM-CR) gold standards. Results According to ICM, 24 patients were defined as PJI positive and the remaining 45 were negative. The overall accuracy of the lateral flow test compared to ICM was 75.36% (52/69, 95% CI 63.51% to 84.95%), sensitivity and specificity were 75.00% (18/24, 95% CI 53.29% to 90.23%) and 75.56% (34/45, 95% CI 60.46% to 87.12%), respectively, positive predictive value (PPV) was 62.07% (18/29, 95% CI 48.23% to 74.19%) and negative predictive value (NPV) was 85.00% (34/40, 95% CI 73.54% to 92.04%), and area under the receiver operating characteristic (ROC) curve (AUC) was 0.78 (95% CI 0.66 to 0.87). Patient data from discordant cases were reviewed by the clinical team to develop the ICM-CR gold standard. The lateral flow test performance improved significantly when compared to ICM-CR, with accuracy increasing to 82.61% (57/69, 95% CI 71.59% to 90.68%), sensitivity increasing to 94.74% (18/19, 95% CI 73.97% to 99.87%), NPV increasing to 97.50% (39/40, 95% CI 85.20% to 99.62%), and AUC increasing to 0.91 (95% CI 0.81 to 0.96). Test performance was better in knees (100.00% accurate (17/17, 95% CI 80.49% to 100.00%)) compared to hips (76.92% accurate (40/52, 95% CI 63.16% to 87.47%)). Conclusion This study demonstrates that the calprotectin lateral flow assay could be an effective diagnostic test for PJI, however additional prospective studies testing fresh samples are required

A generalizable data-driven multicellular model of pancreatic ductal adenocarcinoma.

BACKGROUND: Mechanistic models, when combined with pertinent data, can improve our knowledge regarding important molecular and cellular mechanisms found in cancer. These models make the prediction of tissue-level response to drug treatment possible, which can lead to new therapies and improved patient outcomes. Here we present a data-driven multiscale modeling framework to study molecular interactions between cancer, stromal, and immune cells found in the tumor microenvironment. We also develop methods to use molecular data available in The Cancer Genome Atlas to generate sample-specific models of cancer. RESULTS: By combining published models of different cells relevant to pancreatic ductal adenocarcinoma (PDAC), we built an agent-based model of the multicellular pancreatic tumor microenvironment, formally describing cell type-specific molecular interactions and cytokine-mediated cell-cell communications. We used an ensemble-based modeling approach to systematically explore how variations in the tumor microenvironment affect the viability of cancer cells. The results suggest that the autocrine loop involving EGF signaling is a key interaction modulator between pancreatic cancer and stellate cells. EGF is also found to be associated with previously described subtypes of PDAC. Moreover, the model allows a systematic exploration of the effect of possible therapeutic perturbations; our simulations suggest that reducing bFGF secretion by stellate cells will have, on average, a positive impact on cancer apoptosis. CONCLUSIONS: The developed framework allows model-driven hypotheses to be generated regarding therapeutically relevant PDAC states with potential molecular and cellular drivers indicating specific intervention strategies

Racial and ethnic disparities in access to liver transplantation

Access to liver transplantation is reportedly inequitable for racial/ethnic minorities, but inadequate adjustments for geography and disease progression preclude any meaningful conclusions. We aimed to evaluate the association between candidate race/ethnicity and liver transplant rates after thorough adjustments for these factors and to determine how uniform racial/ethnic disparities were across Model for End-Stage Liver Disease (MELD) scores. Chronic end-stage liver disease candidates initially wait-listed between February 28, 2002 and February 27, 2007 were identified from Scientific Registry for Transplant Recipients data. The primary outcome was deceased donor liver transplantation (DDLT); the primary exposure covariate was race/ethnicity (white, African American, Hispanic, Asian, and other). Cox regression was used to estimate the covariate-adjusted DDLT rates by race/ethnicity, which were stratified by the donation service area and MELD score. With averaging across all MELD scores, African Americans, Asians, and others had similar adjusted DDLT rates in comparison with whites. However, Hispanics had an 8% lower DDLT rate versus whites [hazard ratio (HR) = 0.92, P = 0.011]. The disparity among Hispanics was concentrated among patients with MELD scores < 20, with HR = 0.84 ( P = 0.021) for MELD scores of 6 to 14 and HR = 0.85 ( P = 0.009) for MELD scores of 15 to 19. Asians with MELD scores < 15 had a 24% higher DDLT rate with respect to whites (HR = 1.24, P = 0.024). However, Asians with MELD scores of 30 to 40 had a 46% lower DDLT rate (HR = 0.54, P = 0.004). In conclusion, although African Americans did not have significantly different DDLT rates in comparison with similar white candidates, race/ethnicity-based disparities were prominent among subgroups of Hispanic and Asian candidates. By precluding the survival benefit of liver transplantation, this inequity may lead to excess mortality for minority candidates. Liver Transpl 16:1033–1040, 2010. © 2010 AASLD.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78074/1/22108_ftp.pd

Phage Orf family recombinases:conservation of activities and involvement of the central channel in DNA binding

Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redβ recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized. As with its λ relative, YbcN showed a preference for binding ssDNA over duplex. Neither Orf nor YbcN displayed a significant preference for duplex DNA containing mismatches or 1-3 nucleotide bulges. YbcN also bound E. coli SSB, although unlike Orf, it failed to associate with an SSB mutant lacking the flexible C-terminal tail involved in coordinating heterologous protein-protein interactions. Residues conserved in the Orf family that flank the central cavity in the λ Orf crystal structure were targeted for mutagenesis to help determine the mode of DNA binding. Several of these mutant proteins showed significant defects in DNA binding consistent with the central aperture being important for substrate recognition. The widespread conservation of Orf-like proteins highlights the importance of targeting SSB coated ssDNA during lambdoid phage recombination

Integrative network modeling reveals mechanisms underlying T cell exhaustion.

Failure to clear antigens causes CD8+ T cells to become increasingly hypo-functional, a state known as exhaustion. We combined manually extracted information from published literature with gene expression data from diverse model systems to infer a set of molecular regulatory interactions that underpin exhaustion. Topological analysis and simulation modeling of the network suggests CD8+ T cells undergo 2 major transitions in state following stimulation. The time cells spend in the earlier pro-memory/proliferative (PP) state is a fixed and inherent property of the network structure. Transition to the second state is necessary for exhaustion. Combining insights from network topology analysis and simulation modeling, we predict the extent to which each node in our network drives cells towards an exhausted state. We demonstrate the utility of our approach by experimentally testing the prediction that drug-induced interference with EZH2 function increases the proportion of pro-memory/proliferative cells in the early days post-activation

Boron isotope composition of the cold-water coral Lophelia pertusa along the Norwegian margin: Zooming into a potential pH-proxy by combining bulk and high-resolution approaches

High-latitude cold-water coral reefs are particularly vulnerable to climate change due to enhanced CO2 uptake in these regions. To evaluate their physiological functioning and potential application as pH archives, we retrieved both recent and fossil samples of Lophelia pertusa along the Norwegian margin from Oslofjord (59°N), over to Trondheimsfjord, Sula and Lopphavet (70.6°N). Boron isotope analyses (δ11B) were undertaken using solution-based and laser ablation multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS; LA-ICP-MS), and secondary ion mass spectrometry (SIMS). Epi-fluorescence microscopy was employed to provide a rapid pre-screening routine for structure-specific subsampling in the coral skeleton. This integrated approach enabled us to assess heterogeneities within single specimens, as well as to investigate the role of local environmental influences including recent and past variations. All three mass spectrometry methods show substantial differences in the δ11B of the theca wall (TW) and the centres of calcification (COC's). Micro-bulk subsamples milled from the theca wall of modern specimens originating from different habitats but with comparable seawater pH (8–8.16) gave consistent δ11B values averaging 26.7 (±0.2‰, 2σ, n = 4), while COC subsamples systematically deviated towards lower B/Ca (by ~40%) and depleted δ11B values (minimum 22.7 ± 0.3‰, 2σ), implying a difference of at least 4‰ between TW and COC. SIMS and LA-ICP-MS measurements identified much larger internal heterogeneities with maximum variation of ~10‰ between the distinct skeletal structures; minimal SIMS δ11B values of ~17.3 ± 1.2‰ (2σ) were associated with the pure COC material. Our findings may be interpreted in terms of the occurrence of two main, but likely different, biomineralisation mechanisms in L. pertusa, with the COC's generally exhibiting minimal pH up-regulation, potentially supporting the use of bicarbonate in the early stages of biomineralisation. Furthermore, we highlight the potential utility of L. pertusa for palaeo-proxy studies if targeting the compositionally homogenous TW zones devoid of COC admixtures, which appear to provide highly reproducible measurements

Twin peaks: the Omicron SARS-CoV-2 BA.1 and BA.2 epidemics in England

BACKGROUND Rapid transmission of the SARS-CoV-2 Omicron variant has led to record-breaking incidence rates around the world. Sub-lineages have been detected in many countries with BA.1 replacing Delta and BA.2 replacing BA.1. METHODS The REal-time Assessment of Community Transmission-1 (REACT-1) study has tracked SARS-CoV-2 infection in England using RT-PCR results from self-administered throat and nose swabs from randomly-selected participants aged 5+ years. Rounds of data collection were approximately monthly from May 2020 to March 2022. RESULTS In March 2022, weighted prevalence was 6.37% (N=109,181), more than twice that in February 2022 following an initial Omicron peak in January 2022. Of the lineages determined by viral genome sequencing, 3,382 (99.97%) were Omicron, including 346 (10.2%) BA.1, 3035 (89.7%) BA.2 and one (0.03%) BA.3 sub-lineage; the remainder (1, 0.03%) was Delta AY.4. The BA.2 Omicron sub-lineage had a growth rate advantage (compared to BA.1 and sub-lineages) of 0.11 (95% credible interval [CrI], 0.10, 0.11). Prevalence was increasing overall (reproduction number R=1.07, 95% CrI, 1.06, 1.09), with the greatest increase in those aged 55+ years (R=1.12, 95% CrI, 1.09, 1.14) among whom estimated prevalence on March 31, 2022 was 8.31%, nearly 20-fold the median prevalence since May 1, 2020. CONCLUSIONS We observed unprecedented levels of SARS-CoV-2 infection in England in March 2022 and an almost complete replacement of Omicron BA.1 by BA.2. The high and increasing prevalence in older adults may increase hospitalizations and deaths despite high levels of vaccination. (Funded by the Department of Health and Social Care in England.

Baryon-baryon interactions in the SU6 quark model and their applications to light nuclear systems

Interactions between the octet-baryons (B8) in the spin-flavor SU6 quark model are investigated in a unified coupled-channels framework of the resonating-group method (RGM). The interaction Hamiltonian for quarks consists of the phenomenological confinement potential, the color Fermi-Breit interaction with explicit flavor-symmetry breaking (FSB), and effective-meson exchange potentials of scalar-, pseudoscalar- and vector-meson types. The model parameters are determined to reproduce the properties of the nucleon-nucleon (NN) system and the low-energy cross section data for the hyperon-nucleon (YN) interactions. The NN phase shifts and many observables for the NN and YN interactions are nicely reproduced. Properties of these B8 B8 interactions are analyzed through the G-matrix calculations. The B8 B8 interactions are then applied to some of few-baryon systems and light Lambda-hypernuclei in a three-cluster Faddeev formalism using two-cluster RGM kernels. An application to the three-nucleon system shows that the quark-model NN interaction can give a sufficient triton binding energy with little room for the three-nucleon force. The hypertriton Faddeev calculation indicates that the attraction of the Lambda N interaction in the 1S0 state is only slightly more attractive than that in the 3S1 state. In the application to the alpha alpha Lambda system, the energy spectrum of 9 Lambda Be is well reproduced using the alpha alpha RGM kernel. The very small spin-orbit splitting of the 9 Lambda Be excited states is also discussed. In the Lambda Lambda alpha Faddeev calculation, the NAGARA event for 6 Lambda Lambda He is found to be consistent with the quark-model Lambda Lambda interaction.Comment: 77 pages, 33 figures, review article to be published in Prog. Part. Nucl. Phy

Exponential growth, high prevalence of SARS-CoV-2, and vaccine effectiveness associated with the Delta variant.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections were rising during early summer 2021 in many countries as a result of the Delta variant. We assessed reverse transcription polymerase chain reaction swab positivity in the Real-time Assessment of Community Transmission–1 (REACT-1) study in England. During June and July 2021, we observed sustained exponential growth with an average doubling time of 25 days, driven by complete replacement of the Alpha variant by Delta and by high prevalence at younger, less-vaccinated ages. Prevalence among unvaccinated people [1.21% (95% credible interval 1.03%, 1.41%)] was three times that among double-vaccinated people [0.40% (95% credible interval 0.34%, 0.48%)]. However, after adjusting for age and other variables, vaccine effectiveness for double-vaccinated people was estimated at between ~50% and ~60% during this period in England. Increased social mixing in the presence of Delta had the potential to generate sustained growth in infections, even at high levels of vaccination.The study was funded by the Department of Health and Social Care in England. Sequencing was provided through funding from the COVID-19 Genomics UK (COG-UK) Consortium. P.E. is Director of the Medical Research Council (MRC) Centre for Environment and Health (MR/L01341X/1, MR/S019669/1). P.E. acknowledges support from Health Data Research UK (HDR UK); the National Institute for Health Research (NIHR) Imperial Biomedical Research Centre; NIHR Health Protection Research Units (HPRUs) in Chemical and Radiation Threats and Hazards, and Environmental Exposures and Health; the British Heart Foundation Centre for Research Excellence at Imperial College London (RE/18/4/34215); and the UK Dementia Research Institute at Imperial (MC_PC_17114). S.R., C.A.D. acknowledge support: MRC Centre for Global Infectious Disease Analysis, NIHR HPRU in Modelling and Health Economics, Wellcome Trust (200861/Z/16/Z, 200187/Z/15/Z), and Centres for Disease Control and Prevention (US, U01CK0005-01-02). G.C. is supported by an NIHR Professorship. H.War. acknowledges support from an NIHR Senior Investigator Award and the Wellcome Trust (205456/Z/16/Z). We thank The Huo Family Foundation for their support of our work on COVID-19. Quadram authors gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council (BBSRC); their research was funded by the BBSRC Institute Strategic Programme Microbes in the Food Chain BB/R012504/1 and its constituent project BBS/E/F/000PR10352. We thank members of the COVID-19 Genomics Consortium UK (COG-UK) for their contributions to generating the genomic data used in this study. COG-UK is supported by funding from the MRC, part of UK Research & Innovation (UKRI), NIHR and Genome Research Limited, operating as the Wellcome Sanger Institute