109 research outputs found

    Cyclobutanone Inhibitor of Cobalt-Functionalized Metallo-γ-Lactonase AiiA with Cyclobutanone Ring Opening in the Active Site

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    An α-amido cyclobutanone possessing a C10 hydrocarbon tail was designed as a potential transition-state mimetic for the quorum-quenching metallo-γ-lactonase autoinducer inactivator A (AiiA) with the support of in-house modeling techniques and found to be a competitive inhibitor of dicobalt(II) AiiA with an inhibition constant of Ki = 0.007 ± 0.002 mM. The catalytic mechanism of AiiA was further explored using our product-based transition-state modeling (PBTSM) computational approach, providing substrate-intermediate models arising during enzyme turnover and further insight into substrate–enzyme interactions governing native substrate catalysis. These interactions were targeted in the docking of cyclobutanone hydrates into the active site of AiiA. The X-ray crystal structure of dicobalt(II) AiiA cocrystallized with this cyclobutanone inhibitor unexpectedly revealed an N-(2-oxocyclobutyl)decanamide ring-opened acyclic product bound to the enzyme active site (PDB 7L5F). The C10 alkyl chain and its interaction with the hydrophobic phenylalanine clamp region of AiiA adjacent to the active site enabled atomic placement of the ligand atoms, including the C10 alkyl chain. A mechanistic hypothesis for the ring opening is proposed involving a radical-mediated process

    Survey and Molecular Characterization of Sarcocystidae protozoa in Wild Cricetid Rodents from Central and Southern Chile

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    In Chile, studies of parasites from the family Sarcocystidae (Apicomplexa) have mostly been related to domestic animals. We aimed to assess the presence of Sarcocystidae taxa in cricetid rodents from Central and Southern Chile. We studied 207 rodents, encompassing six species, from 13 localities. We isolated DNA from tissue samples, amplified the Sarcocystidae 18S rRNA gene with polymerase chain reaction, and performed phylogenetic analyses using maximum likelihood and Bayesian inferences. In addition, we examined blood smears and performed histological studies in organs from Sarcocystidae DNA-positive animals. Three specimens were DNA-positive and three genotypes were retrieved and named: Sarcocystis sp. P61, related to Sarcocystis strixi, was detected in two Abrothrix olivacea. Toxoplasmatinae gen. sp. P99 was retrieved from those same two specimens, and was related to Toxoplasma and other genera, although it branched independently. Besnoitia sp. R34 was detected in one Abrothrix hirta, and was clustered with congeneric species associated with rodents. No protozoa were found during microscopic studies; thus, it was not possible to confirm parasitic interactions rather than accidental encounters. However, the close relatedness of the retrieved genotypes to parasites of rodents supports the hypothesis of host–parasite associations. All three genotypes are suggested as potential new taxa, including a putative new genus.This research was funded by the ANID Programa Becas Doctorado Nacional under grant numbers 2019–21190078, 2020–21200182, and 2022-21220118; and Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) under grant number 11170294.Peer reviewe

    Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

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    There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed

    Large-Scale Meta-GWAS Reveals Common Genetic Factors Linked to Radiation-Induced Acute Toxicities across Cancers

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    BACKGROUND: This study was designed to identify common genetic susceptibility and shared genetic variants associated with acute radiation-induced toxicity (RIT) across four cancer types (prostate, head and neck, breast, and lung).METHODS: A GWAS meta-analysis was performed using 19 cohorts including 12,042 patients. Acute standardized total average toxicity (rSTATacute) was modelled using a generalized linear regression model for additive effect of genetic variants adjusted for demographic and clinical covariates. LD score regression estimated shared SNP-based heritability of rSTATacute in all patients and for each cancer type.RESULTS: Shared SNP-based heritability of STATacute among all cancer types was estimated at 10% (se = 0.02), and was higher for prostate (17%, se = 0.07), head and neck (27%, se = 0.09), and breast (16%, se = 0.09) cancers. We identified 130 suggestive associated SNPs with rSTATacute (5.0x10-8&lt;P-value&lt;1.0x10-5) across 25 genomic regions. rs142667902 showed the strongest association (effect allele A; effect size -0.17; P-value=1.7x10-7), which is located near DPPA4, encoding a protein involved in pluripotency in stem cells, which are essential for repair of radiation-induced tissue injury. Gene-set enrichment analysis identified 'RNA splicing via endonucleolytic cleavage and ligation' (P = 5.1 x10-6, Pcorrected =0.079) as the top gene set associated with rSTATacute among all patients. In-silico gene expression analysis showed the genes associated with rSTATacute were statistically significantly up-regulated in skin (not sun exposed Pcorrected=0.004; sun exposed Pcorrected=0.026).CONCLUSIONS: There is shared SNP-based heritability for acute RIT across and within individual cancer sites. Future meta-GWAS among large radiotherapy patient cohorts are worthwhile to identify the common causal variants for acute radiotoxicity across cancer types.</p

    REQUITE: A prospective multicentre cohort study of patients undergoing radiotherapy for breast, lung or prostate cancer

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    Purpose: REQUITE aimed to establish a resource for multi-national validation of models and biomarkers that predict risk of late toxicity following radiotherapy. The purpose of this article is to provide summary descriptive data. Methods: An international, prospective cohort study recruited cancer patients in 26 hospitals in eight countries between April 2014 and March 2017. Target recruitment was 5300 patients. Eligible patients had breast, prostate or lung cancer and planned potentially curable radiotherapy. Radiotherapy was prescribed according to local regimens, but centres used standardised data collection forms. Pre-treatment blood samples were collected. Patients were followed for a minimum of 12 (lung) or 24 (breast/prostate) months and summary descriptive statistics were generated. Results: The study recruited 2069 breast (99% of target), 1808 prostate (86%) and 561 lung (51%) cancer patients. The centralised, accessible database includes: physician-(47,025 forms) and patient-(54,901) reported outcomes; 11,563 breast photos; 17,107 DICOMs and 12,684 DVHs. Imputed genotype data are available for 4223 patients with European ancestry (1948 breast, 1728 prostate, 547 lung). Radiation-induced lymphocyte apoptosis (RILA) assay data are available for 1319 patients. DNA (n = 4409) and PAXgene tubes (n = 3039) are stored in the centralised biobank. Example prevalences of 2-year (1-year for lung) grade >= 2 CTCAE toxicities are 13% atrophy (breast), 3% rectal bleeding (prostate) and 27% dyspnoea (lung). Conclusion: The comprehensive centralised database and linked biobank is a valuable resource for the radiotherapy community for validating predictive models and biomarkers. Patient summary: Up to half of cancer patients undergo radiation therapy and irradiation of surrounding healthy tissue is unavoidable. Damage to healthy tissue can affect short-and long-term quality-of-life. Not all patients are equally sensitive to radiation "damage" but it is not possible at the moment to identify those who are. REQUITE was established with the aim of trying to understand more about how we could predict radiation sensitivity. The purpose of this paper is to provide an overview and summary of the data and material available. In the REQUITE study 4400 breast, prostate and lung cancer patients filled out questionnaires and donated blood. A large amount of data was collected in the same way. With all these data and samples a database and biobank were created that showed it is possible to collect this kind of information in a standardised way across countries. In the future, our database and linked biobank will be a resource for research and validation of clinical predictors and models of radiation sensitivity. REQUITE will also enable a better understanding of how many people suffer with radiotherapy toxicity

    Continuous glucose monitoring in pregnant women with type 1 diabetes (CONCEPTT): a multicentre international randomised controlled trial.

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    BACKGROUND: Pregnant women with type 1 diabetes are a high-risk population who are recommended to strive for optimal glucose control, but neonatal outcomes attributed to maternal hyperglycaemia remain suboptimal. Our aim was to examine the effectiveness of continuous glucose monitoring (CGM) on maternal glucose control and obstetric and neonatal health outcomes. METHODS: In this multicentre, open-label, randomised controlled trial, we recruited women aged 18-40 years with type 1 diabetes for a minimum of 12 months who were receiving intensive insulin therapy. Participants were pregnant (≤13 weeks and 6 days' gestation) or planning pregnancy from 31 hospitals in Canada, England, Scotland, Spain, Italy, Ireland, and the USA. We ran two trials in parallel for pregnant participants and for participants planning pregnancy. In both trials, participants were randomly assigned to either CGM in addition to capillary glucose monitoring or capillary glucose monitoring alone. Randomisation was stratified by insulin delivery (pump or injections) and baseline glycated haemoglobin (HbA1c). The primary outcome was change in HbA1c from randomisation to 34 weeks' gestation in pregnant women and to 24 weeks or conception in women planning pregnancy, and was assessed in all randomised participants with baseline assessments. Secondary outcomes included obstetric and neonatal health outcomes, assessed with all available data without imputation. This trial is registered with ClinicalTrials.gov, number NCT01788527. FINDINGS: Between March 25, 2013, and March 22, 2016, we randomly assigned 325 women (215 pregnant, 110 planning pregnancy) to capillary glucose monitoring with CGM (108 pregnant and 53 planning pregnancy) or without (107 pregnant and 57 planning pregnancy). We found a small difference in HbA1c in pregnant women using CGM (mean difference -0·19%; 95% CI -0·34 to -0·03; p=0·0207). Pregnant CGM users spent more time in target (68% vs 61%; p=0·0034) and less time hyperglycaemic (27% vs 32%; p=0·0279) than did pregnant control participants, with comparable severe hypoglycaemia episodes (18 CGM and 21 control) and time spent hypoglycaemic (3% vs 4%; p=0·10). Neonatal health outcomes were significantly improved, with lower incidence of large for gestational age (odds ratio 0·51, 95% CI 0·28 to 0·90; p=0·0210), fewer neonatal intensive care admissions lasting more than 24 h (0·48; 0·26 to 0·86; p=0·0157), fewer incidences of neonatal hypoglycaemia (0·45; 0·22 to 0·89; p=0·0250), and 1-day shorter length of hospital stay (p=0·0091). We found no apparent benefit of CGM in women planning pregnancy. Adverse events occurred in 51 (48%) of CGM participants and 43 (40%) of control participants in the pregnancy trial, and in 12 (27%) of CGM participants and 21 (37%) of control participants in the planning pregnancy trial. Serious adverse events occurred in 13 (6%) participants in the pregnancy trial (eight [7%] CGM, five [5%] control) and in three (3%) participants in the planning pregnancy trial (two [4%] CGM and one [2%] control). The most common adverse events were skin reactions occurring in 49 (48%) of 103 CGM participants and eight (8%) of 104 control participants during pregnancy and in 23 (44%) of 52 CGM participants and five (9%) of 57 control participants in the planning pregnancy trial. The most common serious adverse events were gastrointestinal (nausea and vomiting in four participants during pregnancy and three participants planning pregnancy). INTERPRETATION: Use of CGM during pregnancy in patients with type 1 diabetes is associated with improved neonatal outcomes, which are likely to be attributed to reduced exposure to maternal hyperglycaemia. CGM should be offered to all pregnant women with type 1 diabetes using intensive insulin therapy. This study is the first to indicate potential for improvements in non-glycaemic health outcomes from CGM use. FUNDING: Juvenile Diabetes Research Foundation, Canadian Clinical Trials Network, and National Institute for Health Research

    Finishing the euchromatic sequence of the human genome

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    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
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