Probing conformational dynamics of DNA binding by CO-sensing transcription factor, CooA.

The transcription factor CooA is a CRP/FNR (cAMP receptor protein/ fumarate and nitrate reductase) superfamily protein that uses heme to sense carbon monoxide (CO). Allosteric activation of CooA in response to CO binding is currently described as a series of discrete structural changes, without much consideration for the potential role of protein dynamics in the process of DNA binding. This work uses site-directed spin-label electron paramagnetic resonance spectroscopy (SDSL-EPR) to probe slow timescale (µs-ms) conformational dynamics of CooA with a redox-stable nitroxide spin label, and IR spectroscopy to probe the environment at the CO-bound heme. A series of cysteine substitution variants were created to selectively label CooA in key functional regions, the heme-binding domain, the 4/5-loop, the hinge region, and the DNA binding domain. The EPR spectra of labeled CooA variants are compared across three functional states: Fe(III) "locked off", Fe(II)-CO "on", and Fe(II)-CO bound to DNA. We observe changes in the multicomponent EPR spectra at each location; most notably in the hinge region and DNA binding domain, broadening the description of the CooA allosteric mechanism to include the role of protein dynamics in DNA binding. DNA-dependent changes in IR vibrational frequency and band broadening further suggest that there is conformational heterogeneity in the active WT protein and that DNA binding alters the environment of the heme-bound CO.

Local nuclear to cytoplasmic ratio regulates chaperone-dependent H3 variant incorporation during zygotic genome activation.

Early embryos often have relatively unstructured chromatin that lacks active and inactive domains typical of differentiated cells. In many species, these regulatory domains are established during zygotic genome activation (ZGA). In Drosophila, ZGA occurs after 13 fast, reductive, syncytial nuclear divisions during which the nuclear to cytoplasmic (N/C) ratio grows exponentially. These divisions incorporate maternally-loaded, cytoplasmic pools of histones into chromatin. Previous work found that chromatin incorporation of replication-coupled histone H3 decreases while its variant H3.3 increases in the cell cycles leading up to ZGA. In other cell types, H3.3 is associated with sites of active transcription as well as heterochromatin, suggesting a link between H3.3 incorporation and ZGA. Here, we examine the factors that contribute to H3.3 incorporation at ZGA. We identify a more rapid decrease in the nuclear availability of H3 than H3.3 over the final pre-ZGA cycles. We also observe an N/C ratio-dependent increase in H3.3 incorporation in mutant embryos with non-uniform local N/C ratios. We find that chaperone binding, not gene expression, controls incorporation patterns using H3/H3.3 chimeric proteins at the endogenous H3.3A locus. We test the specificity of the H3.3 chaperone pathways for H3.3 incorporation using Hira (H3.3 chaperone) mutant embryos. Overall, we propose a model in which local N/C ratios and specific chaperone binding regulate differential incorporation of H3.3 during ZGA.

Characterization of microbiota and histology of cultured sea cucumber Isostichopus badionotus juveniles during an outbreak of skin ulceration syndrome.

Due to the dramatic reduction of sea cucumber Isostichopus badionotus populations in the Yucatan Peninsula by overfishing and poaching, aquaculture has been encouraged as an alternative to commercial catching and restoring wild populations. However, the scarcity of broodstock, the emergence of a new disease in the auricularia larvae stage, and the development of skin ulceration syndrome (SUS) in the culture have limited aquaculture development. This study presents the changes in the intestine and skin microbiota observed in early and advanced stages of SUS disease in cultured juvenile I. badionotus obtained during an outbreak in experimental culture through 16S rRNA gene sequencing and histological evidence. Our results showed inflammation in the intestines of juveniles at both stages of SUS. However, more severe tissue damage and the presence of bacterial clusters were detected only in the advanced stages of SUS. Differences in the composition and structure of the intestinal and skin bacterial community from early and advanced stages of SUS were detected, with more evident changes in the intestinal microbial communities. These findings suggest that SUS was not induced by a single pathogenic bacterium. Nevertheless, a decrease in the abundance of Vibrio and an increase in Halarcobacter (syn. Arcobacter) was observed, suggesting that these two bacterial groups could be keystone genera involved in SUS disease.

Perioperative Exercise Testing in Pregnant and Non-Pregnant Women of Reproductive Age: A Systematic Review.

BACKGROUND: Women have classically been excluded from the development of normal data and reference ranges, with pregnant women experiencing further neglect. The incidence of Caesarean section in pregnant women, and of general operative management in young women (both pregnant and non-pregnant), necessitates the formal development of healthy baseline data in these cohorts to optimise their perioperative management. This systematic review assesses the representation of young women in existing reference ranges for several functional exercise tests in common use to facilitate functional assessment in this cohort. METHODS: Existing reference range data for the exercise tests the Six Minute Walk Test (6MWT), the Incremental Shuttle Walk Test (ISWT) and Cardiopulmonary Exercise Testing (CPET) in young women of reproductive age were assessed using the MEDLINE (Ovid) database, last searched December 2023. Results were comparatively tabulated but not statistically analysed given underlying variances in data. RESULTS: The role of exercise testing in the perioperative period as an assessment tool, as well as its safety during pregnancy, was evaluated using 65 studies which met inclusion criteria. CONCLUSION: There is a significant lack of baseline data regarding these tests in this population, especially amongst the pregnant cohort, which limits the application of exercise testing clinically.

Postoperative Radiographic Outcomes Following Primary Open Coracoid Transfer (Bristow-Latarjet) Vary in Definition, Classification, and Imaging Modality: A Systematic Review.

PURPOSE: To analyze radiographic outcomes by conventional radiography, computed tomography (CT), or both and complication rates of open coracoid transfer at a minimum of 12-months follow-up. METHODS: A literature search was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, using PubMed, Medline (Ovid), and EMBASE library databases. Inclusion criteria were clinical studies reporting on open Latarjet as the primary surgical procedure(revision coracoid transfer after failed prior stabilization excluded) with postoperative radiographic outcomes at a minimum mean 1-year follow-up. Patient demographics, type of postoperative imaging modality, and radiographic outcomes and complications including graft union, osteoarthritis, and osteolysis were systematically reviewed. Data were summarized as ranges of reported values for each outcome metric. Each radiographic outcome was graphically represented in a Forest plot with point estimates of the incidence of radiographic outcomes with corresponding 95% confidence intervals and I2. RESULTS: Thirty-three studies met inclusion criteria, with a total of 1,456 shoulders. The most common postoperative imaging modality was plain radiography only (n = 848 [58.2%]), both CT and radiography (n = 287 [19.7%]), and CT only (n = 321 [22.1%]). Overall, the reported graft union rate ranged from 75% to 100%, of which 79.8% (n = 395) were detected on plain radiography. The most common reported postoperative radiographic complications after the open coracoid transfer were osteoarthritis (range, 0%-100%, pooled mean 28%), graft osteolysis (range, 0%-100%, pooled mean 30%), nonunion (range, 0%-32%, pooled mean 5.1%), malpositioned graft (range, 0%-75%, pooled mean 14.75%), hardware issues (range, 0%-9.1%, pooled mean 5%), and bone block fracture (range, 0%-8%, pooled mean 2.1%). Graft healing was achieved in a majority of cases (range, 75%-100%). CONCLUSION: Postoperative radiographic outcomes after open coracoid transfer vary greatly in definition, classification, and imaging modality of choice. Greater consistency in postoperative radiographic outcomes is essential to evaluate graft healing, osteolysis, and nonunion. LEVEL OF EVIDENCE: Level IV, systematic review of Level III-IV studies.

Vav proteins do not influence dengue virus replication but are associated with induction of phospho-ERK, IL-6, and viperin mRNA following DENV infection in vitro.

IMPORTANCE: Dengue disease is characterized by an inflammatory-mediated immunopathology, with elevated levels of circulating factors including TNF-α and IL-6. If the damaging inflammatory pathways could be blocked without loss of antiviral responses or exacerbating viral replication, then this would be of potential therapeutic benefit. The study here has investigated the Vav guanine exchange factors as a potential alternative signaling pathway that may drive dengue virus (DENV)-induced inflammatory responses, with a focus on Vav1 and 2. While Vav proteins were positively associated with mRNA for inflammatory cytokines, blocking Vav signaling didn't affect DENV replication but prevented DENV-induction of p-ERK and enhanced IL-6 (inflammatory) and viperin (antiviral) mRNA. These initial data suggest that Vav proteins could be a target that does not compromise control of viral replication and should be investigated further for broader impact on host inflammatory responses, in settings such as antibody-dependent enhancement of infection and in different cell types.

One-Step Selective Labeling of Native Cell Surface Sialoglycans by Exogenous α2,8-Sialylation.

Exo-enzymatic glycan labeling strategies have emerged as versatile tools for efficient and selective installation of terminal glyco-motifs onto live cell surfaces. Through employing specific enzymes and nucleotide-sugar probes, cells can be equipped with defined glyco-epitopes for modulating cell function or selective visualization and enrichment of glycoconjugates. Here, we identifyCampylobacter jejunisialyltransferase Cst-II I53S as a tool for cell surface glycan modification, expanding the exo-enzymatic labeling toolkit to include installation of α2,8-disialyl epitopes. Labeling with Cst-II was achieved with biotin- and azide-tagged CMP-Neu5Ac derivatives on a model glycoprotein and native sialylated cell surface glycans across a panel of cell lines. The introduction of modified Neu5Ac derivatives onto cells by Cst-II was also retained on the surface for 6 h. By examining the specificity of Cst-II on cell surfaces, it was revealed that the α2,8-sialyltransferase primarily labeled N-glycans, with O-glycans labeled to a lesser extent, and there was an apparent preference for α2,3-linked sialosides on cells. This approach thus broadens the scope of tools for selective exo-enzymatic labeling of native sialylated glycans and is highly amenable for the construction of cell-based arrays.

Peptide-mimetic treatment of Pseudomonas aeruginosa in a mouse model of respiratory infection.

The rise of drug resistance has become a global crisis, with >1 million deaths due to resistant bacterial infections each year. Pseudomonas aeruginosa, in particular, remains a serious problem with limited solutions due to complex resistance mechanisms that now lead to more than 32,000 multidrug-resistant (MDR) infections and over 2,000 deaths annually. While the emergence of resistant bacteria has become concerningly common, identification of useful new drug classes has been limited over the past 40+ years. We found that a potential novel therapeutic, the peptide-mimetic TM5, is effective at killing P. aeruginosa and displays sufficiently low toxicity for mammalian cells to allow for use in treatment of infections. Interestingly, TM5 kills P. aeruginosa more rapidly than traditional antibiotics, within 30-60 minutes in vitro , and is effective against a range of clinical isolates. In vivo , TM5 significantly reduced bacterial load in the lungs within 24 hours compared to untreated mice and demonstrated few adverse effects. Taken together, these observations suggest that TM5 shows promise as an alternative therapy for MDR P. aeruginosa respiratory infections.

Bacillus Calmette-Guérin vaccination as defense against SARS-CoV-2 (BADAS): a randomized controlled trial to protect healthcare workers in the USA by enhanced trained immune responses.

BACKGROUND: A large epidemic, such as that observed with SARS-CoV-2, seriously challenges available hospital capacity, and this would be augmented by infection of healthcare workers (HCW). Bacillus Calmette-Guérin (BCG) is a vaccine against tuberculosis, with protective non-specific effects against other respiratory tract infections in vitro and in vivo. Preliminary analyses suggest that regions of the world with existing BCG vaccination programs have lower incidence and mortality from COVID-19. We hypothesize that BCG vaccination can reduce SARS-CoV-2 infection and disease severity. METHODS: This will be a placebo-controlled adaptive multi-center randomized controlled trial. A total of 1800 individuals considered to be at high risk, including those with comorbidities (hypertension, diabetes, obesity, reactive airway disease, smokers), racial and ethnic minorities, elderly, teachers, police, restaurant wait-staff, delivery personnel, health care workers who are defined as personnel working in a healthcare setting, at a hospital, medical center or clinic (veterinary, dental, ophthalmology), and first responders (paramedics, firefighters, or law enforcement), will be randomly assigned to two treatment groups. The treatment groups will receive intradermal administration of BCG vaccine or placebo (saline) with groups at a 1:1 ratio. Individuals will be tracked for evidence of SARS-CoV-2 infection and severity as well as obtaining whole blood to track immunological markers, and a sub-study will include cognitive function and brain imaging. The majority of individuals will be followed for 6 months, with an option to extend for another 6 months, and the cognitive sub-study duration is 2 years. We will plot Kaplan-Meier curves that will be plotted comparing groups and hazard ratios and p-values reported using Cox proportional hazard models. DISCUSSION: It is expected this trial will allow evaluation of the effects of BCG vaccination at a population level in high-risk healthcare individuals through a mitigated clinical course of SARS-CoV-2 infection and inform policy making during the ongoing epidemic. TRIAL REGISTRATION: ClinicalTrials.gov NCT04348370. Registered on April 16, 2020.

Pulmonary Expression of Interleukin-17 Contributes to Neutrophil Infiltration into the Lungs during Pneumonic Plague.

Inhalation of respiratory droplets infected with Yersinia pestis results in a rapidly progressing and lethal necrotic pneumonia called primary pneumonic plague. Disease manifests as biphasic, with an initial preinflammatory phase with rapid bacterial replication in the lungs absent readily detectable host immune responses. This is followed by the onset of a proinflammatory phase that sees the dramatic upregulation of proinflammatory cytokines and extensive neutrophil accumulation in the lungs. The plasminogen activator protease (Pla) is an essential virulence factor that is responsible for survival of Y. pestis in the lungs. Our lab recently showed that Pla functions as an adhesin that promotes binding to alveolar macrophages to facilitate translocation of effector proteins called Yops into the cytosol of target host cells via a type 3 secretion system (T3SS). Loss of Pla-mediated adherence disrupted the preinflammatory phase of disease and resulted in early neutrophil migration to the lungs. While it is established that Yersinia broadly suppresses host innate immune responses, it is not clear precisely which signals need to be inhibited to establish a preinflammatory stage of infection. Here, we show that early Pla-mediated suppression of Interleukin-17 (IL-17) expression in alveolar macrophages and pulmonary neutrophils limits neutrophil migration to the lungs and aids in establishing a preinflammatory phase of disease. In addition, IL-17 ultimately contributes to neutrophil migration to the airways that defines the later proinflammatory phase of infection. These results suggest that the pattern of IL-17 expression contributes to the progression of primary pneumonic plague.

Carbon Monoxide-Sensing Transcription Factors: Regulators of Microbial Carbon Monoxide Oxidation Pathway Gene Expression.

Carbon monoxide (CO) serves as a source of energy and carbon for a diverse set of microbes found in anaerobic and aerobic environments. The enzymes that bacteria and archaea use to oxidize CO depend upon complex metallocofactors that require accessory proteins for assembly and proper function. This complexity comes at a high energetic cost and necessitates strict regulation of CO metabolic pathways in facultative CO metabolizers to ensure that gene expression occurs only when CO concentrations and redox conditions are appropriate. In this review, we examine two known heme-dependent transcription factors, CooA and RcoM, that regulate inducible CO metabolism pathways in anaerobic and aerobic microorganisms. We provide an analysis of the known physiological and genomic contexts of these sensors and employ this analysis to contextualize known biochemical properties. In addition, we describe a growing list of putative transcription factors associated with CO metabolism that potentially use cofactors other than heme to sense CO.

Healthcare system contact following ureteroscopy: does discharge instruction readability matter?

INTRODUCTION: We aimed to assess the impact of discharge instruction (DCI) readability on 30-day postoperative contact with the healthcare system. MATERIALS AND METHODS: Utilizing a multidisciplinary team, DCI were modified for patients undergoing cystoscopy, retrograde pyelogram, ureteroscopy, laser lithotripsy, and stent placement (CRULLS) from a 13th grade to a 7th grade reading level. We retrospectively reviewed 100 patients including 50 consecutive patients with original DCI (oDCI) and 50 consecutive patients with improved readability DCI (irDCI). Clinical and demographic data collected including healthcare system contact (communications [phone or electronic message], emergency department [ED], and unplanned clinic visits) within 30 days of surgery. Uni/multivariate logistic regression analyses used to identify factors, including DCI-type, associated with increased healthcare system contact. Findings reported as odds ratios with 95% confidence intervals and p values (< 0.05 significant). RESULTS: There were 105 contacts to the healthcare system within 30 days of surgery: 78 communications, 14 ED visits and 13 clinic visits. There were no significant differences between cohorts in the proportion of patients with communications (p = 0.16), ED visits (p =1.0) or clinic visits (p = 0.37). On multivariable analysis, older age and psychiatric diagnosis were associated with significantly increased odds of overall healthcare contact (p = 0.03 and p = 0.04) and communications (p = 0.02 and p = 0.03). Prior psychiatric diagnosis was also associated with significantly increased odds of unplanned clinic visits (p = 0.003). Overall, irDCI were not significantly associated with the endpoints of interest. CONCLUSIONS: Increasing age and prior psychiatric diagnosis, but not irDCI, were significantly associated with an increased rate of healthcare system contact following CRULLS.

Leaf decomposing fungi influence Saccharomyces paradoxus growth across carbon environments.

Saccharomyces paradoxus is a model organism in ecology and evolution. However, its metabolism in its native habitat remains mysterious: it is frequently found growing on leaf litter, a habitat with few carbon sources that S. paradoxus can metabolize. We hypothesized that leaf-decomposing fungi from the same habitat break down the cellulose in leaf litter extracellularly and release glucose, supporting S. paradoxus growth. We found that facilitation by leaf-decomposing fungi was possible on cellulose and inhibition was common on glucose, suggesting diverse interactions between S. paradoxus and other fungi that have the potential to support S. paradoxus in nature.

Validation of Ligand Tetramers for the Detection of Antigen-Specific Lymphocytes.

The study of Ag-specific lymphocytes has been a key advancement in immunology over the past few decades. The development of multimerized probes containing Ags, peptide:MHC complexes, or other ligands was one innovation allowing the direct study of Ag-specific lymphocytes by flow cytometry. Although these types of study are now common and performed by thousands of laboratories, quality control and assessment of probe quality are often minimal. In fact, many of these types of probe are made in-house, and protocols vary between laboratories. Although peptide:MHC multimers can often be obtained from commercial sources or core facilities, few such services exist for Ag multimers. To ensure high quality and consistency with ligand probes, we have developed an easy and robust multiplexed approach using commercially available beads able to bind Abs specific for the ligand of interest. Using this assay, we have sensitively assessed the performance of peptide:MHC and Ag tetramers and have found considerable batch-to-batch variability in performance and stability over time more easily than using murine or human cell-based assays. This bead-based assay can also reveal common production errors such as miscalculation of Ag concentration. This work could set the stage for the development of standardized assays for all commonly used ligand probes to limit laboratory-to-laboratory technical variation and experimental failure caused by probe underperformance.

A review of the impacts of air pollution on terrestrial birds.

Air pollution has a ubiquitous impact on ecosystem functioning through myriad processes, including the acidification and eutrophication of soil and water, deposition of heavy metals and direct (and indirect) effects on flora and fauna. Describing the impacts of air pollution on organisms in the field is difficult because levels of exposure do not occur in a uniform manner across space and time, and species responses tend to be nuanced and difficult to isolate from other environmental stressors. However, given its far-reaching effects on human and ecosystem health, the impacts of air pollution on species are expected to be substantial, and could be direct or indirect, acting via a range of mechanisms. Here, we expand on previous reviews, to evaluate the existing evidence for the impacts of air pollution on avian species in the field, and to identify knowledge gaps to guide future research. We identified 203 studies that have investigated the impacts of air pollution (including nitrogen and heavy metal deposition) on wild populations of birds, considering 231 species from ten feeding guilds. The majority of studies (82 %) document at least one species trait leading to an overall fitness value that is negatively correlated with pollution concentrations, including deleterious effects on reproductive output, molecular (DNA) damage and overall survival, and effects on foraging behaviour, plumage colouration and body size that may show adaptation. Despite this broad range of trait effects, biases in the literature towards certain species (Parus major and Ficeluda hypoleuca), geographical regions (Western Europe) and pollutants (heavy metal deposition), mean that many unknowns remain in our current understanding of the impacts of air pollution on avian species. We discuss these findings in context of future work, and propose research approaches that could help to provide a more holistic understanding of how avian species are impacted by air pollution.

Examining the Effects of Hibernation on Germline Mutation Rates in Grizzly Bears.

A male mutation bias is observed across vertebrates, and, where data are available, this bias is accompanied by increased per-generation mutation rates with parental age. While continuing mitotic cell division in the male germline post puberty has been proposed as the major cellular mechanism underlying both patterns, little direct evidence for this role has been found. Understanding the evolution of the per-generation mutation rate among species requires that we identify the molecular mechanisms that change between species. Here, we study the per-generation mutation rate in an extended pedigree of the brown (grizzly) bear, Ursus arctos horribilis. Brown bears hibernate for one-third of the year, a period during which spermatogenesis slows or stops altogether. The reduction of spermatogenesis is predicted to lessen the male mutation bias and to lower the per-generation mutation rate in this species. However, using whole-genome sequencing, we find that both male bias and per-generation mutation rates are highly similar to that expected for a non-hibernating species. We also carry out a phylogenetic comparison of substitution rates along the lineage leading to brown bear and panda (a non-hibernating species) and find no slowing of the substitution rate in the hibernator. Our results contribute to accumulating evidence that suggests that male germline cell division is not the major determinant of mutation rates and mutation biases. The results also provide a quantitative basis for improved estimates of the timing of carnivore evolution.

Density-dependence produces spurious relationships among demographic parameters in a harvested species.

Harvest of wild organisms is an important component of human culture, economy, and recreation, but can also put species at risk of extinction. Decisions that guide successful management actions therefore rely on the ability of researchers to link changes in demographic processes to the anthropogenic actions or environmental changes that underlie variation in demographic parameters. Ecologists often use population models or maximum sustained yield curves to estimate the impacts of harvest on wildlife and fish populations. Applications of these models usually focus exclusively on the impact of harvest and often fail to consider adequately other potential, often collinear, mechanistic drivers of the observed relationships between harvest and demographic rates. In this study, we used an integrated population model and long-term data (1973-2016) to examine the relationships among hunting and natural mortality, the number of hunters, habitat conditions, and population size of blue-winged teal Spatula discors, an abundant North American dabbling duck with a relatively fast-paced life history strategy. Over the last two and a half decades of the study, teal abundance tripled, hunting mortality probability increased slightly ( < 0.02 ), and natural mortality probability increased substantially ( > 0.1 ) at greater population densities. We demonstrate strong density-dependent effects on natural mortality and fecundity as population density increased, indicative of compensatory harvest mortality and compensatory natality. Critically, an analysis that only assessed the relationship between survival and hunting mortality would spuriously indicate depensatory mortality due to multicollinearity between abundance, natural mortality and hunting mortality. Our findings demonstrate that models that only consider the direct effect of hunting on survival or natural mortality can fail to accurately assess the mechanistic impact of hunting on population dynamics due to multicollinearity among demographic drivers. This multicollinearity limits inference and may have strong impacts on applied management actions globally.

Long-term trends and drought: Spatiotemporal variation in juvenile sex ratios of North American ducks.

Sex ratios affect population dynamics and individual fitness, and changing sex ratios can be indicative of shifts in sex-specific survival at different life stages. While climate and landscape changes alter sex ratios of wild bird populations, long-term, landscape scale assessments of sex ratios are rare. Further, little work has been done to understand changes in sex ratios in avian communities. In this manuscript, we analyze long-term (1961-2015) data on five species of ducks across five broad climatic regions of the United States to estimate the effects of drought and long-term trends on the proportion of juvenile females captured at banding. As waterfowl have a 1:1 sex ratio at hatch, we interpret changes in sex ratios of captured juveniles as changes in sex-specific survival rates during early life. Seven of 12 species-region pairs exhibited evidence for long-term trends in the proportion of juvenile females at banding. The proportion of juvenile females at banding increased for duck populations in the western United States and typically declined for duck populations in the eastern United States. We only observed evidence for an effect of drought in two of the 12 species-region pairs, where the proportion of females declined during drought. As changes to North American landscapes and climate continue and intensify, we expect continued changes in sex-specific juvenile survival rates. More broadly, we encourage further research examining the mechanisms underlying long-term trends in juvenile sex ratios in avian communities.