Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes

The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here we investigated the properties and mechanisms of clag3 mutual exclusion using transgenic parasite lines with extra copies of clag3 promoters located either in stable episomes or integrated in the parasite genome. We found that the additional clag3 promoters in these transgenic lines are silenced by default, but under strong selective pressure parasites with more than one clag3 promoter simultaneously active are observed, demonstrating that clag3 mutual exclusion is strongly favored but it is not strict. We show that silencing of clag3 genes is associated with the repressive histone mark H3K9me3 even in parasites with unusual clag3 expression patterns, and we provide direct evidence for heterochromatin spreading in P. falciparum. We also found that expression of a neighbor ncRNA correlates with clag3.1 expression. Altogether, our results reveal a scenario where fitness costs and non-deterministic molecular processes that favor mutual exclusion shape the expression patterns of this important gene family

Improvement of the cryopreservation protocols for the dusky grouper, Epinephelus marginatus

The dusky grouper, Epinephelus marginatus, is a potential species for aquaculture production although the limited number of males kept in captivity has been the cause of some constraints in its production. Sperm cryopreservation emerged as a solution for this problem. However, spermatozoa can be severely affected by freezing/thawing processes and poor sperm quality is a limiting factor in reproduction success. The present study aimed at evaluating two main aspects in the design of a cryopreservation protocol-extender additives (taurine, glucose, cholesterol, BSA) and sperm containers (0.5 mL straws, 2 mL cryovials and 5 mL macrotubes). Sperm quality was assessed through the evaluation of the percentage of motile cells, viable cells, DNA fragmentation, lipid peroxidation and apoptosis. Some specific techniques, such as Caspase 3/7 detection, which provides information on membrane integrity and cell death, detecting early and late apoptotic and necrotic events, were required to establish an optimized cryopreservation protocol for this species. Taurine was the most suitable cryopreservation additive in terms of viable cells and cholesterol presented the highest percentage of necrotic cells in this study. Caspase 3/7 assay enabled us to detect necrotic damage induced by cryopreservation. Statement of relevance: The development of reproductive tools in dusky grouper, a potential species for aquaculture production, emerges as important tool to decrease the number of wild males maintained in captivity. A cryopreservation protocol was previously described for this species although several constraints in terms of sperm quality were detected. Our work provided new evidences that cryopreservation protocols can be improved through the addition of certain additives and use of appropriate sperm containers. Specific sperm analysis was crucial to identify and establish the most suitable conditions for breeders management and species conservation purposes. (C) 2016 Elsevier B.V. All rights reserved.European Social Fund [IF/00482/2014/CP1217/CT0005]Operational Programme Human PotentialFoundation for Science and Technology of Portugal (FCT)POCTEP programme under the European territorial cooperation objective [0251_ECOAQUA_5_E]European Regional Development Fund (ERDF) Portugal Spain[CCMAR/SC/BPD/09/2016

Expression of the Plasmodium falciparum Clonally Variant clag3 Genes in Human Infections

Background.: Many genes of the malaria parasite Plasmodium falciparum show clonally variant expression regulated at the epigenetic level. These genes participate in fundamental host-parasite interactions and contribute to adaptive processes. However, little is known about their expression patterns during human infections. A peculiar case of clonally variant genes are the 2 nearly identical clag3 genes, clag3.1 and clag3.2, which mediate nutrient uptake and are linked to resistance to some toxic compounds. Methods.: We developed a procedure to characterize the expression of clag3 genes in naturally infected patients and in experimentally infected human volunteers. Results.: We provide the first description of clag3 expression during human infections, which revealed mutually exclusive expression and identified the gene predominantly expressed. Adaptation to culture conditions or selection with a toxic compound resulted in isolate-dependent changes in clag3 expression. We also found that clag3 expression patterns were reset during transmission stages. Conclusions.: Different environment conditions select for parasites with different clag3 expression patterns, implying functional differences between the proteins encoded. The epigenetic memory is likely erased before parasites start infection of a new human host. Altogether, our findings support the idea that clonally variant genes facilitate the adaptation of parasite populations to changing conditions through bet-hedging strategies

Phaeobacter gallaeciensis genomes from globally opposite locations reveal high similarity of adaptation to surface life

Phaeobacter gallaeciensis, a member of the abundant marine Roseobacter clade, is known to be an effective colonizer of biotic and abiotic marine surfaces. Production of the antibiotic tropodithietic acid (TDA) makes P. gallaeciensis a strong antagonist of many bacteria, including fish and mollusc pathogens. In addition to TDA, several other secondary metabolites are produced, allowing the mutualistic bacterium to also act as an opportunistic pathogen. Here we provide the manually annotated genome sequences of the P. gallaeciensis strains DSM 17395 and 2.10, isolated at the Atlantic coast of north western Spain and near Sydney, Australia, respectively. Despite their isolation sites from the two different hemispheres, the genome comparison demonstrated a surprisingly high level of synteny (only 3% nucleotide dissimilarity and 88% and 93% shared genes). Minor differences in the genomes result from horizontal gene transfer and phage infection. Comparison of the P. gallaeciensis genomes with those of other roseobacters revealed unique genomic traits, including the production of iron-scavenging siderophores. Experiments supported the predicted capacity of both strains to grow on various algal osmolytes. Transposon mutagenesis was used to expand the current knowledge on the TDA biosynthesis pathway in strain DSM 17395. This first comparative genomic analysis of finished genomes of two closely related strains belonging to one species of the Roseobacter clade revealed features that provide competitive advantages and facilitate surface attachment and interaction with eukaryotic hosts

Intubation Practices and Adverse Peri-intubation Events in Critically Ill Patients from 29 Countries

Importance: Tracheal intubation is one of the most commonly performed and high-risk interventions in critically ill patients. Limited information is available on adverse peri-intubation events. Objective: To evaluate the incidence and nature of adverse peri-intubation events and to assess current practice of intubation in critically ill patients. Design, Setting, and Participants: The International Observational Study to Understand the Impact and Best Practices of Airway Management in Critically Ill Patients (INTUBE) study was an international, multicenter, prospective cohort study involving consecutive critically ill patients undergoing tracheal intubation in the intensive care units (ICUs), emergency departments, and wards, from October 1, 2018, to July 31, 2019 (August 28, 2019, was the final follow-up) in a convenience sample of 197 sites from 29 countries across 5 continents. Exposures: Tracheal intubation. Main Outcomes and Measures: The primary outcome was the incidence of major adverse peri-intubation events defined as at least 1 of the following events occurring within 30 minutes from the start of the intubation procedure: cardiovascular instability (either: systolic pressure <65 mm Hg at least once, <90 mm Hg for >30 minutes, new or increase need of vasopressors or fluid bolus >15 mL/kg), severe hypoxemia (peripheral oxygen saturation <80%) or cardiac arrest. The secondary outcomes included intensive care unit mortality. Results: Of 3659 patients screened, 2964 (median age, 63 years; interquartile range [IQR], 49-74 years; 62.6% men) from 197 sites across 5 continents were included. The main reason for intubation was respiratory failure in 52.3% of patients, followed by neurological impairment in 30.5%, and cardiovascular instability in 9.4%. Primary outcome data were available for all patients. Among the study patients, 45.2% experienced at least 1 major adverse peri-intubation event. The predominant event was cardiovascular instability, observed in 42.6% of all patients undergoing emergency intubation, followed by severe hypoxemia (9.3%) and cardiac arrest (3.1%). Overall ICU mortality was 32.8%. Conclusions and Relevance: In this observational study of intubation practices in critically ill patients from a convenience sample of 197 sites across 29 countries, major adverse peri-intubation events - in particular cardiovascular instability - were observed frequently

Epidemiology and outcomes of hospital-acquired bloodstream infections in intensive care unit patients: the EUROBACT-2 international cohort study

Purpose In the critically ill, hospital-acquired bloodstream infections (HA-BSI) are associated with significant mortality. Granular data are required for optimizing management, and developing guidelines and clinical trials. Methods We carried out a prospective international cohort study of adult patients (≥ 18 years of age) with HA-BSI treated in intensive care units (ICUs) between June 2019 and February 2021. Results 2600 patients from 333 ICUs in 52 countries were included. 78% HA-BSI were ICU-acquired. Median Sequential Organ Failure Assessment (SOFA) score was 8 [IQR 5; 11] at HA-BSI diagnosis. Most frequent sources of infection included pneumonia (26.7%) and intravascular catheters (26.4%). Most frequent pathogens were Gram-negative bacteria (59.0%), predominantly Klebsiella spp. (27.9%), Acinetobacter spp. (20.3%), Escherichia coli (15.8%), and Pseudomonas spp. (14.3%). Carbapenem resistance was present in 37.8%, 84.6%, 7.4%, and 33.2%, respectively. Difficult-to-treat resistance (DTR) was present in 23.5% and pan-drug resistance in 1.5%. Antimicrobial therapy was deemed adequate within 24 h for 51.5%. Antimicrobial resistance was associated with longer delays to adequate antimicrobial therapy. Source control was needed in 52.5% but not achieved in 18.2%. Mortality was 37.1%, and only 16.1% had been discharged alive from hospital by day-28. Conclusions HA-BSI was frequently caused by Gram-negative, carbapenem-resistant and DTR pathogens. Antimicrobial resistance led to delays in adequate antimicrobial therapy. Mortality was high, and at day-28 only a minority of the patients were discharged alive from the hospital. Prevention of antimicrobial resistance and focusing on adequate antimicrobial therapy and source control are important to optimize patient management and outcomes

Risk of COVID-19 after natural infection or vaccinationResearch in context

Background: While vaccines have established utility against COVID-19, phase 3 efficacy studies have generally not comprehensively evaluated protection provided by previous infection or hybrid immunity (previous infection plus vaccination). Individual patient data from US government-supported harmonized vaccine trials provide an unprecedented sample population to address this issue. We characterized the protective efficacy of previous SARS-CoV-2 infection and hybrid immunity against COVID-19 early in the pandemic over three-to six-month follow-up and compared with vaccine-associated protection. Methods: In this post-hoc cross-protocol analysis of the Moderna, AstraZeneca, Janssen, and Novavax COVID-19 vaccine clinical trials, we allocated participants into four groups based on previous-infection status at enrolment and treatment: no previous infection/placebo; previous infection/placebo; no previous infection/vaccine; and previous infection/vaccine. The main outcome was RT-PCR-confirmed COVID-19 >7–15 days (per original protocols) after final study injection. We calculated crude and adjusted efficacy measures. Findings: Previous infection/placebo participants had a 92% decreased risk of future COVID-19 compared to no previous infection/placebo participants (overall hazard ratio [HR] ratio: 0.08; 95% CI: 0.05–0.13). Among single-dose Janssen participants, hybrid immunity conferred greater protection than vaccine alone (HR: 0.03; 95% CI: 0.01–0.10). Too few infections were observed to draw statistical inferences comparing hybrid immunity to vaccine alone for other trials. Vaccination, previous infection, and hybrid immunity all provided near-complete protection against severe disease. Interpretation: Previous infection, any hybrid immunity, and two-dose vaccination all provided substantial protection against symptomatic and severe COVID-19 through the early Delta period. Thus, as a surrogate for natural infection, vaccination remains the safest approach to protection. Funding: National Institutes of Health

Risk of COVID-19 after natural infection or vaccinationResearch in context

Summary: Background: While vaccines have established utility against COVID-19, phase 3 efficacy studies have generally not comprehensively evaluated protection provided by previous infection or hybrid immunity (previous infection plus vaccination). Individual patient data from US government-supported harmonized vaccine trials provide an unprecedented sample population to address this issue. We characterized the protective efficacy of previous SARS-CoV-2 infection and hybrid immunity against COVID-19 early in the pandemic over three-to six-month follow-up and compared with vaccine-associated protection. Methods: In this post-hoc cross-protocol analysis of the Moderna, AstraZeneca, Janssen, and Novavax COVID-19 vaccine clinical trials, we allocated participants into four groups based on previous-infection status at enrolment and treatment: no previous infection/placebo; previous infection/placebo; no previous infection/vaccine; and previous infection/vaccine. The main outcome was RT-PCR-confirmed COVID-19 >7–15 days (per original protocols) after final study injection. We calculated crude and adjusted efficacy measures. Findings: Previous infection/placebo participants had a 92% decreased risk of future COVID-19 compared to no previous infection/placebo participants (overall hazard ratio [HR] ratio: 0.08; 95% CI: 0.05–0.13). Among single-dose Janssen participants, hybrid immunity conferred greater protection than vaccine alone (HR: 0.03; 95% CI: 0.01–0.10). Too few infections were observed to draw statistical inferences comparing hybrid immunity to vaccine alone for other trials. Vaccination, previous infection, and hybrid immunity all provided near-complete protection against severe disease. Interpretation: Previous infection, any hybrid immunity, and two-dose vaccination all provided substantial protection against symptomatic and severe COVID-19 through the early Delta period. Thus, as a surrogate for natural infection, vaccination remains the safest approach to protection. Funding: National Institutes of Health