Transcriptomic Analysis of Toxoplasma Development Reveals Many Novel Functions and Structures Specific to Sporozoites and Oocysts

Sexual reproduction of Toxoplasma gondii occurs exclusively within enterocytes of the definitive felid host. The resulting immature oocysts are excreted into the environment during defecation, where in the days following, they undergo a complex developmental process. Within each oocyst, this culminates in the generation of two sporocysts, each containing 4 sporozoites. A single felid host is capable of shedding millions of oocysts, which can survive for years in the environment, are resistant to most methods of microbial inactivation during water-treatment and are capable of producing infection in warm-blooded hosts at doses as low as 1–10 ingested oocysts. Despite its extremely interesting developmental biology and crucial role in initiating an infection, almost nothing is known about the oocyst stage beyond morphological descriptions. Here, we present a complete transcriptomic analysis of the oocyst from beginning to end of its development. In addition, and to identify genes whose expression is unique to this developmental form, we compared the transcriptomes of developing oocysts with those of in vitro-derived tachyzoites and in vivo-derived bradyzoites. Our results reveal many genes whose expression is specifically up- or down-regulated in different developmental stages, including many genes that are likely critical to oocyst development, wall formation, resistance to environmental destruction and sporozoite infectivity. Of special note is the up-regulation of genes that appear “off” in tachyzoites and bradyzoites but that encode homologues of proteins known to serve key functions in those asexual stages, including a novel pairing of sporozoite-specific paralogues of AMA1 and RON2, two proteins that have recently been shown to form a crucial bridge during tachyzoite invasion of host cells. This work provides the first in-depth insight into the development and functioning of one of the most important but least studied stages in the Toxoplasma life cycle

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

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

The Genetic Consequences of Habitat Fragmentation

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The hops (Humulus lupulus) genome contains a mid-sized terpene synthase family that shows wide functional and allelic diversity

Abstract Background Hops (Humulus lupulus L.) are a dioecious climbing perennial, with the dried mature “cones” (strobili) of the pistillate/female inflorescences being widely used as both a bittering agent and to enhance the flavour of beer. The glandular trichomes of the bract and bracteole flowering structures of the cones produce an abundance of secondary metabolites, such as terpenoids, bitter acids and prenylated phenolics depending on plant genetics, developmental stage and environment. More knowledge is required on the functional and allelic diversity of terpene synthase (TPS) genes responsible for the biosynthesis of volatile terpenes to assist in flavour-directed hop breeding. Results Major volatile terpene compounds were identified using gas chromatography–mass spectrometry (GC–MS) in the ripe cones of twenty-one hop cultivars grown in New Zealand. All cultivars produced the monoterpene β-myrcene and the sesquiterpenes α-humulene and β-caryophyllene, but the quantities varied broadly. Other terpenes were found in large quantities in only a smaller subset of cultivars, e.g. β-farnesene (in seven cultivars) and α-pinene (in four). In four contrasting cultivars (Wakatu™, Wai-iti™, Nelson Sauvin™, and ‘Nugget’), terpene production during cone development was investigated in detail, with concentrations of some of the major terpenes increasing up to 1000-fold during development and reaching maximal levels from 50–60 days after flowering. Utilising the published H. lupulus genome, 87 putative full-length and partial terpene synthase genes were identified. Alleles corresponding to seven TPS genes were amplified from ripe cone cDNA from multiple cultivars and subsequently functionally characterised by transient expression in planta. Alleles of the previously characterised HlSTS1 produced humulene/caryophyllene as the major terpenes. HlRLS alleles produced (R)-(-)-linalool, whilst alleles of two sesquiterpene synthase genes, HlAFS1 and HlAFS2 produced α-farnesene. Alleles of HlMTS1, HlMTS2 and HlTPS1 were inactive in all the hop cultivars studied. Conclusions Alleles of four TPS genes were identified and shown to produce key aroma volatiles in ripe hop cones. Multiple expressed but inactive TPS alleles were also identified, suggesting that extensive loss-of-function has occurred during domestication and breeding of hops. Our results can be used to develop hop cultivars with novel/improved terpene profiles using marker-assisted breeding strategies to select for, or against, specific TPS alleles

Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE7 Is Involved in the Production of Negative and Positive Branching Signals in Petunia1[W][OA]

One of the key factors that defines plant form is the regulation of when and where branches develop. The diversity of form observed in nature results, in part, from variation in the regulation of branching between species. Two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes, CCD7 and CCD8, are required for the production of a branch-suppressing plant hormone. Here, we report that the decreased apical dominance3 (dad3) mutant of petunia (Petunia hybrida) results from the mutation of the PhCCD7 gene and has a less severe branching phenotype than mutation of PhCCD8 (dad1). An analysis of the expression of this gene in wild-type, mutant, and grafted petunia suggests that in petunia, CCD7 and CCD8 are coordinately regulated. In contrast to observations in Arabidopsis (Arabidopsis thaliana), ccd7ccd8 double mutants in petunia show an additive phenotype. An analysis using dad3 or dad1 mutant scions grafted to wild-type rootstocks showed that when these plants produce adventitious mutant roots, branching is increased above that seen in plants where the mutant roots are removed. The results presented here indicate that mutation of either CCD7 or CCD8 in petunia results in both the loss of an inhibitor of branching and an increase in a promoter of branching

The Decreased apical dominance1/Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE8 Gene Affects Branch Production and Plays a Role in Leaf Senescence, Root Growth, and Flower Development

Carotenoids and carotenoid cleavage products play an important and integral role in plant development. The Decreased apical dominance1 (Dad1)/PhCCD8 gene of petunia (Petunia hybrida) encodes a hypothetical carotenoid cleavage dioxygenase (CCD) and ortholog of the MORE AXILLARY GROWTH4 (MAX4)/AtCCD8 gene. The dad1-1 mutant allele was inactivated by insertion of an unusual transposon (Dad-one transposon), and the dad1-3 allele is a revertant allele of dad1-1. Consistent with its role in producing a graft-transmissible compound that can alter branching, the Dad1/PhCCD8 gene is expressed in root and shoot tissue. This expression is upregulated in the stems of the dad1-1, dad2, and dad3 increased branching mutants, indicating feedback regulation of the gene in this tissue. However, this feedback regulation does not affect the root expression of Dad1/PhCCD8. Overexpression of Dad1/PhCCD8 in the dad1-1 mutant complemented the mutant phenotype, and RNA interference in the wild type resulted in an increased branching phenotype. Other differences in phenotype associated with the loss of Dad1/PhCCD8 function included altered timing of axillary meristem development, delayed leaf senescence, smaller flowers, reduced internode length, and reduced root growth. These data indicate that the substrate(s) and/or product(s) of the Dad1/PhCCD8 enzyme are mobile signal molecules with diverse roles in plant development

Analysis of the ARTIC V4 and V4.1 SARS-CoV-2 primers and their impact on the detection of Omicron BA.1 and BA.2 lineage-defining mutations

The ARTIC protocol uses a multiplexed PCR approach with two primer pools tiling the entire SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genome. Primer pool updates are necessary for accurate amplicon sequencing of evolving SARS-CoV-2 variants with novel mutations. The suitability of the ARTIC V4 and updated V4.1 primer scheme was assessed using whole genome sequencing of Omicron from clinical samples using Oxford Nanopore Technology. Analysis of Omicron BA.1 genomes revealed that 93.22 % of clinical samples generated improved genome coverage at 50× read depth with V4.1 primers when compared to V4 primers. Additionally, the V4.1 primers improved coverage of BA.1 across amplicons 76 and 88, which resulted in the detection of the variant-defining mutations G22898A, A26530G and C26577G. The Omicron BA.2 sub-variant (VUI-22JAN-01) replaced BA.1 as the dominant variant by March 2022, and analysis of 168 clinical samples showed reduced coverage across amplicons 15 and 75. Upon further interrogation of primer binding sites, a mutation at C4321T [present in 163/168 (97 %) of samples] was identified as a possible cause of complete dropout of amplicon 15. Furthermore, two mutations were identified within the primer binding regions for amplicon 75: A22786C (present in 90 % of samples) and C22792T (present in 12.5 % of samples). Together, these mutations may result in reduced coverage of amplicon 75, and further primer updates would allow the identification of the two BA.2-defining mutations present in amplicon 75: A22688G and T22679C. This work highlights the need for ongoing surveillance of primer matches as circulating variants evolve and change.Publisher PDFPeer reviewe