Solution structure of a bacterial microcompartment targeting peptide and its application in the construction of an ethanol bioreactor

Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The solution structure reveals the peptide to have a well-defined helical conformation along its whole length. Saturation transfer difference and transferred NOE NMR has highlighted the observed interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alcohol dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains containing the modified BMCs produce elevated levels of alcohol

Australian rural adolescents’ experiences of accessing psychological help for a mental health problem

Objective: This study aims to explore Australian rural adolescents’ experiences of accessing help for a mental health problem in the context of their rural communities. Design and setting: A qualitative research design was used whereby university students who had sought help for a mental health problem during their adolescence were interviewed about their experiences. Interviews were conducted face-to-face at the university. Main outcome measures: A semi-structured interview schedule was designed around the study’s main research questions. Audio-taped interviews were transcribed and thematically coded using a constant comparative method. Participants: Participants were first-year undergraduate psychology students between the ages of 17 and 21 years who sought help for a mental health issue during their adolescence and who at that time resided in a rural area. Results: Participants highlighted various barriers to seeking help for mental health problems in the context of a rural community, including: social visibility, lack of anonymity, a culture of self-reliance, and social stigma of mental illness. Participants’ access to help was primarily school-based, and participants expressed a preference for supportive counselling over structured interventions. Characteristics of school-based helpers that made them approachable included: ‘caring’, ‘nonjudgemental’, ‘genuine’, ‘young’, and able to maintain confidentiality. Conclusions: The findings support previous research that reveals barriers to help seeking for mental health problems that are unique to the culture of rural communities. The study raises questions about the merit of delivery of primary mental health care to young people via GPs alone and suggests that school-based counsellors be considered as the first step in a young person’s access to mental health care.C

Intrauterine Candida albicans infection causes systemic fetal candidiasis with progressive cardiac dysfunction in a sheep model of early pregnancy

Introduction: Several recent studies have identified a potential role for intrauterine Candida albicans in adverse pregnancy outcomes, including preterm birth. There is, however, a limited understanding of the impact of intrauterine candida infection on fetal well-being in early pregnancy. Using a sheep model of early pregnancy, the aims of this study were to determine (1) the ability of experimentally induced intrauterine C albicans to infect the fetus and (2) whether C albicans exposure in early pregnancy is associated with alterations in fetal cardiac function, as measured by spectral tissue Doppler imaging analysis of fetal cardiac function. Methods: Merino ewes carrying singleton pregnancies at 89 days’ gestation (term is ∼150 days) received C albicans (n = 8) via ultrasound-guided intra-amniotic injection. Saline-exposed fetuses served as controls (n = 6). Spectral tissue Doppler imaging echocardiography and amniotic fluid collection were performed at baseline and 24 and 72 hours after intrauterine C albicans injection. Fetal tissues were collected at postmortem for analysis of infection and inflammation. Results: Relative to saline control, intrauterine C albicans infection resulted in pronounced increases in amniotic fluid tumor necrosis factor α (TNF-α; P < .05) and cytokine/chemokine messenger RNA (interleukin [IL] 1β, IL-6, TNF-α, and monocyte chemoattractant protein 1; P < .05) in the fetal myocardium, lung, skin, and liver at 72 and 96 hours postinfection. Spectral tissue Doppler imaging showed diastolic dysfunction at 24 hours and severe biventricular diastolic dysfunction 72 hours postinfection. Conclusion: Intrauterine C albicans infection in a sheep model of early pregnancy causes systemic fetal candidiasis, which is associated with a robust systemic inflammatory response and progressive cardiac dysfunction detectable by spectral tissue Doppler imaging

Low-dose betamethasone-acetate for fetal lung maturation in preterm sheep

BackgroundAntenatal steroids are standard of care for women who are at risk of preterm delivery; however, antenatal steroid dosing and formulation have not been evaluated adequately. The standard clinical 2-dose treatment with betamethasone-acetate+betamethasone-phosphate is more effective than 2 doses of betamethasone-phosphate for the induction of lung maturation in preterm fetal sheep. We hypothesized that the slowly released betamethasone-acetate component induces similar lung maturation to betamethasone-phosphate+betamethasone-acetate with decreased dose and fetal exposure.ObjectiveThe purpose of this study was to investigate pharmacokinetics and fetal lung maturation of antenatal betamethasone-acetate in preterm fetal sheep.Study designGroups of 10 singleton-pregnant ewes received 1 or 2 intramuscular doses 24 hours apart of 0.25 mg/kg/dose of betamethasone-phosphate+betamethasone-acetate (the standard of care dose) or 1 intramuscular dose of 0.5 mg/kg, 0.25 mg/kg, or 0.125 mg/kg of betamethasone-acetate. Fetuses were delivered 48 hours after the first injection at 122 days of gestation (80% of term) and ventilated for 30 minutes, with ventilator settings, compliance, vital signs, and blood gas measurements recorded every 10 minutes. After ventilation, we measured static lung pressure-volume curves and sampled the lungs for messenger RNA measurements. Other groups of pregnant ewes and fetuses were catheterized and treated with intramuscular injections of betamethasone-phosphate 0.125 mg/kg, betamethasone-acetate 0.125 mg/kg, or betamethasone-acetate 0.5 mg/kg. Maternal and fetal betamethasone concentrations in plasma were measured for 24 hours.ResultsAll betamethasone-treated groups had increased messenger RNA expression of surfactant proteins A, B, and C, ATP-binding cassette subfamily A member 3, and aquaporin-5 compared with control animals. Treatment with 1 dose of intramuscular betamethasone-acetate 0.125mg/kg improved dynamic and static lung compliance, gas exchange, and ventilation efficiency similarly to the standard treatment of 2 doses of 0.25 m/kg of betamethasone-acetate+betamethasone-phosphate. Betamethasone-acetate 0.125 mg/kg resulted in lower maternal and fetal peak plasma concentrations and decreased fetal exposure to betamethasone compared with betamethasone-phosphate 0.125 mg/kg.ConclusionA single dose of betamethasone-acetate results in similar fetal lung maturation as the 2-dose clinical formulation of betamethasone-phosphate+betamethasone-acetate with decreased fetal exposure to betamethasone. A lower dose of betamethasone-acetate may be an effective alternative to induce fetal lung maturation with less risk to the fetus

The AEDUCATE Collaboration. Comprehensive antenatal education birth preparation programmes to reduce the rates of caesarean section in nulliparous women. Protocol for an individual participant data prospective meta-analysis

Introduction: Rates of medical interventions in normal labour and birth are increasing. This prospective meta-analysis (PMA) proposes to assess whether the addition of a comprehensive multicomponent birth preparation programme reduces caesarean section (CS) in nulliparous women compared with standard hospital care. Additionally, do participant characteristics, intervention components or hospital characteristics modify the effectiveness of the programme? Methods and analysis: Population: women with singleton vertex pregnancies, no planned caesarean section (CS) or epidural. Intervention: in addition to hospital-based standard care, a comprehensive antenatal education programme that includes multiple components for birth preparation, addressing the three objectives: preparing women and their birth partner/support person for childbirth through education on physiological/hormonal birth (knowledge and understanding); building women’s confidence through psychological preparation (positive mindset) and support their ability to birth without pain relief using evidence-based tools (tools and techniques). The intervention could occur in a hospital-based or community setting. Comparator: standard care alone in hospital-based maternity units. Outcomes: Primary: CS. Secondary: epidural analgesia, mode of birth, perineal trauma, postpartum haemorrhage, newborn resuscitation, psychosocial well-being. Subgroup analysis: parity, model of care, maternal risk status, maternal education, maternal socio-economic status, intervention components. Study design: An individual participant data (IPD) prospective meta-analysis (PMA) of randomised controlled trials, including cluster design. Each trial is conducted independently but share core protocol elements to contribute data to the PMA. Participating trials are deemed eligible for the PMA if their results are not yet known outside their Data Monitoring Committees. Ethics and dissemination: Participants in the individual trials will consent to participation, with respective trials receiving ethical approval by their local Human Research Ethics Committees. Individual datasets remain the property of trialists, and can be published prior to the publication of final PMA results. The overall data for meta-analysis will be held, analysed and published by the collaborative group, led by the Cochrane PMA group. Trial registration number: CRD42020103857

Daratumumab, Cyclophosphamide, Bortezomib, Lenalidomide, and Dexamethasone as Induction and Extended Consolidation Improves Outcome in Ultra-High-Risk Multiple Myeloma

Purpose: The multicenter OPTIMUM (MUKnine) phase II trial investigated daratumumab, low-dose cyclophosphamide, lenalidomide, bortezomib, and dexamethasone (Dara-CVRd) before and after autologous stem-cell transplant (ASCT) in newly diagnosed patients with molecularly defined ultra–high-risk (UHiR) multiple myeloma (NDMM) or plasma cell leukemia (PCL). To provide clinical context, progression-free survival (PFS) and overall survival (OS) were referenced to contemporaneous outcomes seen in patients with UHiR NDMM treated in the recent Myeloma XI (MyeXI) trial.Methods: Transplant-eligible all-comers NDMM patients were profiled for UHiR disease, defined by presence of ≥2 genetic risk markers t(4;14)/t(14;16)/t(14;20), del(1p), gain(1q), and del(17p), and/or SKY92 gene expression risk signature. Patients with UHiR MM/PCL were offered treatment with Dara-CVRd induction, V-augmented ASCT, extended Dara-VR(d) consolidation, and Dara-R maintenance. UHiR patients treated in MyeXI with carfilzomib, lenalidomide, dexamethasone, and cyclophosphamide, or lenalidomide, dexamethasone, and cyclophosphamide, ASCT, and R maintenance or observation were identified by mirrored molecular screening. OPTIMUM PFS at 18 months (PFS18m) was compared against MyeXI using a Bayesian framework, and patients were followed up to the end of consolidation for PFS and OS.Results: Of 412 screened NDMM OPTIMUM patients, 103 were identified as UHiR or PCL and subsequently treated on trial with Dara-CVRd; 117 MyeXI patients identified as UHiR formed the external comparator arm, with comparable clinical and molecular characteristics to OPTIMUM. Comparison of PFS18m per Bayesian framework resulted in a 99.5% chance of OPTIMUM being superior to MyeXI. At 30 months' follow-up, PFS was 77% for OPTIMUM versus 39.8% for MyeXI, and OS 83.5% versus 73.5%, respectively. Extended post-ASCT Dara-VRd consolidation therapy was highly deliverable, with limited toxicity.Conclusion: Our results suggest that Dara-CVRd induction and extended post-ASCT Dara-VRd consolidation markedly improve PFS for UHiR NDMM patients over conventional management, supporting further evaluation of this strategy.</p

Outside-in?:Acute fetal systemic inflammation in very preterm chronically catheterized sheep fetuses is not driven by cells in the fetal blood

Background The preterm birth syndrome (delivery before 37 weeks gestation) is a major contributor to the global burden of perinatal morbidity and death. The cause of preterm birth is complex, multifactorial, and likely dependent, at least in part, on the gestational age of the fetus. Intrauterine infection is frequent in preterm deliveries that occur at <32 weeks gestation; understanding how the fetus responds to proinflammatory insult will be an important step towards early preterm birth prevention. However, animal studies of infection and inflammation in prematurity commonly use older fetuses that possess comparatively mature immune systems. Objective Aiming to characterize acute fetal responses to microbial agonist at a clinically relevant gestation, we used 92-day-old fetuses (62% of term) to develop a chronically catheterized sheep model of very preterm pregnancy. We hypothesized that any acute fetal systemic inflammatory responses would be driven by signaling from the tissues exposed to Escherichia coli lipopolysaccharide that is introduced into the amniotic fluid. Study Design Eighteen ewes that were carrying a single fetus at 92 days of gestation had recovery surgery to place fetal tracheal, jugular, and intraamniotic catheters. Animals were recovered for 24 hours before being administered either intraamniotic E coli lipopolysaccharide (n = 9) or sterile saline solution (n = 9). Samples were collected for 48 hours before euthanasia and necroscopy. Fetal inflammatory responses were characterized by microarray analysis, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. Results Intraamniotic lipopolysaccharide reached the distal trachea within 2 hours. Lipopolysaccharide increased tracheal fluid interleukin-8 within 2 hours and generated a robust inflammatory response that was characterized by interleukin-6 signaling pathway activation and up-regulation of cell proliferation but no increases in inflammatory mediator expression in cord blood RNA. Conclusions In very preterm sheep fetuses, lipopolysaccharide stimulates inflammation in the fetal lung and fetal skin and stimulates a systemic inflammatory response that is not generated by fetal blood cells. These data argue for amniotic fluid-exposed tissues that play a key role in driving acute fetal and intrauterine inflammatory responses

Maternal Administration of Solithromycin, a New, Potent, Broad-Spectrum Fluoroketolide Antibiotic, Achieves Fetal and Intra-Amniotic Antimicrobial Protection in a Pregnant Sheep Model

Solithromycin (CEM-101) is a new antibiotic that is highly potent against Ureaplasma and Mycoplasma spp. and active against many other antibiotic-resistant organisms. We have explored the maternal-amniotic-fetal pharmacokinetics of CEM-101 in a pregnant sheep model to assess its potential for treating intrauterine and antenatal infection. Chronically catheterized pregnant ewes (n = 6 or 7) received either a single maternal intravenous (i.v.) infusion of CEM-101 (10 mg/kg of body weight), a single intra-amniotic (i.a.) injection (1.4 mg/kg of estimated fetal weight), or a combined i.v. and i.a. dose. Maternal plasma (MP), fetal plasma (FP), and amniotic fluid (AF) samples were taken via catheter at intervals of 0 to 72 h postadministration, and concentrations of solithromycin and its bioactive polar metabolites (N-acetyl [NAc]–CEM-101 and CEM-214) were determined. Following maternal i.v. infusion, peak CEM-101 concentrations in MP, FP, and AF were 1,073, 353, and 214 ng/ml, respectively, representing a maternal-to-fetal plasma transfer efficiency of 34%. A single maternal dose resulted in effective concentrations (>30 ng/ml) in MP, FP, and AF sustained for >12 h. NAc–CEM-101 and CEM-214 exhibited delayed accumulation and clearance in FP and AF, resulting in an additive antimicrobial effect (>48 h). Intra-amniotic solithromycin injection resulted in elevated (∼50 μg/ml) and sustained CEM-101 concentrations in AF and significant levels in FP, although the efficiency of amniotic-to-fetal transfer was low (∼1.5%). Combined i.v. and i.a. administration resulted in primarily additive concentrations of CEM-101 in all three compartments. Our findings suggest that CEM-101 may provide, for the first time, an effective antimicrobial approach for the prevention and treatment of intrauterine infection and early prevention of preterm birth

Chronic intra-uterine Ureaplasma parvum infection induces injury of the enteric nervous system in ovine fetuses

Background: Chorioamnionitis, inflammation of the fetal membranes during pregnancy, is often caused by intra-amniotic (IA) infection with single or multiple microbes. Chorioamnionitis can be either acute or chronic, and is associated with adverse postnatal outcomes of the intestine, including necrotizing enterocolitis (NEC). Neonates with NEC have structural and functional damage to the intestinal mucosa and the enteric nervous system (ENS), with loss of enteric neurons and glial cells. Yet, the impact of acute, chronic or repetitive antenatal inflammatory stimuli on the development of the intestinal mucosa and ENS has not been studied. The aim of this study is therefore to investigate the effect of acute, chronic and repetitive microbial exposure on the intestinal mucosa, submucosa and ENS in premature lambs. Materials and Methods: A sheep model of pregnancy was used in which the ileal mucosa, submucosa and ENS were assessed following IA exposure to lipopolysaccharide (LPS) for 2 or 7 days (acute), Ureaplasma parvum (UP) for 42 days (chronic) or repetitive microbial exposure (42 days UP with 2 or 7 days LPS). Results: IA LPS exposure for 7 days or IA UP exposure for 42 days caused intestinal injury and inflammation in the mucosal and submucosal layer of the gut. Repetitive microbial exposure did not further aggravate injury of the terminal ileum. Chronic IA UP exposure caused significant structural ENS alterations characterized by loss of PGP9.5 and S100β immunoreactivity whereas these changes were not found after re-exposure of chronic UP-exposed fetuses to LPS for 2 or 7 days. Conclusion: The in utero loss of PGP9.5 and S100β immunoreactivity following chronic UP exposure corresponds with intestinal changes in neonates with NEC, and may therefore form a novel mechanistic explanation for the association of chorioamnionitis and NEC