Production and Purification of the Heavy Chain Fragment C of Botulinum Neurotoxin, Serotype A, Expressed in the Methylotrophic Yeast \u3ci\u3ePichia pastoris\u3c/i\u3e

A recombinant Hc fragment of botulinum neurotoxin, serotype A (rBoNTA(Hc)), has been successfully expressed in a Mutt strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine. Fermentation employed glycerol batch, glycerol-fed batch, and methanol-fed batch phases to achieve high cell density. Induction times were short to maximize rBoNTA(Hc) production while minimizing proteolytic degradation. Concentration of rBoNTA(Hc) in yeast cell lysates was generally 1-2% of the total protein based on ELISA analysis. The Hc fragment was purified from cell lysates using a multistep ion-exchange (IEC) chromatographic process, including SP, Q, and HS resins. The zwitterionic detergent Chaps was included in the buffer system to combat possible interactions, such as protein-protein or protein-DNA interactions. Following IEC was a hydrophobic interaction chromatography (HIC) polishing step, using phenyl resin. The Hc fragment was purified to \u3e95% purity with yields up to 450 mg/kg cells based on ELISA and Bradford protein assay. The purified Hc fragment of serotype A was stable, elicited an immune response in mice, and was protected upon challenge with native botulinum type A neurotoxin

Resolving the contributions of the membrane-bound and periplasmic nitrate reductase systems to nitric oxide and nitrous oxide production in Salmonella enterica serovar Typhimurium

The production of cytotoxic nitric oxide (NO) and conversion into the neuropharmacological agent and potent greenhouse gas nitrous oxide (N2O) is linked with anoxic nitrate catabolism by Salmonella enterica serovar Typhimurium. Salmonella can synthesize two types of nitrate reductase: a membrane-bound form (Nar) and a periplasmic form (Nap). Nitrate catabolism was studied under nitrate-rich and nitrate-limited conditions in chemostat cultures following transition from oxic to anoxic conditions. Intracellular NO production was reported qualitatively by assessing transcription of the NO-regulated genes encoding flavohaemoglobin (Hmp), flavorubredoxin (NorV) and hybrid cluster protein (Hcp). A more quantitative analysis of the extent of NO formation was gained by measuring production of N2O, the end-product of anoxic NO-detoxification. Under nitrate-rich conditions, the nar, nap, hmp, norV and hcp genes were all induced following transition from the oxic to anoxic state, and 20% of nitrate consumed in steady-state was released as N2O when nitrite had accumulated to millimolar levels. The kinetics of nitrate consumption, nitrite accumulation and N2O production were similar to those of wild-type in nitrate-sufficient cultures of a nap mutant. In contrast, in a narG mutant, the steady-state rate of N2O production was ~30-fold lower than that of the wild-type. Under nitrate-limited conditions, nap, but not nar, was up-regulated following transition from oxic to anoxic metabolism and very little N2O production was observed. Thus a combination of nitrate-sufficiency, nitrite accumulation and an active Nar-type nitrate reductase leads to NO and thence N2O production, and this can account for up to 20% of the nitrate catabolized

Parity and Components of the Metabolic Syndrome Among US Hispanic/Latina Women

BACKGROUND: Physiological adaptations occurring across successive pregnancies may increase the risk of adverse cardiovascular health outcomes in later life. METHODS AND RESULTS: The association between parity and metabolic syndrome was examined among 7467 Hispanic/Latina women of diverse backgrounds, aged 18 to 74 years, who participated in the Hispanic Community Health Study/Study of Latinos (HCHS/SOL) from 2008 to 2011. Metabolic syndrome components were defined according to American Heart Association/National Heart, Lung, and Blood Institute criteria and included abdominal obesity, elevated triglycerides, low high-density lipoprotein cholesterol, high blood pressure, and elevated fasting glucose. Logistic regression models estimated odds ratios (ORs) adjusting for sociodemographic, behavioral, and reproductive characteristics. At HCHS/SOL baseline, women reported none (21.1%), 1 (19.9%), 2 (25.7%), 3 (18.6%), 4 (8.8%), and ≥ 5 (5.9%) live births. When compared with women with 1 birth, those with 4 births had the highest odds of abdominal obesity (OR, 2.0; 95% confidence interval, 1.5-2.8) and overall metabolic syndrome (OR, 1.4; 95% confidence interval, 1.0-2.0) and those with ≥ 5 births had the highest odds of low high-density lipoprotein cholesterol (OR, 1.5; 95% confidence interval, 1.2-2.0) and elevated fasting glucose (OR, 1.6; 95% confidence interval, 1.1-2.4), after adjusting for age, background, education, marital status, income, nativity, smoking, physical activity, menopause, oral contraceptive use, hormone therapy, and field center. Further adjustment for percent body fat attenuated these associations. No associations were observed between parity and elevated triglycerides or high blood pressure. CONCLUSIONS: Higher parity is associated with an increased prevalence of selected components of the metabolic syndrome among Hispanic/Latina women in the US. High parity among Hispanics/Latinas with a high prevalence of abdominal obesity suggests high risk for metabolic dysregulation

A calcium channel or angiotensin converting enzyme inhibitor/angiotensin receptor blocker regime to reduced blood pressure variability in acute ischaemic stroke (CAARBS): A feasibility trial

Background: Trials of lowering blood pressure in patients with acute ischaemic stroke not undergoing thrombolysis have not demonstrated improved outcomes with intervention. Rather than absolute levels, it may be that blood pressure variability is important. However, there are no prospective randomised trials investigating the benefit of reducing blood pressure variability in this patient group. Aims: The primary aim of this trial was to determine the feasibility of recruitment to a randomised trial investigating the effect of different antihypertensive medications on blood pressure variability. Methods: CAARBS was a multi-centre, open-label, randomised parallel group controlled feasibility trial. Adults with a first mild-moderate ischaemic stroke or transient ischaemic attack, requiring antihypertensive therapy for secondary prevention, were randomised to a calcium channel blocker or angiotensin-converting enzyme inhibitor/angiotensin receptor blocker. Blood pressure and variability were measured at baseline, three weeks, and three months. Compliance with measurements and treatment was monitored. Results: Fourteen patients were recruited to the trial (0.6% of those screened), nine of whom completed follow-up. The majority of patients screened (98.1%) were ineligible. Compliance with the intervention was good, as were measurement completion rates (88.9% or higher in all cases except ambulatory measurements). No major adverse events were recorded. Conclusions: Recruitment to the trial was difficult due to patient ineligibility, suggesting that the current protocol is unlikely to be successful if scaled for a definitive trial. However, the intervention was safe, and compliance was good, suggesting a future trial with modified eligibility criteria could be successful. Trial registration: ISRCTN10853487

Habitability of hydrothermal systems at Jezero and Gusev Craters as constrained by hydrothermal alteration of a terrestrial mafic dike

NASA’s search for habitable environments has focused on alteration mineralogy of the Martian crust and the formation of hydrous minerals, because they reveal information about the fluid and environmental conditions from which they precipitated. Extensive work has focused on the formation of alteration minerals at low temperatures, with limited work investigating metamorphic or high-temperature alteration. We have investigated such a site as an analog for Mars: a mafic dike on the Colorado Plateau that was hydrothermally altered from contact with groundwater as it was emplaced in the porous and permeable Jurassic Entrada sandstone. Our results show evidence for fluid mobility removing Si and K but adding S, Fe, Ca, and possibly Mg to the system as alteration progresses. Mineralogically, all samples contain calcite, hematite, and kaolinite; with most samples containing minor anatase, barite, halite, and dolomite. The number of alteration minerals increase with alteration. The hydrothermal system that formed during interaction of the magma (heat source) and groundwater would have been a habitable environment once the system cooled below ∼120 °C. The mineral assemblage is similar to alteration minerals seen within the Martian crust from orbit, including those at Gusev and Jezero Craters. Therefore, based on our findings, and extrapolating them to the Martian crust, these sites may represent habitable environments which would call for further exploration and sample return of such hydrothermally altered igneous materials

Activity of a Carbohydrate-Binding Module Therapy, Neumifil, against SARS-CoV-2 Disease in a Hamster Model of Infection.

The rapid global spread of severe acute respiratory coronavirus 2 (SARS-CoV-2) has resulted in an urgent effort to find efficacious therapeutics. Broad-spectrum therapies which could be used for other respiratory pathogens confer advantages, as do those based on targeting host cells that are not prone to the development of resistance by the pathogen. We tested an intranasally delivered carbohydrate-binding module (CBM) therapy, termed Neumifil, which is based on a CBM that has previously been shown to offer protection against the influenza virus through the binding of sialic acid receptors. Using the recognised hamster model of SARS-CoV-2 infection, we demonstrate that Neumifil significantly reduces clinical disease severity and pathological changes in the nasal cavity. Furthermore, we demonstrate Neumifil binding to the human angiotensin-converting enzyme 2 (ACE2) receptor and spike protein of SARS-CoV-2. This is the first report describing the testing of this type of broad-spectrum antiviral therapy in vivo and provides evidence for the advancement of Neumifil in further preclinical and clinical studies

Psychosocial Response to Uncertain Newborn Screening Results for Cystic Fibrosis

Objective To explore the psychosocial implications of diagnostic uncertainty that result from inconclusive results generated by newborn bloodspot screening (NBS) for cystic fibrosis (CF). Study design Using a mixed methods prospective cohort study of children who received NBS for CF, we compared psychosocial outcomes of parents whose children who received persistently inconclusive results with those whose children received true positive or screen-negative results. Results Mothers of infants who received inconclusive results (n = 17), diagnoses of CF (n = 15), and screen-negative results (n = 411) were surveyed; 23 parent interviews were completed. Compared with mothers of infants with true positive/screen-negative results, mothers of infants with inconclusive results reported greater perceived uncertainty (P .05). Qualitatively, parents valued being connected to experts but struggled with the meaning of an uncertain diagnosis, worried about their infant's health-related vulnerability, and had mixed views about surveillance. Conclusion Inconclusive CF NBS results were not associated with anxiety or vulnerability but led to health-related uncertainty and qualitative concerns. Findings should be considered alongside efforts to optimize protocols for CF screening and surveillance. Educational and psychosocial supports are warranted for these families.Peer reviewe

Parent experience with false-positive newborn screening results for cystic fibrosis

BACKGROUND: The risk of psychosocial harm in families of infants with false-positive (FP) newborn bloodspot screening (NBS) results for cystic fibrosis (CF) is a longstanding concern. Whether well designed retrieval and confirmatory testing systems can mitigate risks remains unknown. METHODS: Using a mixed-methods cohort design, we obtained prospective self-report data from mothers of infants with FP CF NBS results 2 to 3 months after confirmatory testing at Ontario\u27s largest follow-up center, and from a randomly selected control sample of mothers of screen negative infants from the same region. Mothers completed a questionnaire assessing experience and psychosocial response. A sample of mothers of FP infants completed qualitative interviews. RESULTS: One hundred thirty-four mothers of FP infants (response rate, 55%) and 411 controls (response rate, 47%) completed questionnaires; 54 mothers of FP infants were interviewed. Selected psychosocial response measures did not detect psychosocial distress in newborns or 1 year later (P \u3e .05). Mothers recalled distress during notification of the positive result and in the follow-up testing period related to fear of chronic illness, but valued the screening system of care in mitigating concerns. CONCLUSIONS: Although immediate distress was reported among mothers of FP infants, selected psychometric tools did not detect these concerns. The NBS center from which mothers were recruited minimizes delay between notification and confirmatory testing and ensures trained professionals are communicating results and facilitating follow-up. These factors may explain the presence of minimal psychosocial burden. The screening system reflected herein may be a model for NBS programs working to minimize FP-related psychosocial harm