Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

<p>Abstract</p> <p>Background</p> <p><it>Trigonopsis variabilis </it><smcaps>D</smcaps>-amino acid oxidase (<it>Tv</it>DAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of <it>Tv</it>DAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active <it>Pichia pastoris Tv</it>DAO whole-cell biocatalyst.</p> <p>Results</p> <p>As compared to the native <it>T. variabilis </it>host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in <it>P. pastoris </it>through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 × 10³U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of ≥ 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of <it>P. pastoris </it>using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used <it>Tv</it>DAO expressing <it>Escherichia coli </it>strain) under conditions of <smcaps>D</smcaps>-methionine conversion using vigorous aeration.</p> <p>Conclusions</p> <p>Stepwise optimization using a multi-level engineering approach has delivered a new <it>P. pastoris </it>whole cell <it>Tv</it>DAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this <it>P. pastoris </it>catalyst has been evaluated for industrial purposes.</p

Nonsteroidal Antiinflammatory Drug Administration and Postpartum Blood Pressure in Women With Hypertensive Disorders of Pregnancy

OBJECTIVE: To evaluate whether postpartum nonsteroidal antiinflammatory drug (NSAID) administration is associated with increased blood pressure in women with hypertensive disorders of pregnancy and to estimate the association between NSAID administration and use of opioid medication. METHODS: We conducted a retrospective cohort study of women with hypertensive disorders of pregnancy. Patients were analyzed in two groups according to whether they received NSAIDs postpartum. Study participants were women delivered at a tertiary care center from 2008 to 2015. The primary outcome was change in mean arterial pressure during the postpartum period. Secondary outcomes were postpartum pain scores, cumulative postpartum opioid requirement, initiation or dose escalation of antihypertensive agents, and adverse postpartum outcomes including acute renal failure, change in hematocrit, and maternal readmission for hypertensive disorder. RESULTS: Two hundred seventy-six women with hypertensive disorders of pregnancy were included (129 NSAID-unexposed and 147 NSAID-exposed). Postpartum NSAID administration was not associated with a statistically significant change in mean arterial pressure compared with no NSAID administration (-0.7 vs -1.8; mean difference 1.10, 95% CI -1.44 to 3.64). Similarly, no difference was observed between the cohorts in terms of need for initiation or escalation in dose of antihypertensive agents or maternal readmission for hypertensive disorder. The study was underpowered to determine whether NSAID administration was associated with any difference in less frequent secondary outcomes (eg, incidence of acute renal insufficiency, need for postpartum transfusion) or cumulative opioid use. CONCLUSION: Nonsteroidal antiinflammatory drug administration to postpartum patients with hypertensive disorders of pregnancy is not associated with a change in blood pressure or requirement for antihypertensive medication

The strength of the meridional overturning circulation of the stratosphere.

The distribution of gases such as ozone and water vapour in the stratosphere - which affect surface climate - is influenced by the meridional overturning of mass in the stratosphere, the Brewer-Dobson circulation. However, observation-based estimates of its global strength are difficult to obtain. Here we present two calculations of the mean strength of the meridional overturning of the stratosphere. We analyze satellite data that document the global diabatic circulation between 2007- 2011, and compare these to three re-analysis data sets and to simulations with a state-of-the-art chemistry-climate model. Using measurements of sulfur hexafluoride (SF6) and nitrous oxide, we calculate the global mean diabatic overturning mass flux throughout the stratosphere. In the lower stratosphere, these two estimates agree, and at a potential temperature level of 460 K (about 20 km or 60 hPa in tropics), the global circulation strength is 6.3-7.6 × 109 kg/s. Higher in the atmosphere, only the SF6-based estimate is available, and it diverges from the re-analysis data and simulations. Interpretation of the SF6 data-based estimate is limited because of a mesospheric sink of SF6; however, the reanalyses also differ substantially from each other. We conclude that the uncertainty in the mean meridional overturning circulation strength at upper levels of the stratosphere amounts to at least 100 %

Senescence and Inflammatory Markers for Predicting Clinical Progression in Parkinson's Disease: The ICICLE-PD Study.

BACKGROUND: Cognitive decline is a frequent complication of Parkinson's disease (PD) and the identification of predictive biomarkers for it would help in its management. OBJECTIVE: Our aim was to analyse whether senescence markers (telomere length, p16 and p21) or their change over time could help to better predict cognitive and motor progression of newly diagnosed PD patients. We also compared these senescence markers to previously analysed markers of inflammation for the same purpose. METHODS: This study examined the association of blood-derived markers of cell senescence and inflammation with motor and cognitive function over time in an incident PD cohort (the ICICLE-PD study). Participants (154 newly diagnosed PD patients and 99 controls) underwent physical and cognitive assessments over 36 months of follow up. Mean leukocyte telomere length and the expression of senescence markers p21 and p16 were measured at two time points (baseline and 18 months). Additionally, we selected five inflammatory markers from existing baseline data. RESULTS: We found that PD patients had shorter telomeres at baseline and 18 months compared to age-matched healthy controls which also correlated to dementia at 36 months. Baseline p16 levels were associated with faster rates of motor and cognitive decline over 36 months in PD cases, while a simple inflammatory summary score at baseline best predicted cognitive score over this same time period in PD patients. CONCLUSION: Our study suggests that both inflammatory and senescence markers (p16) are valuable predictors of clinical progression in PD patients.This study was supported by a Newcastle upon Tyne Hospital Trust (Brain Research Unit PD0612) grant to GS. ICICLE-PD is funded by Parkinson’s UK (grant no J-0802, G-1301) and supported by the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre in Ageing and Chronic Disease and the Biomedical Research Unit in Lewy Body Dementia based at Newcastle upon Tyne Hospitals NHS Foundation Trust and Newcastle University (CM-R) and the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre (146281). This work was also supported by grants from the Academy of Medical Sciences, UK, the Rosetrees Trust, and the Stevenage Biosciences Catalyst. CHWG is supported by a RCUK/UKRI Research Innovation Fellowship awarded by the Medical Research Council (MR/R007446/1). RAB is an NIHR Senior Investigator (NF-SI-0616-10011) and is supported by the WT/MRC Stem Cell Institute (203151/Z/16/Z

Predictive modeling of the virtual Hemi-Fontan operation for second stage single ventricle palliation: Two patient-specific cases

Single ventricle hearts are congenital cardiovascular defects in which the heart has only one functional pumping chamber. The treatment for these conditions typically requires a three-staged operative process where Stage 1 is typically achieved by a shunt between the systemic and pulmonary arteries, and Stage 2 by connecting the superior venous return to the pulmonary circulation. Surgically, the Stage 2 circulation can be achieved through a procedure called the Hemi-Fontan, which reconstructs the right atrium and pulmonary artery to allow for an enlarged confluence with the superior vena cava. Based on pre-operative data obtained from two patients prior to Stage 2 surgery, we developed two patient-specific multi-scale computational models, each including the 3D geometrical model of the surgical junction constructed from magnetic resonance imaging, and a closed-loop systemic lumped-parameter network derived from clinical measurements. “Virtual” Hemi-Fontan surgery was performed on the 3D model with guidance from clinical surgeons, and a corresponding multi-scale simulation predicts the patient\u27s post-operative hemodynamic and physiologic conditions. For each patient, a post-operative active scenario with an increase in the heart rate (HR) and a decrease in the pulmonary and systemic vascular resistance (PVR and SVR) was also performed. Results between the baseline and this “active” state were compared to evaluate the hemodynamic and physiologic implications of changing conditions. Simulation results revealed a characteristic swirling vortex in the Hemi-Fontan in both patients, with flow hugging the wall along the SVC to Hemi-Fontan confluence. One patient model had higher levels of swirling, recirculation, and flow stagnation. However, in both models, the power loss within the surgical junction was less than 13% of the total power loss in the pulmonary circulation, and less than 2% of the total ventricular power. This implies little impact of the surgical junction geometry on the SVC pressure, cardiac output, and other systemic parameters. In contrast, varying HR, PVR, and SVR led to significant changes in theses clinically relevant global parameters. Adopting a work-flow of customized virtual planning of the Hemi-Fontan procedure with patient-specific data, this study demonstrates the ability of multi-scale modeling to reproduce patient specific flow conditions under differing physiological states. Results demonstrate that the same operation performed in two different patients can lead to different hemodynamic characteristics, and that modeling can be used to uncover physiologic changes associated with different clinical conditions

Going beyond ‘regular and casual’: development of a classification of sexual partner types to enhance partner notification for STIs

OBJECTIVES To develop a classification of sexual partner types for use in partner notification (PN) for STIs. METHODS A four-step process: (1) an iterative synthesis of five sources of evidence: scoping review of social and health sciences literature on partner types; analysis of relationship types in dating apps; systematic review of PN intervention content; and review of PN guidelines; qualitative interviews with public, patients and health professionals to generate an initial comprehensive classification; (2) multidisciplinary clinical expert consultation to revise the classification; (3) piloting of the revised classification in sexual health clinics during a randomised controlled trial of PN; (4) application of the Theoretical Domains Framework (TDF) to identify index patients' willingness to engage in PN for each partner type. RESULTS Five main partner types emerged from the evidence synthesis and consultation: 'established partner', 'new partner', 'occasional partner', 'one-off partner' and 'sex worker'. The types differed across several dimensions, including likely perceptions of sexual exclusivity, likelihood of sex reoccurring between index patient and sex partner. Sexual health professionals found the classification easy to operationalise. During the trial, they assigned all 3288 partners described by 2223 index patients to a category. The TDF analysis suggested that the partner types might be associated with different risks of STI reinfection, onward transmission and index patients' engagement with PN. CONCLUSIONS We developed an evidence-informed, useable classification of five sexual partner types to underpin PN practice and other STI prevention interventions. Analysis of biomedical, psychological and social factors that distinguish different partner types shows how each could warrant a tailored PN approach. This classification could facilitate the use of partner-centred outcomes. Additional studies are needed to determine the utility of the classification to improve measurement of the impact of PN strategies and help focus resources