Functional characterisation of alkane-degrading monooxygenases in Rhodococcus jostii strain 8

Short-chain alkanes are gaseous hydrocarbons that contribute to photochemical pollution and ozone production in the atmosphere. A number of studies have shown that Rhodococcus species possess the ability to metabolite a wide range of hydrocarbons since they contain multiple hydrocarbon-degrading enzymes such as soluble diiron monooxygenases (SDIMOs) and alkB-type alkane monooxygenases. This study aimed to investigate the role of multiple alkane-degrading enzymes in the metabolism of gaseous alkanes in Rhodococcus jostii strain 8. R. jostii strain 8 was isolated from a propane enrichment culture using petroleum-contaminated soil as an inoculum. R. jostii strain 8 could grow on ethane, propane, butane, octane, naphthalene and some potential intermediates in alkane metabolism. Oxidation studies showed that R. jostii strain 8 is likely to oxidise propane via both terminal and sub-terminal oxidation of propane and that these activities are induced in propane-grown cells. Alcohol dehydrogenase assays were carried out in order to determine cofactor and substrate ranges of these enzymes. Results showed that alcohol dehydrogenases involved in the metabolism of gaseous alkanes are NDMA-dependent. The size of the genome sequence of R. jostii strain 8 is 8.5 Mbp with a G+C content of 67%. The closest relative of R. jostii strain 8, based on 16S rRNA sequence, is Rhodococcus jostii RHA1 with 99% identity. However, growth profiles and a number of catabolic genes in the genome of R. jostii strain 8 clearly indicated that this bacterium is different from R. jostii RHA1. R. jostii strain 8 contains two alkane-degrading enzyme systems – a propane monooxygenase and an alkB-type alkane monooxygenase. Polypeptide analysis on cell-free extracts from cells grown on gaseous alkanes using SDS-PAGE indicated that propane monooxygenase is inducible during growth on propane. Expression studies using RT-qPCR of prmA and alkB showed that prmA was highly expressed during growth on propane. The exact involvement of alkB-type alkane monooxygenase in the degradation of alkanes was still unclear. A gene transfer system for R. jostii strain 8 was established. Marker-exchanged mutagenesis of prmA and alkB was attempted. Construction of mutated-prmA and mutated-alkB plasmids was achieved. Electroporation conditions were successfully optimised in order to transfer linear DNA into R. jostii strain 8. However, mutants lacking active prmA or alkB are still needed to further study their phenotypes and to provide more evidence supporting the role of these two enzymes

Pharmacokinetics of single low dose primaquine in Ugandan and Congolese children with falciparum malaria

Background: There are no pharmacokinetic data of single low dose primaquine (SLDPQ) as transmission blocking in African children with acute Plasmodium falciparum and glucose-6-phosphate dehydrogenase deficiency (G6PDd). Methods: Primaquine pharmacokinetics of age-dosed SLDPQ (shown previously to be gametocytocidal with similar tolerability as placebo) were characterised in falciparum-infected Ugandan and Congolese children aged 6 months to 11 years, treated on admission with standard 3-day dihydroartemisinin-piperaquine or artemether-lumefantrine plus SLDPQ: 6 m–<1 y: 1.25 mg, 1–5 y: 2.5 mg, 6–9 y: 5 mg, 10–11 y: 7.5 mg. LC-MS/MS-measured plasma primaquine and carboxyprimaquine (baseline, 1, 1.5, 2, 4, 8, 12, 24 h) were analysed by noncompartmental analysis. Multivariable linear regression modelled associations between covariates, including cytochrome-P450 2D6 metaboliser status, and outcomes. Findings: 258 children (median age 5 [interquartile range (IQR) 3–7]) were sampled; 8 (3.1%) with early vomiting were excluded. Primaquine doses of 0.10–0.40 (median 0.21, IQR 0.16–0.25) mg base/kg resulted in primaquine maximum plasma concentrations (Cmax) of 2.3–447 (median 103.0, IQR 72.1–140.0) ng/mL between 1.0 and 8.0 (median 2) hours (Tmax) and median areas under the drug concentration curves (AUC0-last) 730.2 (6 m–<1 y, n = 12), 582.8 (1–5 y, n = 126), 871.1 (6–9 y, n = 80), and 931.0 (10–11 y, n = 32) ng∗h/mL. Median elimination half-live (T½) was 4.7 (IQR 3.8–5.6) hours. Primaquine clearance/kg peaked at 18 months, plateauing at 4 y. Increasing CYP2D6 metaboliser activity score [poor (3/250), intermediate (52/250), normal (150/250), ultrarapid (5/250), indeterminate (40/250)] and baseline haemoglobin were significantly associated with a lower primaquine AUC0-last,which increased with increasing mg/kg dose and age but was independent of the artemisinin treatment used. Interpretation: Age-dosed SLDPQ resulted in variable primaquine exposure that depended on bodyweight-adjusted dose, age, baseline haemoglobin and CYP2D6 metaboliser status, but not on dihydroartemisinin-piperaquine or artemether-lumefantrine. These data support age-dosed SLDPQ for transmission blocking in sub-Saharan Africa. Funding: This work was cofunded by the UK Medical Research Council, Wellcome Trust, and UK Aid through the Global Health Trials (grant reference MR/P006973/1). The funders had no role in the study design, execution, and analysis and decisions regarding publication

The role of TNFa and IFNy on CXCL10 regulation and beta-2 agonist inhibition in human airway smooth muscle cells

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Solubility and diffusion of organic vapors in silicone polymers

Study of solubility and diffusion of organic vapors in polymer membranes is important for the development of membrane processes for the removal of organic vapors from air for environmental applications. The solubility and absorption/desorption kinetics of seven organic vapors, namely, n-pentane, n-hexane, n-heptane, benzene, cyclohexane, methanol, and 1-propanol in two polymer membranes, i.e. poly(dimethyl siloxane), PDMS, and poly(trifluoropropyl methyl siloxane), PTFPMS, have been studied at 35.0\sp\circC at different vapor activities. Solubilities of seven vapors in both polymers increase exponentially with increasing vapor activity. S(0) of the nonpolar vapors in PDMS are considerably higher than the corresponding values of these vapors in PTFPMS because of the lower glass-transition temperature, T\sb{g}, and hence the larger mean free volume of the former polymer. S(0) of polar vapors (methanol and propanol) are of similar magnitude for both silicones polymers. The Flory-Huggins equation describes satisfactorily the solubilities of nonpolar penetrant vapors but fails to describe the solubilities of polar vapors in both polymers. However, solubilities of all vapors in both polymers can be represented by either Flory-Rehner equation or modified Flory-Huggins equation. Values of the Flory interaction parameters for seven organic vapors in both polymers decrease with increasing solubility of these vapors. Diffusion coefficients obtained from absorption measurements were corrected for heat and swelling effects caused by the very high solubilities of the penetrant vapors in both polymers. Diffusion coefficients determined from the desorption measurements were corrected only for swelling effects, because the heat effects were negligible under the experimental conditions of this study. Heat-effect corrections were made using theory of Armstrong et al. with pertinent initial and boundary conditions. Corrected mutual diffusion coefficients of nonpolar vapors increase exponentially with increasing vapor concentration. By contrast, diffusion coefficients of polar vapors decrease with increasing concentration because of the clustering. Temperature changes of membrane sample (increase during absorption and decrease during desorption) obtained from measurements and those obtained from heat-effect models are in satisfactory agreement. The magnitude of the heat effects decreases with increasing membrane thickness. The correlation of diffusion coefficients and concentration using Fujita\u27s and Vrentas and Duda\u27s free-volume models must be considered as semiempirical

The Prevalence and Severity of Tooth Wear in Type 2 Diabetic Patients

Objective. To assess the prevalence and severity of tooth wear in type 2 diabetic patients. Methods. Attendees at a diabetic clinic at Wiang Pa Pao Hospital in Chiang Rai province, Thailand, were invited to take part in this cross-sectional study. All participants were aged 35–74 and had type 2 diabetes. Participants were required to have been diagnosed with diabetes for at least three months. 179 subjects accepted a clinical oral examination and completed the questionnaire. Tooth wear was assessed clinically using the Smith and Knight Tooth Wear Index. Results. The mean age of diabetic patients was 56.5 ± 7.8 years. The majority (44.1%) had diabetes more than 5 years. The average years of having had diabetes was 6.5 ± 6.3 years. The most prevalent type of tooth wear was attrition (99.4%). The prevalence of erosion, abrasion, and abfraction were 64.8%, 31.3%, and 7.3%, respectively. The majority of the tooth wear was moderate to high severity (62.1%). Erosion and abfraction showed significant association with age group (p<0.05). Age group was significantly associated with the severity level (p=0.017). Mild tooth wear severity was the highest in age groups 35–44 and 45–54 (53.8% and 41.2%, respectively). Moderate tooth wear was the highest proportion in age groups 55–65 and 65–74 (52.2% and 44.0%, respectively). There were no significant differences between specific diabetic symptoms and types of tooth wear. Conclusion. There was a high prevalence of tooth wear among diabetic patients. The role of prevention is vital in maintaining the integrity of the teeth and to avoid treating these worn teeth in diabetic patients

Factors influencing resilience in patients with advanced cancer: A cross-sectional study

Background: Advanced cancer and its treatments lead to various detrimental impacts on patients. Resilience is an important ability to adapt to such adversity, but there is limited information about its influencing factors, specifically in patients with advanced cancer. Objective: This study aimed to examine the influence of social support, depression, anxiety, hope, optimism, spiritual well-being, religious belief,and hardiness on resilience among adults with advanced cancer. Methods: This cross-sectional research used multi-stage sampling to select 288 participants from a university hospital and three tertiary hospitalsin northern Thailand. Data were collected using a demographic data collection form, the Hospital Anxiety and Depression Scale (HADS), the Thai version of the Social Support Questionnaire (SSQ), the Herth Hope Index (HHI), Life Orientation Test-Revised (LOT-R), Buddhist Belief Questionnaire, Health-Related Hardiness Scale (HRHS), and Connor-Davidson Resilience Scale (CD-RISC), from February 2021 to February 2022. Data were analyzed using descriptive statistics, Pearson correlation, and regression analysis. Results: Depression (r = -0.47, p <0.01) and anxiety (r = -0.39, p <0.01) had a significant negative relationship with resilience. Spiritual well-being(r = 0.74, p <0.01), hope (r = 0.67, p <0.01), religious belief (r = 0.53, p <0.01), optimism (r = 0.40, p <0.01), social support (r = 0.33, p <0.01), and hardiness (r = 0.21, p <0.01) had significant positive relationships with resilience. Only hope (β = 0.29, p <0.01) and spiritual well-being (β = 0.59, p<0.01) together influenced resilience by 64.70%. Conclusion: Spiritual well-being and hope are crucial to resilience in patients with advanced cancer. Nurses should provide spiritual support to strengthen patients’ ability to adapt successfully to life with advanced cancer

Patterns of Health-Related Quality of Life among Stroke Survivors: A Longitudinal Study

Six months poststroke is critical for optimizing stroke survivors’ recovery and improving their health-related quality of life (HRQoL). Understanding HRQoL profiles in specific domains can provide a more typical plan for stroke survivors. This pilot study aimed to explore the HRQoL patterns among 39 stroke survivors within 6 months following diagnosis. The results revealed that there were four patterns based on HRQoL domains. Pattern 1: energy, family role, mobility, self-care, social role, and work/productivity domains likely to improve; Pattern 2: language and vision domains likely to improve and be stable; Pattern 3: thinking and upper extremity function domains likely to worsen and improve later; and Pattern 4: mood and personality domains likely to worsen and be constant. Health care providers can apply these results by continuing to assess and provide care to stroke survivors focusing on functional status, cognitive status, and depression over time. Further study should explore the factors predicting HRQoL

Development of simple kinetic models and parameter estimation for simulation of recombinant human serum albumin production by Pichia pastoris

In order to describe and predict the growth and expression of recombinant proteins by using a genetically modified Pichia pastoris, we developed a number of unstructured models based on growth kinetic equation, fed-batch mass balance and the assumptions of constant cell and protein yields. The growth of P. pastoris on both glycerol and methanol could be represented by Monod kinetic equation. A simple simulation methodology and developed models were shown to satisfactorily describe both growth and production of recombinant human serum albumin (rHSA) using a genetically modified P. pastoris MutS strain. The obtained parameters from curve fitting were reasonable and could be acceptable. Moreover, the same parameter sets obtained by the experiments indicated the rigidity and consistency of the developed models and fermentation approach of this study. With correlation coefficients (r2) exceeding 0.99, the models were able to simulate and predict the cell growth behavior and recombinant protein production by P. pastoris without requiring complex models.Key words: Exponential feed, growth modeling, Monod kinetic equation, Pichia pastoris, recombinant human serum albumin

On-line methanol sensor system development for recombinant human serum albumin production by Pichia pastoris

An on-line methanol sensor system was developed using a methanol probe, methanol sensor unit and peristaltic pump. The system was commanded using data acquisition (DAQ) and LabVIEW software. Calibration of the methanol sensor system was done in a medium environment with yeast cells during cells adaptation to methanol metabolism after glycerol feeding was stopped. The correlation equations between voltage output signal from the methanol sensor unit and residual methanol in culture broth were created with third order polynomial regression. This developed system was implemented for online methanol control in recombinant human serum albumin (rHSA) protein production by P. pastoris KM71 at methanol levels of 4 and 10 g/l with controlled fluctuations at 13.0 and 11.3% of oscillation, respectively. The accumulated amounts of recombinant protein from two levels of methanol concentration controls (4 and 10 g/l) were similar but the proteins were produced at a different rate related with methanol concentration in the broth. Therefore, the control at 10 g/l methanol had a higher production rate (0.53 mg-protein/g dry-cell-h) than 4 g/l methanol control (0.38 mg-protein/g dry-cell-h) as it reached the maximum protein concentration in a shorter time, even though its cell yield was less than that of 4 g/l methanol control. At the end of the experiments, the high cell density environment caused both cell and protein reduction by cell autolysis and protease degradation. However, the protein decrease could be prevented by taking protein induction at a low temperature and a pH where protease does not function.Key words: Methanol monitoring, methanol sensor, on-line methanol, Pichia pastoris, recombinant human serum albumin