CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16.

BACKGROUND: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. RESULTS: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB- 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. CONCLUSION: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.

Angular dynamics in vector coding: a new approach based on angular velocity.

The assessment of coordination variability in multi-joint human movements has traditionally started from angle-angle representations, and then used the angle change between subsequent time points as input for further analysis through vector coding. We propose an improvement to this approach, and suggest employing angular velocities as input data (Velocity Ellipse Method, VEM). We used experimental data and theoretical principles to contrast VEM with an existing standard (Difference Ellipse Method) and discuss its advantages and potential issues. Normalised cross-correlation was used to compare VEM and DEM in 36 angle couplings, from 20 participants running at 12 km/h on a treadmill. The hip flexion/extension-knee flexion/extension data were further investigated to discuss the robustness of the approach to measurement noise and outliers. Although DEM and VEM generally exhibited similar patterns (cross-correlation between 0.851 and 0.999), the variability curves from the two methods were noticeably different in some intervals. Also, using angular velocities as input appeared more robust to potential noise from raw data whilst retaining the following features: (a) more coherent with biomechanical conventions for calculating three-dimensional angular dynamics; (b) still suitable for coordination analysis; and, (c) more easily interpretable by practitioners when represented as relative motion plots.

Structure of the Reductase Domain of a Fungal Carboxylic Acid Reductase and Its Substrate Scope in Thioester and Aldehyde Reduction.

The synthesis of aldehydes from carboxylic acids has long been a challenge in chemistry. In contrast to the harsh chemically driven reduction, enzymes such as carboxylic acid reductases (CARs) are considered appealing biocatalysts for aldehyde production. Although structures of single- and didomains of microbial CARs have been reported, to date no full-length protein structure has been elucidated. In this study, we aimed to obtain structural and functional information regarding the reductase (R) domain of a CAR from the fungus Neurospora crassa (Nc). The NcCAR R-domain revealed activity for N-acetylcysteamine thioester (S-(2-acetamidoethyl) benzothioate), which mimics the phosphopantetheinylacyl-intermediate and can be anticipated as the minimal substrate for thioester reduction by CARs. The determined crystal structure of the NcCAR R-domain reveals a tunnel that putatively harbors the phosphopantetheinylacyl-intermediate, which is in good agreement with docking experiments performed with the minimal substrate. In vitro studies were performed with this highly purified R-domain and NADPH, demonstrating carbonyl reduction activity. The R-domain was able to accept not only a simple aromatic ketone but also benzaldehyde and octanal, which are typically considered to be the final product of carboxylic acid reduction by CAR. Also, the full-length NcCAR reduced aldehydes to primary alcohols. In conclusion, aldehyde overreduction can no longer be attributed exclusively to the host background.

Applying circular statistics can cause artefacts in the calculation of vector coding variability: A bivariate solution.

BACKGROUND: Coordination variability is thought to provide meaningful insights into motor learning, skill level and injury prevention. Current analytical techniques, based on vector coding (VC) methods, use calculations from circular statistics. However a statistical artefact associated with the application of circular statistics may artificially increase the estimated coordination variability, especially when VC vectors are short. RESEARCH QUESTION: Are two popular methods for calculating vector coding coordination variability susceptible to contamination by statistical artefacts and if so, how can coordination variability be calculated without statistical artefact? METHODS: A combination of simulated and experimental data was used to prove the existence of the statistical artefact and to understand the extent to which it may affect experimental running gait data, respectively. An alternative approach that uses ellipse area as a bivariate measure of variability was proposed, applied to the same dataset, and compared to two popular methods of coordination variability analysis. RESULTS: The simulated data showed the existence of a statistical artefact, which was greater for shorter VC vector lengths in coordination variability measures that used circular statistics. The statistical artefact typically manifests itself as inflated peaks in the coordination variability trace. The experimental data also indicated that short vector lengths are prevalent in running gait. The Ellipse Area Method of coordination variability was not affected by the VC vector length. SIGNIFICANCE: Researchers using current VC variability measures should be particularly aware of the possible effect of the statistical artefact on their data, which is most likely to occur when vector lengths are short. The novel approach we have suggested for calculating VC coordination variability may provide the foundation for future research into vector coding coordination variability.

The association of gender, age, body mass index, and vital signs in healthy plateletapheresis donors.

In order to determine the association between gender, age, body mass index and vital signs among healthy plateletapheresis donors, the mean and median values from the most recent donations of 236 plateletapheresis donors were calculated for age, body mass index (BMI), oral temperature, blood pressure (BP), and pulse. Values of these parameters were compared after stratification by gender, age, and BMI and multiple regression analysis between parameters performed. Gender, age, and BMI were associated with significant differences in vital signs of healthy plateletapheresis donors.

CMV antibody prevalence and seroincidence in plateletpheresis donors.

OBJECTIVE: To determine the prevalence and seroincidence of CMV seropositivity in plateletpheresis donors of different ages and gender. METHODS: CMV antibody serostatus, birthdate, and date of first and most recent donation between the years 1976 and 2006 were retrieved from 222 plateletpheresis donor records at the Johns Hopkins Hospital Donor Center. CMV antibody serostatus was obtained for 183 donors at the most recent donation for which CMV antibody data were available. CMV antibody status and time interval between first and most recent donation were also obtained from 97 repeat plateletpheresis donors who were CMV antibody negative at time of first donation. RESULTS: Overall CMV antibody positivity was 35.5% for 183 donors (mean age = 46.0 years) at time of most recent donation. CMV seropositivity tended to increase with age, being 37.5, 17.9, 37.5, 39.0, and 61.5% for donors aged 20-29 years (n = 8), 30-39 years (n = 39), 40-49 years (n = 64), 50-59 years (n = 59), and 60+ years (n = 13), respectively. Overall CMV seroincidence was 1.6 seroconversions per 100 person years with a rate of 1.4 seroconversions per 100 years for men and 2.3 for women. CONCLUSION: CMV seroprevalence and seroincidence in this plateletpheresis donor population are relatively low so that a large percentage of donors are likely to be able to provide CMV seronegative platelet components for many years. Our data suggest that targeting groups with lower CMV seroprevalence and seroincidence rates such as young people and possibly men will likely yield the highest percentage of CMV seronegative donors.

Effectiveness of education and individualized counseling in reducing environmental hazards in the homes of community-dwelling older women.

OBJECTIVES: To test the effectiveness of an education and counseling intervention on reducing environmental hazards in the homes of older women. DESIGN: Secondary analysis from a randomized, controlled trial with two arms: fall prevention program and health education program (control). Environmental hazards were assessed at baseline and immediately posttreatment (12-weeks). SETTING: Participants' homes. PARTICIPANTS: Two hundred seventy-two community-dwelling women aged 70 and older at risk for falling. INTERVENTION: The fall prevention program involved a comprehensive fall risk evaluation, exercise, education, individualized counseling, and referrals. The health education program included topics unrelated to fall prevention. With the exception of the fall risk evaluation conducted by a nurse practitioner, baccalaureate-prepared nurses carried out the interventions. MEASUREMENTS: Summed and individual scores for hazards related to the bathroom, floor surfaces, lighting, furniture, stairways, and storage areas. RESULTS: Environmental hazards were found in all homes, with a baseline mean+/-standard deviation of 10.7+/-2.6 total hazards and range of four to 17 hazards. Analysis of within-group changes indicated that the fall prevention group had significantly fewer bathroom, lighting, and total hazards after the intervention, whereas the health education group had significantly fewer bathroom hazards but more floor hazards. At follow-up, the fall prevention group had significantly fewer lighting hazards and total hazards than the health education group. CONCLUSION: Education and counseling have only modest effects in helping older women make recommended home modifications. To be most effective in reducing environmental hazards, fall prevention programs may need to provide and install safety devices.

Exercise behavior in older adults: a test of the transtheoretical model.

The transtheoretical model (TTM) was developed as a guide for understanding behavior change. Little attention has been given, however, to the appropriateness of the TTM for explaining the adoption of exercise behavior in older adults. The purposes of this study were to determine the reliability of the TTM instruments and validate TTM predictions in 86 community-dwelling older adults (mean age 75.1 +/- 7.0 years, 87% women) who were participants in a 16-week walking program. TTM construct scales--self-efficacy, decisional balance (pros and cons), and processes of change (behavioral and cognitive)--were generally reliable (all>.78). Behavioral processes of change increased from baseline to follow-up, but pros, cons, and cognitive processes did not change among participants who became regular exercisers. Stage of change did not predict exercise adoption, but baseline self-efficacy predicted walking behavior. These results lend partial support to the TTM in predicting exercise behavior.

Does the Transtheoretical Model (TTM) explain exercise behavior in a community-based sample of older women?

OBJECTIVE: To determine whether patterns of exercise adoption by older women would conform to the Transtheoretical Model (TTM) of behavior change. METHODS: Participants were randomized into an exercise group (walk 30 minutes per day, 5 days per week, plus balance exercises twice per week) or attention control (health education on topics other than exercise). The intervention was conducted over 28 weeks with 1-year follow-up. RESULTS: Participants included 272 sedentary women aged 70 and above. Exercise adoption was higher in the intervention group (83% vs. 17% among controls). After 1 year, 60% of the intervention group was in action or maintenance, compared to 16% of the control group. Self-efficacy following the intervention predicted long-term exercise adherence. DISCUSSION: Few studies have addressed longitudinal analysis of the TTM for exercise adoption. Most constructs from the TTM were useful in explaining exercise adoption in older sedentary women.