Exact solution of a model DNA-inversion genetic switch with orientational control

DNA inversion is an important mechanism by which bacteria and bacteriophage switch reversibly between phenotypic states. In such switches, the orientation of a short DNA element is flipped by a site-specific recombinase enzyme. We propose a simple model for a DNA inversion switch in which recombinase production is dependent on the switch state (orientational control). Our model is inspired by the fim switch in Escherichia coli. We present an exact analytical solution of the chemical master equation for the model switch, as well as stochastic simulations. Orientational control causes the switch to deviate from Poissonian behaviour: the distribution of times in the on state shows a peak and successive flip times are correlated.Comment: Revised version, accepted for publicatio

Phase variation controls expression of Salmonella lipopolysaccharide modification genes by a DNA methylation-dependent mechanism

The O-antigen of Salmonella lipopolysaccharide is a major antigenic determinant and its chemical composition forms the basis for Salmonella serotyping. Modifications of the O-antigen that can affect the serotype include those carried out by the products of glycosyltransferase operons (gtr), which are present on specific Salmonella and phage genomes. Here we show that expression of the gtr genes encoded by phage P22 that confers the O1 serotype is under the control of phase variation. This phase variation occurs by a novel epigenetic mechanism requiring OxyR in conjunction with the DNA methyltransferase Dam. OxyR is an activator or a repressor of the system depending on which of its two binding sites in the gtr regulatory region is occupied. Binding is decreased by methylation at Dam target sequences in either site, and this confers heritability of the expression state to the system. Most Salmonella gtr operons share the key regulatory elements that are identified here as essential for this epigenetic phase variation

Recovery of protein synthesis to assay DNA repair activity in transcribed genes in living cells and tissues

Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that protects against the negative effects of transcription-blocking DNA lesions. Hereditary TC-NER deficiencies cause pleiotropic and often severe neurodegenerative and progeroid symptoms. While multiple assays have been developed to determine TC-NER activity for clinical and research purposes, monitoring TC-NER is hampered by the low frequency of repair events occurring in transcribed DNA. ’Recovery of RNA Synthesis’ is widely used as indirect TC-NER assay based on the notion that lesion-blocked transcription only resumes after successful TC-NER. Here, we show that measuring novel synthesis of a protein after its compound-induced degradation prior to DNA damage induction is an equally effective but more versatile manner to indirectly monitor DNA repair activity in transcribed genes. This ‘Recovery of Protein Synthesis’ (RPS) assay can be adapted to various degradable proteins and readouts, including imaging and immunoblotting. Moreover, RPS allows real-time monitoring of TC-NER activity in various living cells types and even in differentiated tissues of living organisms. To illustrate its utility, we show that DNA repair in transcribed genes declines in aging muscle tissue of C. elegans. Therefore, the RPS assay constitutes an important novel clinical and research tool to investigate transcription-coupled DNA repair

The pre- and post-authorisation data published by the European Medicines Agency on the use of Biologics during pregnancy and lactation

Aims: The effects of biologics on reproduction/lactation are mostly unknown although many patients that receive biologics are women of reproductive age. The first objective of this study was to investigate the publicly available data on pregnancy/lactation before and after marketing authorization in Europe of biologics for the indications of rheumatologic inflammatory autoimmune diseases and inflammatory bowel disease. Secondary objectives included the assessment of the clinical relevance of the provided data and comparison of initial and post-authorization data. Methods: Initial and post-authorization data were extracted from the European Public Assessment Reports and the latest versions of Summary of Product Characteristics using publicly available documents on the European Medicines Agency's website. Four sections were categorized regarding pregnancy outcomes: pre-clinical/animal studies, human female fertility, pregnancy-related outcomes and congenital malformations in the human fetus. Three sections were categorized regarding lactation outcomes: pre-clinical/animal studies, excretion in human breast milk and absorption in children through breastfeeding. The clinical applicability of each category was scored by specified criteria, based on scientific literature, and further as defined by the authors. Results: For the 16 included biologics, post-authorization data were delivered only for adalimumab, certolizumab pegol, etanercept and infliximab. For the 12 remaining biologics limited data on pregnancy and lactation during the post-marketing period of 2–21 years were available. Conclusions: In this article several suggestions are provided for improving a multidisciplinary approach to these issues. The initiation of suitable registries by marketing authorization holders and data transparency for clinicians and academics are highly endorsed

Training for the HandbikeBattle:an explorative analysis of training load and handcycling physical capacity in recreationally active wheelchair users

Purpose: (1) to analyze training characteristics of recreationally active wheelchair users during handcycle training, and (2) to examine the associations between training load and change in physical capacity. Methods: Former rehabilitation patients (N = 60) with health conditions such as spinal cord injury or amputation were included. Participants trained for five months. A handcycling/arm crank graded exercise test was performed before and after the training period. Outcomes: peak power output per kg (POpeak/kg) and peak oxygen uptake per kg (VO 2peak/kg). Training load was defined as Training Impulse (TRIMP), which is rating of perceived exertion (sRPE) multiplied by duration of the session, in arbitrary units (AU). Training intensity distribution (TID) was also determined (time in zone 1, RPE ≤4; zone 2, RPE 5–6; zone 3, RPE ≥7). Results: Multilevel regression analyses showed that TRIMP sRPE was not significantly associated with change in physical capacity. Time in zone 2 (RPE 5–6) was significantly associated with ΔVO 2peak, %ΔVO 2peak, ΔVO 2peak/kg and %ΔVO 2peak/kg. Conclusion: Training at RPE 5–6 was the only determinant that was significantly associated with improvement in physical capacity. Additional controlled studies are necessary to demonstrate causality and gather more information about its usefulness, and optimal handcycle training regimes for recreationally active wheelchair users.IMPLICATIONS FOR REHABILITATION Monitoring of handcycle training load is important to structure the training effort and intensity over time and to eventually optimize performance capacity. This is especially important for relatively untrained wheelchair users, who have a low physical capacity and a high risk of overuse injuries and shoulder pain. Training load can be easily calculated by multiplying the intensity of the training (RPE 0–10) with the duration of the training in minutes. Results on handcycle training at RPE 5–6 intensity in recreationally active wheelchair users suggests to be promising and should be further investigated with controlled studies

Secondary health conditions in persons with a spinal cord injury for at least 10 years:design of a comprehensive long-term cross-sectional study

Purpose: To describe the prevalence of secondary health conditions (SHCs) (urinary tract and bowel problems, pressure ulcers, spasticity, musculoskeletal and neuropathic pain, sexual dysfunction, respiratory and cardiovascular disorders) in persons with long-term spinal cord injury (SCI), and to explore the impact of SHCs on fitness, active lifestyle, participation and well-being. Methods: A time since injury (TSI)-stratified cros-ssectional study among 300 persons between 28- and 65-year-old with a SCI for at least 10 years. Strata of TSI are 10-19, 20-29, and 30 or more years. All eight Dutch rehabilitation centres with a SCI unit will participate. Participants will be invited for a 1-day visit to the rehabilitation centre for an aftercare check-up by the local SCI rehabilitation physician (neurological impairment, SHCs and management), physical tests by a trained research assistant (lung function, wheelchair skills, physical capacity), and they will be asked to complete a self-report questionnaire in advance. Results: Not applicable. Conclusion: This study will provide knowledge on the health status and functioning of persons aging with SCI living in the Netherlands. This knowledge will help us to develop predictive models for the occurrence of SHCs and to formulate guidelines to improve health care for persons with long-term SCI.</p

Force direction and physical load in dynamic pushing and pulling

In pushing and pulling wheeled carts, the direction of force exertion may, beside the force magnitude, considerably affect musculoskeletal loading. This paper describes how force direction changes as handle height and force level change, and the effects this has on the loads on the shoulder and low back. Eight subjects pushed against or pulled on a stationary bar or movable cart at various handle heights and horizontal force levels while walking on a treadmill. The forces at the hands in the vertical and horizontal direction were measured by a force-transducer. The forces, body movements and anthropometric data were used to calculate the net joint torques in the sagittal plane in the shoulder and the lumbosacral joint. The magnitudes and directions of forces did not differ between the cart and the bar pushing and pulling. Force direction was affected by the horizontal force level and handle height. As handle height and horizontal force level increased, the pushing force direction changed from 45 degrees (SD 3.3 degrees) downward to near horizontal, while the pulling force direction changed from pulling upward by 14 degrees (SD 15.3 degrees) to near horizontal. As a result, it was found that across conditions the changes in force exertion were frequently reflected in changes in shoulder torque and low back torque although of a much smaller magnitude. Therefore, an accurate evaluation of musculoskeletal loads in pushing and pulling requires, besides a knowledge of the force magnitude, knowledge of the direction of force exertion with respect to the bod

Nonselective Bottlenecks Control the Divergence and Diversification of Phase-Variable Bacterial Populations

Phase variation occurs in many pathogenic and commensal bacteria and is a major generator of genetic variability. A putative advantage of phase variation is to counter reductions in variability imposed by nonselective bottlenecks during transmission. Genomes of Campylobacter jejuni, a widespread food-borne pathogen, contain multiple phase-variable loci whose rapid, stochastic variation is generated by hypermutable simple sequence repeat tracts. These loci can occupy a vast number of combinatorial expression states (phasotypes) enabling populations to rapidly access phenotypic diversity. The imposition of nonselective bottlenecks can perturb the relative frequencies of phasotypes, changing both within-population diversity and divergence from the initial population. Using both in vitro testing of C. jejuni populations and a simple stochastic simulation of phasotype change, we observed that single-cell bottlenecks produce output populations of low diversity but with bimodal patterns of either high or low divergence. Conversely, large bottlenecks allow divergence only by accumulation of diversity, while interpolation between these extremes is observed in intermediary bottlenecks. These patterns are sensitive to the genetic diversity of initial populations but stable over a range of mutation rates and number of loci. The qualitative similarities of experimental and in silico modeling indicate that the observed patterns are robust and applicable to other systems where localized hypermutation is a defining feature. We conclude that while phase variation will maintain bacterial population diversity in the face of intermediate bottlenecks, narrow transmission-associated bottlenecks could produce host-to-host variation in bacterial phenotypes and hence stochastic variation in colonization and disease outcomes