Analysis of the sheep MHC using HLA class I,II, and C4 cDNA probes

Four cDNA probes for the human major histocompatibility complex (MHC) were used to investigate the sheep MHC, in conjunction with serological typing for ovine lymphocyte antigen (OLA). Lymphocytes from a family (two parents and five offspring) of Romanov sheep were subjected to genomic DNA digestion by the restriction endonuclease Eco RI, followed by gel electrophoresis. A single Southern blot representing all seven individuals was then consecutively hybridized with the class I, alpha-DC, beta-DR, and C4 probes, which were originally designed to identify HLA class I, class II (DC and DR), and C4 products, respectively. Using each of the three class I/class II probes, several bands showing DNA polymorphism were detected. The segregation of these bands in the five offspring exactly paralleled the OLA haplotype segregation established by serological typing. A further eight individuals carrying haplotypes which were phenotypically identical to those in the above-mentioned family showed bands in the corresponding positions when tested with the same three probes. Using the C4 probe, no polymorphism was detected in these fifteen individuals.Abbreviations used in this paper MHC major histocompatibility complex - OLA ovine lymphocyte antigen - kbp kilobase pair(s) - MLR mixed lymphocyte reaction - RFLP restriction fragment length polymorphism     

Cell free synthesis of some storage protein subunits by polyribosomes and RNA isolated from developing seeds of pea (Pisum sativum L.)

Polyribosomes which have template activity in the wheat germ system have been isolated from developing pea seeds. Some of the translation products have identical mobilities to the vicilin and legumin subunits by SDS-PAGE. Certain products were specifically immunoprecipitated with antisera prepared against purified vicilin and legumin fractions. Various RNA fractions including poly A-rich RNA have also been isolated from polyribosomes and shown to direct the synthesis of polyripeptides whose properties are similar to the storage protein subunits. The results are discussed in relationship to other investigations with seed storage protein biosynthesis in vitro.Abbreviations DTT dithiothreitol - SDS-PAGE SDS-polyacrylamide gel electrophoresis - TCA tricarboxylic acid     

The relationship between the size of mitochondria and the intensity of light that they scatter in different energetic states

The intensity of light scattered at 90° to the incident beam and the effective hydrodynamic radii of mitochondria incubated under a variety of conditions have been measured. Addition of high concentrations of uncouplers to respiring mitochondria resulted in a decrease in scatter which was not due to swelling. Addition of valinomycin to mitochondria depleted of substrate in K⁺-free medium produced an increase in scatter that was not due to shrinking. It is concluded that changes in the intensity of scattered light are not reliable indices of changes of volume of mitochondria, and that changes in conformation with changes in metabolic state dominate changes in light scatter. A molecular mechanism for the effect of metabolic state upon the scattered intensity is suggested.     

The use of preserved tissue in finite element modelling of fresh ovine vertebral behaviour

The aim of this study was to investigate whether the predicted finite element (FE) stiffness of vertebral bone is altered when using images of preserved rather than fresh tissue to generate specimen-specific FE models. Fresh ovine vertebrae were used to represent embalmed (n = 3) and macerated dry-bone (n = 3) specimens and treated accordingly. Specimens were scanned pre- and post-treatment using micro-computed tomography. A constant threshold level derived from these images was used to calculate the respective bone volume fraction (BV/TV) from which the conversion factor validated for fresh tissue was used to determine material properties that were assigned to corresponding FE models. Results showed a definite change in the BV/TV between the fresh and the preserved bone. However, the changes in the predicted FE stiffness were not generally greater than the variations expected from assignment of loading and boundary conditions. In conclusion, images of preserved tissue can be used to generate FE models that are representative of fresh tissue with a tolerable level of error.     

A Computational Model of Lipopolysaccharide-Induced Nuclear Factor Kappa B Activation: A Key Signalling Pathway in Infection-Induced Preterm Labour

Preterm birth is the single biggest cause of significant neonatal morbidity and mortality, and the incidence is rising. Development of new therapies to treat and prevent preterm labour is seriously hampered by incomplete understanding of the molecular mechanisms that initiate labour at term and preterm. Computational modelling provides a new opportunity to improve this understanding. It is a useful tool in (i) identifying gaps in knowledge and informing future research, and (ii) providing the basis for an in silico model of parturition in which novel drugs to prevent or treat preterm labour can be “tested”. Despite their merits, computational models are rarely used to study the molecular events initiating labour. Here, we present the first attempt to generate a dynamic kinetic model that has relevance to the molecular mechanisms of preterm labour. Using published data, we model an important candidate signalling pathway in infection-induced preterm labour: that of lipopolysaccharide (LPS) -induced activation of Nuclear Factor kappa B. This is the first model of this pathway to explicitly include molecular interactions upstream of Nuclear Factor kappa B activation. We produced a formalised graphical depiction of the pathway and built a kinetic model based on ordinary differential equations. The kinetic model accurately reproduced published in vitro time course plots of Lipopolysaccharide-induced Nuclear Factor kappa B activation in mouse embryo fibroblasts. In this preliminary work we have provided proof of concept that it is possible to build computational models of signalling pathways that are relevant to the regulation of labour, and suggest that models that are validated with wet-lab experiments have the potential to greatly benefit the field.     

Hepatitis C Transmission and Treatment in Contact Networks of People Who Inject Drugs

Hepatitis C virus (HCV) chronically infects over 180 million people worldwide, with over 350,000 estimated deaths attributed yearly to HCV-related liver diseases. It disproportionally affects people who inject drugs (PWID). Currently there is no preventative vaccine and interventions feature long treatment durations with severe side-effects. Upcoming treatments will improve this situation, making possible large-scale treatment interventions. How these strategies should target HCV-infected PWID remains an important unanswered question. Previous models of HCV have lacked empirically grounded contact models of PWID. Here we report results on HCV transmission and treatment using simulated contact networks generated from an empirically grounded network model using recently developed statistical approaches in social network analysis. Our HCV transmission model is a detailed, stochastic, individual-based model including spontaneously clearing nodes. On transmission we investigate the role of number of contacts and injecting frequency on time to primary infection and the role of spontaneously clearing nodes on incidence rates. On treatment we investigate the effect of nine network-based treatment strategies on chronic prevalence and incidence rates of primary infection and re-infection. Both numbers of contacts and injecting frequency play key roles in reducing time to primary infection. The change from “less-” to “more-frequent” injector is roughly similar to having one additional network contact. Nodes that spontaneously clear their HCV infection have a local effect on infection risk and the total number of such nodes (but not their locations) has a network wide effect on the incidence of both primary and re-infection with HCV. Re-infection plays a large role in the effectiveness of treatment interventions. Strategies that choose PWID and treat all their contacts (analogous to ring vaccination) are most effective in reducing the incidence rates of re-infection and combined infection. A strategy targeting infected PWID with the most contacts (analogous to targeted vaccination) is the least effective.     

Sleep and Sleepiness of Fishermen on Rotating Schedules

Seafaring is a hazardous occupation with high death and injury rates, but the role of seafarer fatigue in these events is generally not well documented. The International Maritime Organization has identified seafarer fatigue as an important health and safety issue. Most research to date has focused on more regularly scheduled types of operations (e.g., merchant vessels, ferries), but there is relatively little information on commercial fishing, which often involves high day‐to‐day and seasonal variability in work patterns and workload. The present study was designed to monitor the sleep and sleepiness of commercial fishermen at home and during extended periods at sea during the peak of the hoki fishing season, with a view to developing better fatigue management strategies for this workforce. Sleep (wrist actigraphy and sleep diaries) and sleepiness (Karolinska Sleepiness Scale [KSS] before and after each sleep period) of 20 deckhands were monitored for 4–13 days at home and for 5–9 days at sea while working a nominal 12 h on/6 h off schedule. On the 12 h on/6 h off schedule, there was still a clear preference for sleep at night. Comparing the last three days at home and the first three days at sea showed that fishermen were more likely to have split sleep at sea (Wilcoxon signed ranks p<0.001), but the median sleep/24 h did not differ significantly by location (5.9 h at sea vs. 6.7 h at home). However, on 23% of days at sea, fishermen obtained<4 h total sleep/24 h, compared to 3% of days at home (p(χ²)<0.01). Sleep efficiency, mean activity counts/min sleep, and subjective ratings of sleep quality did not differ significantly between the last three days at home and the first three days at sea. However, sleepiness ratings remained higher after sleep at sea (Wilcoxon signed ranks p<0.05), with fishermen having post‐sleep KSS ratings ≥7 on 24% of days at sea vs. 9% of days at home (Wilcoxon signed ranks p<0.01). This work adds to the limited number of studies that objectively monitored the sleep of seafarers. It has the strength of operational fidelity but the weakness that large inter‐ and intra‐individual variability in sleep, combined with the small sample size, limited the power of the study to detect statistically significant differences between sleep at home and at sea. The clear preference for sleep at night during the 12 h on/6 h off schedule at sea is consistent with the expectation that this 18 h duty/rest cycle is outside the range of entrainment of the circadian pacemaker. High levels of acute sleep loss, and residual sleepiness after sleep, were much more common at sea than at home. The longer duration of trips during the peak of the fishing season increases the risk of performance impairment due to greater cumulative sleep loss than would be expected on typical three‐day trips. Key fatigue management strategies in this environment include that fishermen report to work as well rested as possible. Once at sea, the day‐to‐day variability in activities due to uncontrollable factors, such as fishing success, repairing gear, and weather conditions, mean that contingency planning is required for managing situations where the entire crew have experienced long periods of intensive work with minimum recovery opportunities.     

Apolipoproteins C-I and C-III Inhibit Lipoprotein Lipase Activity by Displacement of the Enzyme from Lipid Droplets

Apolipoproteins (apo) C-I and C-III are known to inhibit lipoprotein lipase (LPL) activity, but the molecular mechanisms for this remain obscure. We present evidence that either apoC-I or apoC-III, when bound to triglyceride-rich lipoproteins, prevent binding of LPL to the lipid/water interface. This results in decreased lipolytic activity of the enzyme. Site-directed mutagenesis revealed that hydrophobic amino acid residues centrally located in the apoC-III molecule are critical for attachment to lipid emulsion particles and consequently inhibition of LPL activity. Triglyceride-rich lipoproteins stabilize LPL and protect the enzyme from inactivating factors such as angiopoietin-like protein 4 (angptl4). The addition of either apoC-I or apoC-III to triglyceride-rich particles severely diminished their protective effect on LPL and rendered the enzyme more susceptible to inactivation by angptl4. These observations were seen using chylomicrons as well as the synthetic lipid emulsion Intralipid. In the presence of the LPL activator protein apoC-II, more of apoC-I or apoC-III was needed for displacement of LPL from the lipid/water interface. In conclusion, we show that apoC-I and apoC-III inhibit lipolysis by displacing LPL from lipid emulsion particles. We also propose a role for these apolipoproteins in the irreversible inactivation of LPL by factors such as angptl4.     

Influenza Nucleoprotein Delivered with Aluminium Salts Protects Mice from an Influenza A Virus That Expresses an Altered Nucleoprotein Sequence

Influenza virus poses a difficult challenge for protective immunity. This virus is adept at altering its surface proteins, the proteins that are the targets of neutralizing antibody. Consequently, each year a new vaccine must be developed to combat the current recirculating strains. A universal influenza vaccine that primes specific memory cells that recognise conserved parts of the virus could prove to be effective against both annual influenza variants and newly emergent potentially pandemic strains. Such a vaccine will have to contain a safe and effective adjuvant that can be used in individuals of all ages. We examine protection from viral challenge in mice vaccinated with the nucleoprotein from the PR8 strain of influenza A, a protein that is highly conserved across viral subtypes. Vaccination with nucleoprotein delivered with a universally used and safe adjuvant, composed of insoluble aluminium salts, provides protection against viruses that either express the same or an altered version of nucleoprotein. This protection correlated with the presence of nucleoprotein specific CD8 T cells in the lungs of infected animals at early time points after infection. In contrast, immunization with NP delivered with alum and the detoxified LPS adjuvant, monophosphoryl lipid A, provided some protection to the homologous viral strain but no protection against infection by influenza expressing a variant nucleoprotein. Together, these data point towards a vaccine solution for all influenza A subtypes.