MUTATION LOAD AND THE SURVIVAL OF SMALL POPULATIONS

Previous attempts to model the joint action of selection and mutation in finite populations have treated population size as being independent of the mutation load. However, the accumulation of deleterious mutations is expected to cause a gradual reduction in population size. Consequently, in small populations random genetic drift will progressively overpower selection making it easier to fix future mutations. This synergistic interaction, which we refer to as a mutational melt-down, ultimately leads to population extinction. For many conditions, the coefficient of variation of extinction time is less than 0.1, and for species that reproduce by binary fission, the expected extinction time is quite insensitive to population carrying capacity. These results are consistent with observations that many cultures of ciliated protozoans and vertebrate fibroblasts have characteristic extinction times. The model also predicts that clonal lineages are unlikely to survive more than 10⁴ to 10⁵ generations, which is consistent with existing data on parthenogenetic animals. Contrary to the usual view that Muller's ratchet does more damage when selection is weak, we show that the mean extinction time declines as mutations become more deleterious. Although very small sexual populations, such as self-fertilized lines, are subject to mutational meltdowns, recombination effectively eliminates the process when the effective population size exceeds a dozen or so. The concept of the effective mutation load is developed, and several procedures for estimating it are described. It is shown that this load can be reduced substantially when mutational effects are highly variable.     

Use of 16S rRNA Gene for Identification of a Broad Range of Clinically Relevant Bacterial Pathogens

According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.     

Regulation by glucocorticoids of rat-liver phenylalanine hydroxylase in vivo

Phenylalanine hydroxylase, a liver-associated enzyme, is induced markedly by glucocorticoids in two permanent rat-hepatoma cell lines. In order to gain evidence that this phenomenon also occurs in vivo, we examined the effect of adrenalectomy and/or hormone supplementation on the levels of phenylalanine hydroxylase in the livers of adult rats: glucocorticoid administration increases, and adrenal ablation reduces, the activity of the hepatic enzyme, and the diminution occurring in the latter instance is entirely prevented by concurrent hormone replacement. These results thus corroborate earlier findings from a single experiment and are consistent with the hypothesis that adrenal corticosteroid hormones participate in modulating phenylalanine-hydroxylase levels within the diploid hepatocyte.     

Mortality,Morbidity and Health-Seeking Behaviour during the Ebola Epidemic 2014–2015 in Monrovia Results from a Mobile Phone Survey

Between March 2014 and July 2015 at least 10,500 Ebola cases including more than 4,800 deaths occurred in Liberia, the majority in Monrovia. However, official numbers may have underestimated the size of the outbreak. Closure of health facilities and mistrust in existing structures may have additionally impacted on all-cause morbidity and mortality. To quantify mortality and morbidity and describe health-seeking behaviour in Monrovia, Médecins sans Frontières (MSF) conducted a mobile phone survey from December 2014 to March 2015. We drew a random sample of households in Monrovia and conducted structured mobile phone interviews, covering morbidity, mortality and health-seeking behaviour from 14 May 2014 until the day of the survey. We defined an Ebola-related death as any death meeting the Liberian Ebola case definition. We calculated all-cause and Ebola-specific mortality rates. The sample consisted of 6,813 household members in 905 households. We estimated a crude mortality rate (CMR) of 0.33/10,000 persons/day (95%CI:0.25–0.43) and an Ebola-specific mortality rate of 0.06/10,000 persons/day (95%-CI:0.03–0.11). During the recall period, 17 Ebola cases were reported including those who died. In the 30 days prior to the survey 277 household members were reported sick; malaria accounted for 54% (150/277). Of the sick household members, 43% (122/276) did not visit any health care facility. The mobile phone-based survey was found to be a feasible and acceptable alternative method when data collection in the community is impossible. CMR was estimated well below the emergency threshold of 1/10,000 persons/day. Non-Ebola-related mortality in Monrovia was not higher than previous national estimates of mortality for Liberia. However, excess mortality directly resulting from Ebola did occur in the population. Importantly, the small proportion of sick household members presenting to official health facilities when sick might pose a challenge for future outbreak detection and mitigation. Substantial reported health-seeking behaviour outside of health facilities may also suggest the need for adapted health messaging and improved access to health care.     

Characterization of the human and mouse sphingosine 1-phosphate receptor, S1P5 (Edg-8): structure-activity relationship of sphingosine1-phosphate receptors.

Five G protein-coupled receptors (S1P(1)/Edg-1, S1P(3)/Edg-3, S1P(2)/Edg-5, S1P(4)/Edg-6, and S1P(5)/Edg-8) for the intercellular lipid mediator sphingosine 1-phosphate have been cloned and characterized. We found human and mouse sequences closely related to rat S1P(5) (97% identical amino acids) and report now the characterization of the human and mouse S1P(5) gene products as encoding sphingosine 1-phosphate receptors. When HEK293T cells were cotransfected with S1P(5) and G protein DNAs, prepared membranes showed sphingosine 1-phosphate concentration-dependent increases in [gamma-(35)S]GTP binding (EC(50) = 12.7 nM). The lipid mediator inhibited forskolin-driven rises in cAMP by greater than 80% after introduction of the mouse or human S1P(5) DNAs into rat hepatoma RH7777 cells (IC(50) = 0.22 nM). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Northern blot analysis showed high expression of human S1P(5) mRNA in spleen, corpus collosum, peripheral blood leukocytes, placenta, lung, aorta, and fetal tissues. Mouse S1P(5) mRNA is also expressed in spleen and brain. Finally, we found that one enantiomer of a sphingosine 1-phosphate analogue wherein the 3-hydroxyl and 4,5-olefin are replaced by an amide functionality shows some selectivity as an agonist S1P(1) and S1P(3) vs S1P(2) and S1P(5).     

4-1BB-specific monoclonal antibody promotes the generation of tumor-specific immune responses by direct activation of CD8 T cells in a CD40-dependent manner

4-1BB (CD137) is a member of the TNFR superfamily (TNFRSF9). T cell expression of 4-1BB is restricted to activated cells, and cross-linking has been shown to deliver a costimulatory signal. Here we have shown that treatment of tumor-bearing mice with agonistic 4-1BB-specific Abs can lead to T cell-mediated tumor rejection. In vivo mAb depletion experiments demonstrated that this rejection requires CD8(+) cells but not CD4(+) or NK cells. Both IFN-gamma- and CD40-mediated signals were also required, because no benefit was observed on treatment with 4-1BB mAb in mice in which the genes for these molecules had been knocked out. Interestingly, 4-1BB-mediated stimulation of immune responses in CD40L(-/-) mice is effective (although at a reduced level), and may suggest the existence of an alternative ligand for CD40. Additional experiments in IL-15(-/-) mice indicate that IL-15 is not required for either the generation of the primary tumor-specific immune response or the maintenance of the memory immune response. In contrast, the presence of CD4 cells during the primary immune response appears to play a significant role in the maintenance of effective antitumor memory. Finally, in mice in which the number of dendritic cells had been expanded by Fms-like tyrosine kinase3 ligand treatment, the antitumor effects of 4-1BB ligation were enhanced.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Booster effect of canine distemper, canine parvovirus infection and infectious canine hepatitis combination vaccine in domesticated adult dogs

Domesticated adult dogs with antibody titer classified as below 'high' to one or more of canine distemper virus (CDV), canine parvovirus type-2 (CPV-2) and canine adenovirus type-1 (CAdV-1) were then given an additional inoculation, and the effectiveness of this booster evaluated 2 months later. Consequently, CDV and CAdV-1 antibody titer experienced a significant increase, but the same effect was not observed in the antibody titer of CPV-2. These findings suggest that with additional inoculation, a booster effect may be expected in increasing antibody titers for CDV and CAdV-1, but it is unlikely to give an increase in CPV-2 antibody titer.     

Surgical castration of pigs affects the behavioural response to a low-dose lipopolysaccharide (LPS) challenge after weaning

The objective of the present study was to evaluate the effect of an acute stressor in the early postnatal life of pigs, surgical castration, on post-weaning behaviour, and on the behavioural, endocrine and immune responses elicited by a low-dose lipopolysaccharide (LPS) challenge after weaning. At 5-days-of-age, 64 male piglets were randomly assigned to undergo surgical castration or were left untreated (treatment). Pigs were weaned at 28 days-of-age. Behaviour post-weaning and mixing was assessed during a 1-h period, during which agonistic interactions were recorded. One day post-weaning, pigs were injected intraperitoneally with a single dose of 0 or 5 μg/kg of BW of LPS from Escherichia coli (challenge). Sickness behaviour was studied by scan sampling every 5 min for 45 min at 0, 1, 2, 3, 4, 6 and 8 h after the challenge. Blood samples were taken at 0, 2, 12 or 24 h after injection and were analysed for plasma concentrations of tumor necrosis factor-alpha (TNF-), interleukin-1beta (IL-1β), C-reactive protein (CRP), serum amyloid A (SAA) and cortisol. Results showed that non-castrated pigs were more aggressive than castrated pigs immediately after weaning (P < 0.05). Administration of LPS provoked behaviours characteristic of sickness including a reduction in general activity, as well as decreased eating and exploratory behaviours (P < 0.05). These altered behaviours occurred predominantly 3-h post injection (P < 0.05). Significant treatment by challenge interactions showed that castration reduced the occurrence of sickness behaviours induced by LPS, such as depressed general activity (P < 0.01), anorexia (P < 0.01) and reduced exploratory behaviours (P < 0.05). LPS administration increased TNF- levels (P < 0.05), with peak concentrations 2 h after injection (P < 0.01). CRP levels of LPS-treated pigs were higher than saline-treated animals at 12 h (P < 0.05). LPS administration tended to increase plasma SAA levels (P < 0.1), but did not increase cortisol levels (P > 0.1). However, castration did not affect the response of pro-inflammatory cytokines, acute phase proteins and cortisol to the challenge. These results show that surgical castration reduces aggressiveness at weaning and affects specific sickness behaviours but not the endocrine and immune responses elicited by low-dose endotoxin challenge in weaned pigs.     

Shovelomics: high throughput phenotyping of maize (Zea mays L.) root architecture in the field

We present a method to visually score 10 root architectural traits of the root crown of an adult maize plant in the field in a few minutes. Phenotypic profiling of three recombinant inbred line (RIL) populations of maize (Zea mays L.; B73xMo17, Oh43xW64a, Ny821xH99) was conducted in 2008 in a silt loam soil in Pennsylvania and in a sandy soil in Wisconsin, and again in 2009 in Pennsylvania. Numbers, angles and branching pattern of crown and brace roots were assessed visually at flowering. Depending on the soil type in which plants were grown, sample processing took from three (sand) to 8 min (silt-loam). Visual measurement of the root crown required 2 min per sample irrespective of the environment. Visual scoring of root crowns gave a reliable estimation of values for root architectural traits as indicated by high correlations between measured and visually scored trait values for numbers (r ²?=?0.46?C0.97), angles (r ²?=?0.66?C0.76), and branching (r ²?=?0.54?C0.88) of brace and crown roots. Based on the visual evaluation of root crown traits it was possible to discriminate between populations. RILs derived from the cross NY821 x H99 generally had the greatest number of roots, the highest branching density and the most shallow root angles, while inbred lines from the cross between OH43 x W64a generally had the steepest root angles. The ranking of genotypes remained the same across environments, emphasizing the suitability of the method to evaluate genotypes across environments. Scoring of brace roots was better correlated with the actual measurements compared to crown roots. The visual evaluation of root architecture will be a valuable tool in tailoring crop root systems to specific environments.