Oncolytic Vesicular Stomatitis Virus in an Immunocompetent Model of MUC1-Positive or MUC1-Null Pancreatic Ductal Adenocarcinoma

Vesicular stomatitis virus (VSV) is a promising oncolytic agent against various malignancies. Here, for the first time, we tested VSV in vitro and in vivo in a clinically relevant, immunocompetent mouse model of pancreatic ductal adenocarcinoma (PDA). Our system allows the study of virotherapy against PDA in the context of overexpression (80% of PDA patients) or no expression of human mucin 1 (MUC1), a major marker for poor prognosis in patients. In vitro, we tested three VSV recombinants, wild-type VSV, VSV-green fluorescent protein (VSV-GFP), and a safe oncolytic VSV-ΔM51-GFP, against five mouse PDA cell lines that either expressed human MUC1 or were MUC1 null. All viruses demonstrated significant oncolytic abilities independent of MUC1 expression, although VSV-ΔM51-GFP was somewhat less effective in two PDA cell lines. In vivo administration of VSV-ΔM51-GFP resulted in significant reduction of tumor growth for tested mouse PDA xenografts (+MUC1 or MUC1 null), and antitumor efficacy was further improved when the virus was combined with the chemotherapeutic drug gemcitabine. The antitumor effect was transient in all tested groups. The developed system can be used to study therapies involving various oncolytic viruses and chemotherapeutics, with the goal of inducing tumor-specific immunity while preventing premature virus clearance.     

Impact of phagostimulants on effectiveness of OMRI‐listed insecticides used for control of spotted‐wing drosophila (Drosophila suzukii Matsumura)

Spotted‐wing drosophila, Drosophila suzukii Matsumura, is an invasive pest in the United States that causes considerable damage to fruit crops. It is responsible for many millions of dollars of revenue loss. The female D. suzukii has a heavily sclerotized ovipositor and can lay eggs in ripening or ripe fruit. The arrival of this invasive species has disrupted existing integrated pest management programmes, and growers rely on repeated insecticide applications to protect fruit. Organic growers have few chemical control options, and their reliance on spinosad increases the risk of developing insecticide resistance. We hypothesized that combining phagostimulants with insecticides would increase insecticide efficacy by prompting flies to spend more time in contact with residues. Therefore, the objective of this study was to evaluate the effectiveness of sucrose and the yeast Saccharomyces cerevisiae as phagostimulants in combination with organic biopesticides against D. suzukii in blueberries. Adding sucrose with or without yeast did not improve insecticide efficacy in terms of adult fly mortality or fruit infestation. Spinosad was very effective in all experiments, and for this product, there is little room for improvement. The phagostimulants had no effect on residual activity of any insecticide. The addition of sucrose with or without yeast did not improve the effectiveness of organic insecticides for D. suzukii. Concentrations of these phagostimulants in our experiments (0.36%) may have been too low to elicit a response. Further research is recommended to test different types and concentrations of phagostimulants.     

The regulation of aggrecanase ADAMTS-4 expression in human Achilles tendon and tendon-derived cells

Several members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family have been identified as aggrecanases, whose substrates include versican, the principal large proteoglycan in the tendon extracellular matrix. We have characterized the expression of ADAMTS-4 in human Achilles tendon and tendon-derived cells. ADAMTS-4 mRNA levels were higher in ruptured tendon compared with normal tendon or chronic painful tendinopathy. In tissue extracts probed by Western blotting, mature ADAMTS-4 (68 kDa) was detected only in ruptured tendons, while processed ADAMTS-4 (53 kDa) was detected also in chronic painful tendinopathy and in normal tendon. In cultured Achilles tendon cells, transforming growth factor-β (TGF-β) stimulated ADAMTS-4 mRNA expression (typically 20-fold after 24 h), while interleukin-1 induced a smaller, shorter-term stimulation which synergised markedly with that induced by TGF-β. Increased levels of immunoreactive proteins consistent with mature and processed forms of ADAMTS-4 were detected in TGF-β-stimulated cells. ADAMTS-4 mRNA was expressed at higher levels by tendon cells in collagen gels than in monolayer cultures. In contrast, the expression of ADAMTS-1 and -5 mRNA was lower in collagen gels compared with monolayers, and these mRNA showed smaller or opposite responses to growth factors and cytokines compared with that of ADAMTS-4 mRNA. We conclude that both ADAMTS-4 mRNA and ADAMTS-4 protein processing may be differentially regulated in normal and damaged tendons and that both the matrix environment and growth factors such as TGF-β are potentially important factors controlling ADAMTS aggrecanase activities in tendon pathology.     

Physicians' and nurses' retention of knowledge and skill after training in cardiopulmonary resuscitation.

Physicians and nurses in a community hospital who successfully completed the standard 1-day training program in basic life support cardiopulmonary resuscitation (CPR) were retested 6 and 12 months after training. Their perceptions of their knowledge of and skill in CPR were recorded along with an account of the roles they had taken in CPR incidents. The physicians and nurses initially had the same level of knowledge of CPR, but the physicians learned significantly more and retained it longer. After training, the nurses participated much more in CPR incidents, limiting themselves to basic life support functions. The physicians'' participation, however, remained at about the same level and was limited to advanced life support functions. By 12 months after training the scores in both groups were similar to the pretraining scores, which suggests that practice with feedback is necessary during the 1-year period before retraining and recertification. It may be that the two groups require different training programs.     

Effects of an anthropogenic diet on indicators of physiological challenge and immunity of white ibis nestlings raised in captivity

When wildlife forage and/or live in urban habitats, they often experience a shift in resource availability and dietary quality. Some species even use human handouts, such as bread, as well as human refuse, as a large part of their new diets; yet the influences of this nutritional shift on health and survival remain unclear. American white ibises are increasingly being seen in urban areas in Florida; they collect handouts, such as bread and other food items, from humans in parks, and are also found foraging on anthropogenic sources in trash heaps. We hypothesized that the consumption of these new anthropogenic food sources may trigger increases in indicators of physiological challenge and dampen immune responses. We tested this experimentally by raising 20 white ibis nestlings in captivity, and exposing 10 to a simulated anthropogenic diet (including the addition of white bread and a reduction in seafood content) while maintaining 10 on a diet similar to what ibises consume in more natural environments. We then tested two indicators of physiological challenge (corticosterone and heat shock protein 70), assessed innate immunity in these birds via bactericidal assays and an in vitro carbon clearance assay, and adaptive immunity using a phytohemagglutinin skin test. The anthropogenic diet depressed the development of the ability to kill Salmonella paratyphi in culture. Our results suggest that consuming an anthropogenic diet may be detrimental in terms of the ability to battle a pathogenic bacterial species, but there was little effect on indicators of physiological challenge and other immunological measures.     

A systems approach to improving rural care in Ethiopia

Background

Multiple interventions have been launched to improve the quality, access, and utilization of primary health care in rural, low-income settings; however, the success of these interventions varies substantially, even within single studies where the measured impact of interventions differs across sites, centers, and regions. Accordingly, we sought to examine the variation in impact of a health systems strengthening intervention and understand factors that might explain the variation in impact across primary health care units.

Methodology/Principal Findings

We conducted a mixed methods positive deviance study of 20 Primary Health Care Units (PHCUs) in rural Ethiopia. Using longitudinal data from the Ethiopia Millennium Rural Initiative (EMRI), we identified PHCUs with consistently higher performance (n = 2), most improved performance (n = 3), or consistently lower performance (n = 2) in the provision of antenatal care, HIV testing in antenatal care, and skilled birth attendance rates. Using data from site visits and in-depth interviews (n = 51), we applied the constant comparative method of qualitative data analysis to identify key themes that distinguished PHCUs with different performance trajectories. Key themes that distinguished PHCUs were 1) managerial problem solving capacity, 2) relationship with the woreda (district) health office, and 3) community engagement. In higher performing PHCUs and those with the greatest improvement after the EMRI intervention, health center and health post staff were more able to solve day-to-day problems, staff had better relationships with the woreda health official, and PHCU communities'' leadership, particularly religious leadership, were strongly engaged with the health improvement effort. Distance from the nearest city, quality of roads and transportation, and cultural norms did not differ substantially among PHCUs.

Conclusions/Significance

Effective health strengthening efforts may require intensive development of managerial problem solving skills, strong relationships with government offices that oversee front-line providers, and committed community leadership to succeed.     

Eradication of intracellular Francisella tularensis in THP-1 human macrophages with a novel autophagy inducing agent

Background

Autophagy has been shown recently to play an important role in the intracellular survival of several pathogenic bacteria. In this study, we investigated the effect of a novel small-molecule autophagy-inducing agent, AR-12, on the survival of Francisella tularensis, the causative bacterium of tularemia in humans and a potential bioterrorism agent, in macrophages.

Methods and results

Our results show that AR-12 induces autophagy in THP-1 macrophages, as indicated by increased autophagosome formation, and potently inhibits the intracellular survival of F. tularensis (type A strain, Schu S4) and F. novicida in macrophages in association with increased bacterial co-localization with autophagosomes. The effect of AR-12 on intracellular F. novicida was fully reversed in the presence of the autophagy inhibitor, 3-methyl adenine or the lysosome inhibitor, chloroquine. Intracellular F. novicida were not susceptible to the inhibitory activity of AR-12 added at 12 h post-infection in THP-1 macrophages, and this lack of susceptibility was independent of the intracellular location of bacteria.

Conclusion

Together, AR-12 represents a proof-of-principle that intracellular F. tularensis can be eradicated by small-molecule agents that target innate immunity.     

Plasma membrane calcium pumps and their emerging roles in cancer

Alterations in calcium signaling and/or the expression of calcium pumps and channels are an increasingly recognized property of some cancer cells. Alterations in the expression of plasma membrane calcium ATPase (PMCA) isoforms have been reported in a variety of cancer types, including those of breast and colon, with some studies of cancer cell line differentiation identifying specific PMCA isoforms, which may be altered in some cancers. Some studies have also begun to assess levels of PMCA isoforms in clinical tumor samples and to address mechanisms of altered PMCA expression in cancers. Both increases and decreases in PMCA expression have been reported in different cancer types and in many cases these alterations are isoform specific. In this review, we provide an overview of studies investigating the expression of PMCA in cancer and discuss how both the overexpression and reduced expression of a PMCA isoform in a cancer cell could bestow a growth advantage, through augmenting responses to proliferative stimuli or reducing sensitivity to apoptosis.     

Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism

Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N-terminal membrane-binding amphipathic helix attached to the main ATPase domain. In infected cells, 2C is involved in the formation of membrane vesicles, where it co-localizes with viral RNA replication complexes, but its precise role in virus replication has not been elucidated. We show here that deletion of the predicted N-terminal amphipathic helix enables overexpression in Escherichia coli of a highly soluble truncated protein, 2C(34–318), that has ATPase and RNA binding activity. ATPase activity was abrogated by point mutations in the Walker A (K116A) and B (D160A) motifs and Motif C (N207A) in the active site. Unliganded 2C(34–318) exhibits concentration-dependent self-association to yield oligomeric forms, the largest of which is tetrameric. Strikingly, in the presence of ATP and RNA, FMDV 2C(34–318) containing the N207A mutation, which binds but does not hydrolyze ATP, was found to oligomerize specifically into hexamers. Visualization of FMDV 2C-ATP-RNA complexes by negative stain electron microscopy revealed hexameric ring structures with 6-fold symmetry that are characteristic of AAA+ ATPases. ATPase assays performed by mixing purified active and inactive 2C(34–318) subunits revealed a coordinated mechanism of ATP hydrolysis. Our results provide new insights into the structure and mechanism of picornavirus 2C proteins that will facilitate new investigations of their roles in infection.     

A framework for the analysis of gender,intra-household dynamics,and livestock disease control with examples from Uasin Gishu district,Kenya

As livestock disease control programs in Africa begin to rely more upon para-professionals and livestock producers as deliverers of animal health care services, understanding the role different household members play in providing animal health care becomes increasingly important. This paper presents a framework for the analysis of gender aspects of livestock disease control based on a similar framework developed by Feldstein and Poats (1989). The utility of this framework is illustrated using household-level data collected from a district in central Kenya. Adult women and elderly men in the sample have primary responsibility for livestock care, and are therefore well placed to diagnose illness. Dipping and spraying of animals to prevent tick-borne and other diseases is the primary responsibility of adult males. Decisions regarding use of milk from the morning milking are more likely to be made by adult men. It is morning milk that is most often sold. Adult women, however, make decisions about use of evening milk, which is most often kept for household consumption. Knowledge of livestock diseases did not appear to vary significantly by gender, although some elderly men did possess extensive knowledge of indigenous disease categories and traditional remedies. The importance of recognizing gender issues in planning and implementing livestock disease control programs is discussed.