A community-based survey to assess risk for one health challenges in rural Philippines using a mobile application.

BACKGROUND: Recent emerging and re-emerging diseases in animals and humans show the vulnerability of humans, animals, and crops to disease outbreaks and the large potential impact on health, food security, and economies worldwide. A technology-enabled One Health (OH) surveillance program offers an opportunity for early detection and response as well as prevention of disease outbreaks in resource-limited settings. As an initial step toward developing the surveillance program, we aimed to identify at-risk groups of households for potential shared health challenges at the human-animal-environmental interface in a rural community of the Philippines. METHODS: A cross-sectional household survey was conducted in the municipality of Los Baños in proximity (63 kilometers south) to Metro Manila by enumerators living in the same community. Twenty-four enumerators conducted household interviews asking a) household characteristics including ownership of animals and crops; b) awareness, beliefs and knowledge about OH; c) family-level health practices related to sanitation, hygiene, and food safety; and d) risk factors for potential OH issues. All data collection and transferring process were streamlined using a mobile application. RESULTS: Of 6,055 participating households, 68% reported having one or more of gardens, farms, and animals for various reasons. While only 2% of the households have heard about OH, 97% believed they can get disease from animals, plants or the environment. A latent class analysis with nine risk factors for potential OH issues suggested that 46% of the households were at moderate to high risk for exposure to zoonotic pathogens and environmental contaminants. CONCLUSION: Our findings indicate that there are unaddressed threats to human, animal, and plant health. Given the importance of the interconnections between the health of humans, animals, and plants, further evaluations of the at-risk households would be necessary to mitigate potential shared health threats in the community. Further, our study demonstrates that mHealth technology can provide an opportunity to systematically assess potential one health problems in the rural communities with limited internet connection.

Point stresses during reproductive stage rather than warming seasonal temperature determine yield in temperate rice.

Climate change is predicted to shift temperature regimes in most agricultural areas with temperature changes expected to impact yields of most crops, including rice. These temperature-driven effects can be classified into point stresses, where a temperature event during a sensitive stage drives a reduction in yield, or seasonal warming losses, where raised temperature is thought to increase maintenance energy demands and thereby decrease available resources for yield formation. Simultaneous estimation of the magnitude of each temperature effect on yield has not been well documented due to the inherent difficulty in separating their effects. We simultaneously quantified the magnitude of each effect for a temperate rice production system using a large data set covering multiple locations with data collected from 1995 to 2015, combined with a unique probability-based modeling approach. Point stresses, primarily cold stress during the reproductive stages (booting and flowering), were found to have the largest impact on yield (over 3 Mg/ha estimated yield losses). Contrary to previous reports, yield losses caused by increased temperatures, both seasonal and during grain-filling, were found to be small (approximately 1-2% loss per °C). Occurrences of cool temperature events during reproductive stages were found to be persistent over the study period, and within season, the likelihood of a cool temperature event increased when flowering occurred later in the season. Short and medium grain types, typically recommended for cool regions, were found to be more tolerant of cool temperatures but more sensitive to heat compared to long grain cultivars. These results suggest that for temperate rice systems, the occurrence of periodic stress events may currently overshadow the impacts of general warming temperature on crop production.

Penetrating and Intrastromal Corneal Arcuate Incisions in Rabbit and Human Cadaver Eyes: Manual Diamond Blade and Femtosecond Laser-Created Incisions.

OBJECTIVES: To compare morphologic differences between freehand diamond or femtosecond laser-assisted penetrating and intrastromal arcuate incisions. METHODS: Freehand diamond blade, corneal arcuate incisions (180° apart, 60° arc lengths) and 150 kHz femtosecond laser (80% scheimpflug pachymetry depth corneal thickness) arcuate incisions were performed in rabbits. Intrastromal arcuate incisions (100 µm above Descemet's membrane, 100 µm below epithelium) were performed in rabbit corneas (energy 1.2 µJ, spot line separation 3 × 3 µm, 90° side cut angle). Eyes were examined by slit lamp and light microscopy up to 47 days post-procedure. Freehand diamond blade penetrating incisions, and femtosecond laser penetrating and intrastromal arcuate incisions (energy 1.8 µJ, spot line separation 2 × 2 µm) were performed in cadaver eyes. Optical coherence tomography was performed immediately after surgery and the corneas were fixed for light scanning and transmission electron microscopy. RESULTS: The rabbit model showed anterior stromal inflammation with epithelial hyperplasia in penetrating blade and laser penetrating wounds. The laser intrastromal and penetrating incisions showed localized constriction of the stromal layers of the cornea near the wound. In cadaver eyes, penetrating wound morphology was similar between blade and laser whereas intrastromal wounds did not affect the cornea above or below incisions. CONCLUSION: Penetrating femtosecond laser arcuate incisions have more predictable and controlled outcomes shown by less post-operative scarring than incisions performed with a diamond blade. Intrastromal incisions do not affect uncut corneal layers as demonstrated by histopathology. The femtosecond laser has significant advantages in its ability to make intrastromal incisions which are not achievable by traditional freehand or mechanical diamond blades.

Direct neutralization of type III effector translocation by the variable region of a monoclonal antibody to Yersinia pestis LcrV.

Plague is an acute infection caused by the Gram-negative bacterium Yersinia pestis. Antibodies that are protective against plague target LcrV, an essential virulence protein and component of a type III secretion system of Y. pestis. Secreted LcrV localizes to the tips of type III needles on the bacterial surface, and its function is necessary for the translocation of Yersinia outer proteins (Yops) into the cytosol of host cells infected by Y. pestis. Translocated Yops counteract macrophage functions, for example, by inhibiting phagocytosis (YopE) or inducing cytotoxicity (YopJ). Although LcrV is the best-characterized protective antigen of Y. pestis, the mechanism of protection by anti-LcrV antibodies is not fully understood. Antibodies bind to LcrV at needle tips, neutralize Yop translocation, and promote opsonophagocytosis of Y. pestis by macrophages in vitro. However, it is not clear if anti-LcrV antibodies neutralize Yop translocation directly or if they do so indirectly, by promoting opsonophagocytosis. To determine if the protective IgG1 monoclonal antibody (MAb) 7.3 is directly neutralizing, an IgG2a subclass variant, a deglycosylated variant, F(ab')2, and Fab were tested for the ability to inhibit the translocation of Yops into Y. pestis-infected macrophages in vitro. Macrophage cytotoxicity and cellular fractionation assays show that the Fc of MAb 7.3 is not required for the neutralization of YopJ or YopE translocation. In addition, the use of Fc receptor-deficient macrophages, and the use of cytochalasin D to inhibit actin polymerization, confirmed that opsonophagocytosis is not required for MAb 7.3 to neutralize translocation. These data indicate that the binding of the variable region of MAb 7.3 to LcrV is sufficient to directly neutralize Yop translocation.

Nutrients and sediments in surface runoff water from direct-seeded rice fields: implications for nutrient budgets and water quality.

Nutrient losses from rice fields can have economic and environmental consequences. Little is known about nutrient losses in surface runoff waters from direct-seeded rice systems, which are common in the United States and increasingly more so in Asia. The objectives of this research were to quantify nutrient losses from California rice fields in surface runoff waters and to determine when and under what conditions losses are greatest. Research was conducted in 10 rice fields varying in residue (burned or incorporated) and water management over a 2-yr period. Concentrations of NH-N and NO-N in runoff water across sites, seasons, and management practices averaged <0.1 mg N L. Runoff water PO-P concentration averaged 0.14 mg L and was not affected by season or straw management practices. However, P fluxes were higher in the winter when rice straw was burned (2.59 kg ha) as opposed to incorporated (0.44 kg ha). Average seasonal runoff water K concentrations did not vary with season and straw management, although they were highest at the onset of the winter season. Average total suspended solids (TSS) concentrations did not vary by season but were highest during the winter in the straw-incorporated fields (46 mg L). Rice fields were sinks for K (4.9 kg K ha) during the growing season. Fields were not significant sources of nutrients or TSS during the growing season; however, during the winter fallow they could be sources of NH-N, P, K, and TSS, especially as water fluxes from fields increased.

Regulation of neurotropic signaling by the inducible, NF-kB-sensitive miRNA-125b in Alzheimer's disease (AD) and in primary human neuronal-glial (HNG) cells.

Inducible microRNAs (miRNAs) perform critical regulatory roles in central nervous system (CNS) development, aging, health, and disease. Using miRNA arrays, RNA sequencing, enhanced Northern dot blot hybridization technologies, Western immunoblot, and bioinformatics analysis, we have studied miRNA abundance and complexity in Alzheimer's disease (AD) brain tissues compared to age-matched controls. In both short post-mortem AD and in stressed primary human neuronal-glial (HNG) cells, we observe a consistent up-regulation of several brain-enriched miRNAs that are under transcriptional control by the pro-inflammatory transcription factor NF-kB. These include miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a, and miRNA-155. Of the inducible miRNAs in this subfamily, miRNA-125b is among the most abundant and significantly induced miRNA species in human brain cells and tissues. Bioinformatics analysis indicated that an up-regulated miRNA-125b could potentially target the 3'untranslated region (3'-UTR) of the messenger RNA (mRNA) encoding (a) a 15-lipoxygenase (15-LOX; ALOX15; chr 17p13.3), utilized in the conversion of docosahexaneoic acid into neuroprotectin D1 (NPD1), and (b) the vitamin D3 receptor (VDR; VD3R; chr12q13.11) of the nuclear hormone receptor superfamily. 15-LOX and VDR are key neuromolecular factors essential in lipid-mediated signaling, neurotrophic support, defense against reactive oxygen and nitrogen species (reactive oxygen and nitrogen species), and neuroprotection in the CNS. Pathogenic effects appear to be mediated via specific interaction of miRNA-125b with the 3'-UTR region of the 15-LOX and VDR messenger RNAs (mRNAs). In AD hippocampal CA1 and in stressed HNG cells, 15-LOX and VDR down-regulation and a deficiency in neurotrophic support may therefore be explained by the actions of a single inducible, pro-inflammatory miRNA-125b. We will review the recent data on the pathogenic actions of this up-regulated miRNA-125b in AD and discuss potential therapeutic approaches using either anti-NF-kB or anti-miRNA-125b strategies. These may be of clinical relevance in the restoration of 15-LOX and VDR expression back to control levels and the re-establishment of homeostatic neurotrophic signaling in the CNS.

Fibrin facilitates both innate and T cell-mediated defense against Yersinia pestis.

The Gram-negative bacterium Yersinia pestis causes plague, a rapidly progressing and often fatal disease. The formation of fibrin at sites of Y. pestis infection supports innate host defense against plague, perhaps by providing a nondiffusible spatial cue that promotes the accumulation of inflammatory cells expressing fibrin-binding integrins. This report demonstrates that fibrin is an essential component of T cell-mediated defense against plague but can be dispensable for Ab-mediated defense. Genetic or pharmacologic depletion of fibrin abrogated innate and T cell-mediated defense in mice challenged intranasally with Y. pestis. The fibrin-deficient mice displayed reduced survival, increased bacterial burden, and exacerbated hemorrhagic pathology. They also showed fewer neutrophils within infected lung tissue and reduced neutrophil viability at sites of liver infection. Depletion of neutrophils from wild-type mice weakened T cell-mediated defense against plague. The data suggest that T cells combat plague in conjunction with neutrophils, which require help from fibrin to withstand Y. pestis encounters and effectively clear bacteria.

Surgical specialization and training - its relation to clinical outcome for colorectal cancer surgery.

RATIONALE, AIMS AND OBJECTIVES: Surgical sub-specialization has been considered to be a major factor in improving cancer surgery-related outcomes in terms of 5-year survival and disease-free intervals. In this article we have looked at the evidence supporting the improvement in colorectal cancer outcomes with 'colorectal specialists' performing colon and rectal surgery. METHODS: A literature review was carried out using search engines such as Pubmed, Ovid and Cochrane Databases. Only studies looking at colorectal cancer outcome related to surgery were included in our review. RESULTS: Specialist surgeons performing a high volume of colorectal cancer surgery demonstrated better 5-year survival rates in patients, with less local recurrence. This was most evident in surgery for rectal cancer, where an association with increased sphincter saving surgery was also seen. Total mesorectal excision is now the accepted treatment for rectal cancer and has markedly improved survival rates and decreased local recurrence. CONCLUSION: The outcomes in colorectal surgery continue to steadily improve. The training of specialized colorectal surgeons is a major contributing factor towards this improvement.

TNFα and IFNγ contribute to F1/LcrV-targeted immune defense in mouse models of fully virulent pneumonic plague.

Immunization with the Yersinia pestis F1 and LcrV proteins improves survival in mouse and non-human primate models of pneumonic plague. F1- and LcrV-specific antibodies contribute to protection, however, the mechanisms of antibody-mediated defense are incompletely understood and serum antibody titers do not suffice as quantitative correlates of protection. Previously we demonstrated roles for tumor necrosis factor-alpha (TNFα) and gamma-interferon (IFNγ) during defense against conditionally attenuated pigmentation (pgm) locus-negative Y. pestis. Here, using intranasal challenge with fully virulent pgm-positive Y. pestis strain CO92, we demonstrate that neutralizing TNFα and IFNγ interferes with the capacity of therapeutically administered F1- or LcrV-specific antibody to reduce bacterial burden and increase survival. Moreover, using Y. pestis strain CO92 in an aerosol challenge model, we demonstrate that neutralizing TNFα and IFNγ interferes with protection conferred by immunization with recombinant F1-LcrV fusion protein vaccine (p<0.0005). These findings establish that TNFα and IFNγ contribute to protection mediated by pneumonic plague countermeasures targeting F1 and LcrV, and suggest that an individual's capacity to produce these cytokines in response to Y. pestis challenge will be an important co-determinant of antibody-mediated defense against pneumonic plague.

N255 is a key residue for recognition by a monoclonal antibody which protects against Yersinia pestis infection.

Mab7.3 to Yersinia pestis LcrV antigen (LcrV(Ype)) protected J774A.1 macrophages in vitro from killing by a Yersinia pseudotuberculosis strain expressing LcrV(Ype). Of 4 site-directed mutations in the coiled-coil region (148-169) and 7 mutations in the 225-255 sequence of LcrV(Ype), only the mutation of N255 to D255, abrogated the binding of Mab7.3 and reduced its protective capacity against plague. Since the Mab7.3 epitope in LcrV(Ype) (135-275) encompasses a region (136-180) thought to be exposed on the injectisome, we suggest that Mab7.3 protects by binding to LcrV(Ype) and interfering with protein-protein interactions necessary for type three secretion.

Yersinia pestis can bypass protective antibodies to LcrV and activation with gamma interferon to survive and induce apoptosis in murine macrophages.

Yersinia pestis, the agent of plague, uses a type III secretion injectisome to deliver Yop proteins into macrophages to counteract phagocytosis and induce apoptosis. Additionally, internalized Y. pestis can survive in the phagosomes of naïve or gamma interferon (IFN-gamma)-activated macrophages by blocking vacuole acidification. The Y. pestis LcrV protein is a target of protective antibodies. The binding of antibodies to LcrV at the injectisome tip results in neutralization of the apoptosis of Y. pestis-infected macrophages and is used as an in vitro correlate of protective immunity. The cytokines IFN-gamma and tumor necrosis factor alpha can cooperate with anti-LcrV to promote protection against lethal Y. pestis infection in mice. It is not known if these phagocyte-activating cytokines cooperate with anti-LcrV to increase the killing of the pathogen and decrease apoptosis in macrophages. We investigated how anti-LcrV and IFN-gamma impact bacterial survival and apoptosis in cultured murine macrophages infected with Y. pestis KIM5. Y. pestis KIM5 opsonized with polyclonal or monoclonal anti-LcrV was used to infect macrophages treated with or without IFN-gamma. The phagocytosis and survival of KIM5 and the apoptosis of macrophages were measured at different time points postinfection. The results show that anti-LcrV reduced apoptosis at an early time point (5 h) but not at a later time point (24 h). Polyclonal anti-LcrV was unable to inhibit apoptosis at either time point in IFN-gamma-activated macrophages. Additionally, anti-LcrV was ineffective at promoting the killing of KIM5 in naïve or activated macrophages. We conclude that Y. pestis can bypass protective antibodies to LcrV and activation with IFN-gamma to survive and induce apoptosis in murine macrophages.

Small protective fragments of the Yersinia pestis V antigen.

Yersinia pestis is the causative agent of plague. Naturally occurring cases of the disease and the potential use of Y. pestis as a bioweapon fuel the need for efficacious vaccines. The most recent plague vaccine is a killed whole cell preparation that is expensive to manufacture and its side effects are common. The protective antigens F1 and V have been identified and are currently being developed as a combined subunit vaccine. Protective epitopes of the V antigen have previously been shown to reside in the central part of the protein. In order to identify the minimum protective fragment of the V antigen that can provide protection against plague, the structures of several small fragments of the antigen were modelled in silico and recombinant proteins were produced. These fragments were probed for the retention of a protective epitope using a protective monoclonal antibody and protection against Y. pestis in mice was determined. The smallest protective fragment of V antigen identified comprised amino acids 135-262. Finally the ability of this fragment to confer protection when given in the context of a DNA vaccine was confirmed.

Antibodies and cytokines independently protect against pneumonic plague.

Yersinia pestis causes pneumonic plague, an exceptionally virulent disease for which we lack a safe and effective vaccine. Antibodies specific for the Y. pestis F1 and LcrV proteins can protect mice against pulmonary Y. pestis infection. We demonstrate that neutralizing tumor necrosis factor-alpha (TNFalpha) and gamma-interferon (IFNgamma) abrogates this protection at sub-optimal levels of F1- or LcrV-specific antibody, but not at optimal levels. Moreover, we demonstrate that endogenous TNFalpha and IFNgamma confer measurable protection in the complete absence of protective antibodies. These findings indicate that antibodies and cytokines independently protect against pneumonic plague and suggest that surrogate assays for plague vaccine efficacy should consider both the level of vaccine-induced antibody and the capacity of vaccine recipients to produce TNFalpha and IFNgamma upon exposure to Y. pestis.

Concomitant administration of Yersinia pestis specific monoclonal antibodies with plague vaccine has a detrimental effect on vaccine mediated immunity.

Antibodies can be used to confer rapid immunity against infectious agents for short periods of time. By comparison, vaccine induced immunity is more protective, but takes a relatively long time to develop. Concomitant administration of antibody and vaccine by different routes was evaluated as a means of providing both rapid and long-term protection against plague. BALB/c mice were treated intraperitoneally with monoclonal antibodies, with specificities for Yersinia pestis LcrV and F1 antigens. A cohort of these mice was simultaneously vaccinated with rF1 and rLcrV by the intramuscular route. Antibody co-administration with vaccine reduced the level of vaccine mediated protection afforded against a high level Y. pestis challenge. Conversely, antibody-mediated protection was unaffected by vaccine co-administration and lasted for at least 8 weeks post administration. We also evaluated the effect of administering vaccine intradermally and antibody intratracheally and observed that, irrespective of administration route, concomitant administration of antibody reduced the effectiveness of vaccine mediated immunity. The results of passive transfer experiments supported the thesis that the development of protective antibody responses following vaccination is impaired by the presence of circulating monoclonal antibodies with specificities for important B-cell epitopes in the vaccine. We also noted that intradermal injection of LcrV antigen and cholera toxin adjuvant afforded good levels of protection against systemic and aerosol challenge with Y. pestis: intradermal injection might therefore be considered as a potential minimally invasive method of plague vaccine administration. These data have implications for the design of therapeutic strategies against plague infection.

The weak interaction of LcrV and TLR2 does not contribute to the virulence of Yersinia pestis.

Yersinia pestis and the enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica share the virulence-antigen LcrV. Previously, using reverse genetics we have proven that LcrV contributes to the virulence of Y. enterocolitica serotype O:8 by inducing IL-10 via Toll-like receptor 2 (TLR2). However, both the ability of Y. pestis LcrV to activate TLR2 and a possible role of TLR2-dependent IL-10 induction by LcrV in Y. pestis are not yet known. To eliminate interference from additional protein sequences, we produced LcrVs without affinity tags from Y. pestis and from Y. enterocolitica O:8 (LcrVO:8). LcrVO:8 was much more potent in TLR2-activity than Y. pestis LcrV. To analyse the role of TLR2 in plague, we infected both wild-type and TLR2-/- mice subcutaneously with Y. pestis GB. While TLR2-/- mice exhibited lower blood levels of IL-10 (day 2 post-infection) and of the pro-inflammatory cytokines TNF-alpha, IFN-gamma and MCP-1 (day 4) than wild-type mice, there was no significant difference in survival. The low TLR2-activity of Y. pestis LcrV and associated cytokine expression might explain why - in contrast to Y. enterocolitica O:8 infection - TLR2-deficient mice are not more resistant than wild-type mice in a bubonic plague model.

Identification of outer membrane proteins of Yersinia pestis through biotinylation.

The outer membrane of Gram-negative bacteria contains proteins that might be good targets for vaccines, antimicrobials or detection systems. The identification of surface located proteins using traditional methods is often difficult. Yersinia pestis, the causative agent of plague, was labelled with biotin. Tagged proteins were visualised through streptavidin probing of Western blots. Seven biotinylated proteins of Y. pestis were identified including two porins and the putative virulence factor catalase peroxidase.

Administration of antibody to the lung protects mice against pneumonic plague.

Intratracheal delivery of aerosolized monoclonal antibodies with specificity for Yersinia pestis LcrV and F1 antigens protected mice in a model of pneumonic plague. These data support the utility of inhaled antibodies as a fast-acting postexposure treatment for plague.

Analogues with fluorescent leaving groups for screening and selection of enzymes that efficiently hydrolyze organophosphorus nerve agents.

Enzymes that efficiently hydrolyze highly toxic organophosphorus nerve agents could potentially be used as medical countermeasures. As sufficiently active enzymes are currently unknown, we synthesized twelve fluorogenic analogues of organophosphorus nerve agents with the 3-chloro-7-oxy-4-methylcoumarin leaving group as probes for high-throughput enzyme screening. This set included analogues of the pesticides paraoxon, parathion, and dimefox, and the nerve agents DFP, tabun, sarin, cyclosarin, soman, VX, and Russian-VX. Data from inhibition of acetylcholinesterase, in vivo toxicity tests of a representative analogue (cyclosarin), and kinetic studies with phosphotriesterase (PTE) from Pseudomonas diminuta, and a mammalian serum paraoxonase (PON1), confirmed that the analogues mimic the parent nerve agents effectively. They are suitable tools for high-throughput screens for the directed evolution of efficient nerve agent organophosphatases.