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To examine the potential impact of invasive dreissenid mussels on in situ populations of phytoplankton and nutrients in western Lake Erie, we combined mussel population estimates from a 2018 survey, results from mussel excretion, grazing, and in situ growth experiments, along with nutrient measurements on collected lake water. We calculated the proportion of the water column filtered per day, based on both clearance rates from grazing experiments and mussel biomass. In most cases the water column was filtered less than once per day. Based on mussel densities from nearby survey sites, we found that mussels could be expected to clear less than 5% of phytoplankton from the water column each day. We combined measurements of nitrogen and phosphorus excretion by mussels with survey densities and found that concentrations of nitrogen and phosphorus from excretion were much less than the ambient inorganic nitrogen and phosphorus measured throughout the season. Despite the modest potential impact that we measured, spatial variability in mussel density and temporal variability in nutrients and seston suggest that more substantial impact likely occurs in some conditions. Lastly, we used a mass balance approach to compare flows of nitrogen and phosphorus attributable to mussel assimilation, growth, and excretion. The proportion of assimilated nitrogen (0.01-0.21) and phosphorus (0.007-0.08) due to growth changed markedly throughout the season, but the excretion rate sometimes exceeded the apparent assimilation rate. These differences in growth:assimilation suggest changes in food quantity or quality, fluctuations in growth rates over time, or other physiological effects can lead to short-term imbalance in nutrient cycling by mussels, which could lead to locally important impacts on phytoplankton and algal blooms. Moreover, this work underscores the importance of mapping mussel densities at fine spatial scales and across interannual variation. [ABSTRACT FROM AUTHOR]

We measured Quagga Mussel grazing-induced changes in seston concentration in 20-L laboratory mesocosms containing lake water, during May-October, as part of a study to investigate the present role of mussel feeding and nutrient excretion in affecting phytoplankton composition. A variety of measuring methodologies including size-fractionated chlorophyll (< 2, 2-20, and >20 µm), phytoplankton groups by FluoroProbe, cyanobacterial genera by 16S rRNA gene sequencing, and particulate C, N, and P gave us different insights into seasonal phytoplankton dynamics and mussel selective feeding and assimilation. High clearance rates and/or high assimilation rates were seen during late May-early July across all size categories, with high rates seen particularly in cryptophytes and diatoms as revealed by FluoroProbe. Starting in late July cyanophytes dominated the phytoplankton and feeding rate on them was generally zero or low. Sequencing analyses suggested that there was a diverse cyanobacterial community present, with Microcystis, contrary to expectation, dominating only in late June. Cyanobium was the dominant genera at most times during summer-fall, and as expected, Microcystis was rejected by mussels relative to other cyanobacteria. Planktothrix, dominant in spring, was readily ingested as well as Anabaena in late June. We show that a combination of methods is helpful to make progress in understanding plankton succession and grazing. These methods were important adjuncts to microscopic analysis (Carrick et al, this issue). Our results support the hypothesis that dreissenid mussels, when abundant, can affect seasonal succession of phytoplankton shifting composition to cyanobacteria and even changes within the cyanobacterial community; however, impacts are likely modest now due low mussel biomass (Carter et al., this issue). Assimilation of C, N, and P was generally high, which is important for mussel population maintenance. [ABSTRACT FROM AUTHOR]

Herein we provide experimental evidence for the effects invasive mussels (Dreissena) grazing can exert on a natural assemblage of plankton that included both hetero- and photo-trophic components in western Lake Erie. Five mussel feeding experiments were performed seasonally in 2018 (May, June, July August, October). Pre- and post-grazing samples were collected from seven 20-L mesocosms (3 control, 4 mussel), and analyzed using microscope cell counts. Results from our experiments showed that Dreissena were active grazers of plankton on all dates, with significant correspondence between mussel clearance rates measured using microscopy versus size-specific chlorophyll and FluoroProbe based estimates (Spearman rank correlation, r = 0.45, and r = 0.48, respectively, p < 0.05). Clearance rates (ml mg-1 h-1) were variable among taxonomic groups and seemed to track the abundance of ambient plankton assemblage (range 1.70 to 25.00, mean ± SE 11.70 ± 1.33). Dreissena grazed consistently on nano-sized hetero- and phototrophic plankton that constituted key trophic linkages in the Lake Erie foodweb. The most actively grazed plankton were phototrophic cryptophytes (Rhodomonas minuta, Cryptomonas spp.), centric diatoms (Cyclotella sp. and Discotella spp.), and non-pigmented chrysomonads (Chromulina sp., Ochromonas sp.). Conversely, clearance rates were low for cyanobacteria (e.g. Microcystis), dinoflagellates (Gymnodinium varians), and some colonial chlorophytes (Desmodesmus, Pediastrum); these plankton groups occurred during specific temporal windows (one-way ANOVA, p < 0.05). Our results indicate the potential mussel grazing can suppress typical, non-harmful plankton species in the nanoplankton size range, thereby favoring the occurrence of less-edible, larger cyanobacteria and chlorophyte species in western Lake Erie. [ABSTRACT FROM AUTHOR]

Abstract: Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of catfish (ESC). We have shown recently that tricarboxylic acid cycle (TCA) and one-carbon (C1) metabolism are involved in E. ictaluri pathogenesis. However, the effect of multiple mutations in these pathways is unknown. Here, we report four novel E. ictaluri mutants carrying double gene mutations in TCA cycle (EiΔmdhΔsdhC, EiΔfrdAΔsdhC), C1 metabolism (EiΔglyAΔgcvP), and both TCA and C1 metabolism pathways (EiΔgcvPΔsdhC). In-frame gene deletions were constructed by allelic exchange and mutants’ virulence and vaccine efficacy were evaluated using in vivo bioluminescence imaging (BLI) as well as end point mortality counts in catfish fingerlings. Results indicated that all the double gene mutants were attenuated compared to wild-type (wt) E. ictaluri. There was a 1.39-fold average reduction in bioluminescence, and hence bacterial numbers, from all the mutants except for EiΔfrdAΔsdhC at 144h post-infection. Vaccination with mutants was very effective in protecting channel catfish against subsequent infection with virulent E. ictaluri 93-146 strain. In particular, immersion vaccination resulted in complete protection. Our results provide further evidence on the importance of TCA and C1 metabolism pathways in bacterial pathogenesis. [Copyright &y& Elsevier]

Reducing a process to its component units underscores scientific research, yet such a pre-occupation with the details of human cognition may mean that the bigger picture is missed. By adopting a global approach to cognitive processing, the current study aimed to identify cortical locations capable of accurately reflecting global cognitive load; that is, overall cognitive processing. Working on the assumption that higher and lower order cognitive processes have limited processing capacity, and that interactions between these processes will have at least an additive effect on performance degradation, results suggested that global cognitive load is measureable in real time using <5 cortical sites. This thus lays the foundation for future research into suitable metrics for accurately measuring degree of global cognitive load at increasingly finer levels. [ABSTRACT FROM AUTHOR]

Abstract: We characterized Influenza A/H5N1 virus that caused the first outbreak of highly pathogenic avian influenza (HPAI) in chickens in Bhutan in 2010. The virus was highly virulent to chicken, killing them within two days of the experimental inoculation with an intravenous pathogenicity index (IVPI) of 2.88. For genetic and phylogenetic analyses, complete genome sequencing of 4 viral isolates was carried out. The isolates revealed multiple basic amino acids at their hemagglutinin (HA) cleavage site, similar to other “Qinghai-like” H5N1 isolates. The receptor-binding site of HA molecule contained avian-like amino acids (222Q and 224G). The isolates also contained amino acid residue K at position 627 of the PB2 protein, and other markers in NS 1 and PB1 proteins, highlighting the risk to mammals. However, the isolates were sensitive to influenza drugs presently available in the market. The sequence analysis indicated that the Bhutan viruses shared 99.1–100% nucleotide homology in all the eight genes among themselves and 2010 chicken isolate from Bangladesh (A/chicken/Bangladesh/1151-11/2010) indicating common progenitor virus. The phylogenetic analysis indicated that the Bhutan isolates belonged to sub-clade 2.2.3 (EMA 3) and shared common progenitor virus with the 2010 Bangladesh virus. Based on the evidence of phylogeny and molecular markers, it could be concluded that the outbreaks in Bhutan and Bangladesh in 2010 were due to independent introductions of the virus probably through migratory birds. [Copyright &y& Elsevier]

The article presents a study which examined the risk factors in the transmission of foot-and-mouth disease (FMD) in the sedentary livestock herds and villages in Bhutan. It was discovered that the odds of contracting FMD significantly increased when cattle mixed with herds from nearby villages compared with those when mixing did not occur. Quarantining of early cases in affected herds or villages was proposed to prevent the spread of the disease.

Biomaterials synthesized from cross-linked folded proteins have untapped potential for biocompatible, resilient, and responsive implementations, but face challenges due to costly molecular refinement and limited understanding of their mechanical response. Under a stress vector, these materials combine the gel-like response of cross-linked networks with the mechanical unfolding and extension of proteins from well-defined 3D structures to unstructured polypeptides. Yet the nanoscale dynamics governing their viscoelastic response remains poorly understood. This lack of understanding is further exacerbated by the fact that the mechanical stability of protein domains depends not only on their structure, but also on the direction of the force vector. To this end, here we propose a coarse-grained network model based on the physical characteristics of polyproteins and combine it with the mechanical unfolding response of protein domains, obtained from single molecule measurements and steered molecular dynamics simulations, to explain the macroscopic response of protein-based materials to a stress vector. We find that domains are about 10-fold more stable when force is applied along their end-to-end coordinate than along the other tethering geometries that are possible inside the biomaterial. As such, the macroscopic response of protein-based materials is mainly driven by the unfolding of the node-domains and rearrangement of these nodes inside the material. The predictions from our models are then confirmed experimentally using force-clamp rheometry. This model is a critical step toward developing protein-based materials with predictable response and that can enable applications for shape memory and energy storage and dissipation.

Label-free measurement and analysis of single bacterial cells are essential for food safety monitoring and microbial disease diagnosis. We report a microwave flow cytometric sensor with a microstrip sensing device with reduced channel height for bacterial cell measurement. Escherichia coli B and Escherichia coli K-12 were measured with the sensor at frequencies between 500 MHz and 8 GHz. The results show microwave properties of E. coli cells are frequency-dependent. A LightGBM model was developed to classify cell types at a high accuracy of 0.96 at 1 GHz. Thus, the sensor provides a promising label-free method to rapidly detect and differentiate bacterial cells. Nevertheless, the method needs to be further developed by comprehensively measuring different types of cells and demonstrating accurate cell classification with improved machine-learning techniques.

Species residing across elevational gradients display adaptations in response to environmental changes such as oxygen availability, ultraviolet radiation, and temperature. Here, we study genomic variation, gene expression, and long-term adaptation in Tibetan Partridge (Perdix hodgsoniae) populations residing across the elevational gradient of the Tibetan Plateau. We generated a high-quality draft genome and used it to carry out downstream population genomic and transcriptomic analysis. The P. hodgsoniae populations residing across various elevations were genetically distinct, and their phylogenetic clustering was consistent with their geographic distribution. We identified possible evidence of gene flow between populations residing in <3,000 and >4,200 m elevation that is consistent with known habitat expansion of high-altitude populations of P. hodgsoniae to a lower elevation. We identified a 60 kb haplotype encompassing the Estrogen Receptor 1 (ESR1) gene, showing strong genetic divergence between populations of P. hodgsoniae. We identified six single nucleotide polymorphisms within the ESR1 gene fixed for derived alleles in high-altitude populations that are strongly conserved across vertebrates. We also compared blood transcriptome profiles and identified differentially expressed genes (such as GAPDH, LDHA, and ALDOC) that correlated with differences in altitude among populations of P. hodgsoniae. These candidate genes from population genomics and transcriptomics analysis were enriched for neutrophil degranulation and glycolysis pathways, which are known to respond to hypoxia and hence may contribute to long-term adaptation to high altitudes in P. hodgsoniae. Our results highlight Tibetan Partridges as a useful model to study molecular mechanisms underlying long-term adaptation to high altitudes., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Studying the genetic variation among different species distributed across their core and range-edge habitats can provide valuable insights into how genetic variation changes across the species' distribution range. This information can be important for understanding local adaptation, as well as for conservation and management efforts. In this study, we have carried out genomic characterization of six species of Asian Pikas distributed along their core and range-edge habitats in the Himalayas. We utilized a population genomics approach using ~28,000 genome-wide SNP markers obtained from restriction-site associated DNA sequencing. We identified low nucleotide diversity and high inbreeding coefficients in all six species across their core and range-edge habitats. We also identified evidence of gene flow among genetically diverse species. Our results provide evidence of reduced genetic diversity in Asian pikas distributed across the Himalayas and the neighboring regions and indicate that recurrent gene flow is possibly a key mechanism for maintaining genetic diversity and adaptive potential in these pikas. However, full-scale genomics studies that utilize whole-genome sequencing approaches will be needed to quantify the direction and timing of gene flow and functional changes associated with introgressed regions in the genome. Our results represent an important step toward understanding the patterns and consequences of gene flow in species, sampled at the least studied, yet climatically vulnerable part of their habitat that can be further used to inform conservation strategies that promote connectivity and gene flow between populations., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Bacteria represent most of the biodiversity and play key roles in virtually every ecosystem. In doing so, bacteria act as part of complex communities shaped by interactions across all domains of life. Here, we report on direct interactions between bacteria and dreissenid mussels, a group of invasive filter-feeders threatening global aquatic systems due to high filtration rates. Previous studies showed that dreissenids can impact bacterial community structure by changing trait distributions and abundances of specific taxa. However, studies on bacterial community effects were conducted using water from Lake Michigan (an oligotrophic lake) only, and it is unknown whether similar patterns are observed in systems with differing nutrient regimes. We conducted ten short-term dreissenid grazing experiments in 2019 using water from two eutrophic lake regions-the western basin of Lake Erie and Saginaw Bay in Lake Huron. Predation by dreissenids led to decline in overall bacterial abundance and diversity in both lakes. However, feeding on bacteria was not observed during every experiment. We also found that traits related to feeding resistance are less phylogenetically conserved than previously thought. Our results highlight the role of temporal, spatial, and genomic heterogeneity in bacterial response dynamics to a globally important invasive filter feeder., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Two years after the emergence of SARS-CoV-2, there is still a need for better ways to assess the risk of transmission in congregate spaces. We deployed active air samplers to monitor the presence of SARS-CoV-2 in real-world settings across communities in the Upper Midwestern states of Wisconsin and Minnesota. Over 29 weeks, we collected 527 air samples from 15 congregate settings. We detected 106 samples that were positive for SARS-CoV-2 viral RNA, demonstrating that SARS-CoV-2 can be detected in continuous air samples collected from a variety of real-world settings. We expanded the utility of air surveillance to test for 40 other respiratory pathogens. Surveillance data revealed differences in timing and location of SARS-CoV-2 and influenza A virus detection. In addition, we obtained SARS-CoV-2 genome sequences from air samples to identify variant lineages. Collectively, this shows air sampling is a scalable, high throughput surveillance tool that could be used in conjunction with other methods for detecting respiratory pathogens in congregate settings., (© 2022. The Author(s).)

Talin is a force-sensing multidomain protein and a major player in cellular mechanotransduction. Here, we use single-molecule magnetic tweezers to investigate the mechanical response of the R8 rod domain of talin. We find that under various force cycles, the R8 domain of talin can display a memory-dependent behavior: At the same low force (<10 pN), the same protein molecule shows vastly different unfolding kinetics. This history-dependent behavior indicates the evolution of a unique force-induced native state. We measure through mechanical unfolding that talin R8 domain binds one of its ligands, DLC1, with much higher affinity than previously reported. This strong interaction can explain the antitumor response of DLC1 by regulating inside-out activation of integrins. Together, our results paint a complex picture for the mechanical unfolding of talin in the physiological range and a new mechanism of function of DLC1 to regulate inside-out activation of integrins.

Recognizing the imperative to evaluate species recovery and conservation impact, in 2012 the International Union for Conservation of Nature (IUCN) called for development of a "Green List of Species" (now the IUCN Green Status of Species). A draft Green Status framework for assessing species' progress toward recovery, published in 2018, proposed 2 separate but interlinked components: a standardized method (i.e., measurement against benchmarks of species' viability, functionality, and preimpact distribution) to determine current species recovery status (herein species recovery score) and application of that method to estimate past and potential future impacts of conservation based on 4 metrics (conservation legacy, conservation dependence, conservation gain, and recovery potential). We tested the framework with 181 species representing diverse taxa, life histories, biomes, and IUCN Red List categories (extinction risk). Based on the observed distribution of species' recovery scores, we propose the following species recovery categories: fully recovered, slightly depleted, moderately depleted, largely depleted, critically depleted, extinct in the wild, and indeterminate. Fifty-nine percent of tested species were considered largely or critically depleted. Although there was a negative relationship between extinction risk and species recovery score, variation was considerable. Some species in lower risk categories were assessed as farther from recovery than those at higher risk. This emphasizes that species recovery is conceptually different from extinction risk and reinforces the utility of the IUCN Green Status of Species to more fully understand species conservation status. Although extinction risk did not predict conservation legacy, conservation dependence, or conservation gain, it was positively correlated with recovery potential. Only 1.7% of tested species were categorized as zero across all 4 of these conservation impact metrics, indicating that conservation has, or will, play a role in improving or maintaining species status for the vast majority of these species. Based on our results, we devised an updated assessment framework that introduces the option of using a dynamic baseline to assess future impacts of conservation over the short term to avoid misleading results which were generated in a small number of cases, and redefines short term as 10 years to better align with conservation planning. These changes are reflected in the IUCN Green Status of Species Standard., (© 2021 The Authors. Conservation Biology published by Wiley Periodicals LLC on behalf of Society for Conservation Biology.)

Nepal, a small landlocked country in South Asia, holds about 800 km of Himalayan Mountain range including the Earth's highest mountain. Within such a mountain range in the north and plain lowlands in the south, Nepal provides a habitat for about 9% of global avian fauna. However, this diversity is underrated because of the lack of enough studies, especially using molecular tools to quantify and understand the distribution patterns of diversity. In this study, we reviewed the studies in the last two decades (2000‒2019) that used molecular methods to study the biodiversity in Nepal to examine the ongoing research trend and focus. Although Nepalese Himalaya has many opportunities for cutting-edge molecular research, our results indicated that the rate of genetic/genomic studies is much slower compared to the regional trends. We found that genetic research in Nepal heavily relies on resources from international institutes and that too is mostly limited to research on species monitoring, distribution, and taxonomic validations. Local infrastructures to carry out cutting-edge genomic research in Nepal are still in their infancy and there is a strong need for support from national/international scientists, universities, and governmental agencies to expand such genomic infrastructures in Nepal. We particularly highlight avian fauna as a potential future study system in this region that can be an excellent resource to explore key biological questions such as understanding eco-physiology and molecular basis of organismal persistence to changing environment, evolutionary processes underlying divergence and speciation, or mechanisms of endemism and restrictive distribution of species., Supplementary Information: The online version contains supplementary material available at 10.1186/s40657-021-00290-5., Competing Interests: Competing interestsThe authors declare that they have no competing interests related to the subject matter., (© The Author(s) 2021.)

The virus that causes COVID-19, SARS-CoV-2, has a large RNA genome that encodes numerous proteins that might be targets for antiviral drugs. Some of these proteins, such as the RNA-dependent RNA polymerase, helicase, and main protease, are well conserved between SARS-CoV-2 and the original SARS virus, but several others are not. This study examines one of the proteins encoded by SARS-CoV-2 that is most different, a macrodomain of nonstructural protein 3 (nsp3). Although 26% of the amino acids in this SARS-CoV-2 macrodomain differ from those observed in other coronaviruses, biochemical and structural data reveal that the protein retains the ability to bind ADP-ribose, which is an important characteristic of beta coronaviruses and a potential therapeutic target.

Binding-induced mechanical stabilization plays key roles in proteins involved in muscle contraction, cellular mechanotransduction, or bacterial adhesion. Because of the vector nature of force, single-molecule force spectroscopy techniques are ideal for measuring the mechanical unfolding of proteins. However, current approaches are still prone to calibration errors between experiments and geometrical variations between individual tethers. Here, we introduce a single-molecule assay based on magnetic tweezers and heterocovalent attachment, which can measure the binding of the substrate-ligand using the same protein molecule. We demonstrate this approach with protein L, a model bacterial protein which has two binding interfaces for the same region of kappa-light chain antibody ligands. Engineered molecules with eight identical domains of protein L between a HaloTag and a SpyTag were exposed to repeated unfolding-refolding cycles at forces up to 100 pN for several hours at a time. The unfolding behavior of the same protein was measured in solution buffers with different concentrations of antibody ligands. With increasing antibody concentration, an increasing number of protein L domains became more stable, indicative of ligand binding and mechanical reinforcement. Interestingly, the dissociation constant of the mechanically reinforced states coincides with that measured for the low-avidity binding interface of protein L, suggesting a physiological role for the second binding interface. The molecular approach presented here opens the road to a new type of binding experiments, where the same molecule can be exposed to different solvents or ligands.

Pikas ( Ochotona Link, 1795) are high-altitude specialist species making them a useful bioindicator species to warming in high-altitude ecosystem. The Himalayan Mountains are an important part of their range, supporting approximately 23%-25% of total pika species worldwide, yet we lack basic information on the distribution patterns. We combine field-based surveys with genetics-based identification and phylogeny to identify differences in species-environment relationships. Further, we suggest putative evolutionary causes for the observed niche patterns., Location: Himalayan high-altitude region., Methods: We sampled 11 altitudinal transects (ranging from ~2,000 to 5,000 m) in the Himalaya to establish occurrence records. We collected 223 species records using genetic analyses to confirm species' identity (based on some invasive and mostly noninvasive biological samples). Niche and geographic overlap were estimated using kernel density estimates., Results: Most pikas in the Himalaya span wide elevation ranges and exhibit extensive spatial overlap with other species. However, even in areas of high species diversity, we found species to have a distinct environmental niche. Despite apparent overlapping distributions at broad spatial scales, in our field surveys, we encountered few cases of co-occurrence of species in the sampled transects. Deeply diverged sister-species pair had the least environmental niche overlap despite having the highest geographic range overlap. In contrast, sister-species pair with shallow genetic divergence had a higher environmental niche overlap but was geographically isolated. We hypothesize that the extent of environmental niche divergence in pikas is a function of divergence time within the species complex. We assessed vulnerability of species to future climate change using environmental niche and geographic breadth sizes as a proxies. Our findings suggest that O. sikimaria may be the most vulnerable species. Ochotona roylii appears to have the most unique environmental niche space, with least niche overlap with other pika species from the study area., Competing Interests: None declared., (© 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Introduction: Governance is one of the most important aspects for strong primary healthcare (PHC) service delivery. To achieve the targets for the Sustainable Development Goals, good governance may play a prime role in low-income and middle-income countries (LMICs). This evidence gap map (EGM) explored the available evidence in LMICs to identify the knowledge gap concerning PHC policy and governance in these settings., Methods: We followed the standard 3ie EGM protocol, finalising the scope of the EGM through a stakeholder workshop. We searched a total of 32 bibliographic databases, systematic review databases, impact evaluation databases, and donor and bilateral agency databases using a comprehensive search strategy. Two reviewers screened retrieved studies, extracted data and performed quality assessment. We plotted the interventions and outcomes derived from the included studies in a dynamic platform to build the interactive EGM and conducted a stakeholder consultation with nominal group technique methods to prioritise the identified gaps., Results: The EGM included 24 systematic reviews and 7 impact evaluations focusing on PHC policy and governance in LMICs. Most of the sources emphasised workforce management and supervision. There were noticeable evidence gaps regarding accountability and social responsibility. The most highly prioritised themes were the role of accountability, the role of public-private partnerships and the role of user-provider communication in PHC governance., Conclusions: This EGM identified some important aspects of PHC policy and governance such as accountability, social responsibility, public-private partnership, user-provider communication through the methodological approaches of evidence synthesis and stakeholder consultation. Identified gaps will provide directions for an implementation research plan to improve the governance of PHC in LMICs., Competing Interests: Competing interests: None declared.

This study analyses costs and benefits of the selected climate adaptive and equitable water management practices and strategies (CAEWMPS) in Dhulikhel Municipality and Dharan Sub-metropolitan city of Nepal. The CAEWMPS adopted the construction of water recharge pit at household level in Dharan and recharge ponds at community level in Dhulikhel. The results of household survey reveal that households have employed different coping strategies including minimizing consumption, purchasing from market, harvesting rain water and installing equipment for storing and pumping in both cities. In Dhulikhel, a significant number of households (18.56%) minimize consumption during the dry season but this is not the case in Dharan. Rather, around one-fifth (19.27%) of the households harvest rainwater in Dharan. In addition, households are forced to give-up their regular activities in order to implement coping strategies such as household chores, leisure time, meeting and gardening. The average estimated annual coping cost in Dharan (USD 87.5) is eight times higher than in Dhulikhel (USD 11.05); however, per unit coping cost is nearly equal in both the cities. In terms of benefit-cost ration, the community level recharge ponds in Dhulikhel (5.15) were found to be cost effective compared to the household level recharge pits of Dharan (1.72). These results provide policy makers with a comparative basis for adopting appropriate strategies to tackle problems related to water shortage under city-specific contexts.

Proteomics, metabolomics, and transcriptomics generate comprehensive data sets, and current biocomputational capabilities allow their efficient integration for systems biology analysis. Published multiomics studies cover methodological advances as well as applications to biological questions. However, few studies have focused on the development of a high-throughput, unified sample preparation approach to complement high-throughput omic analytics. This report details the automation, benchmarking, and application of a strategy for transcriptomic, proteomic, and metabolomic analyses from a common sample. The approach, sample preparation for multi-omics technologies (SPOT), provides equivalent performance to typical individual omic preparation methods but greatly enhances throughput and minimizes the resources required for multiomic experiments. SPOT was applied to a multiomics time course experiment for zinc-treated HL-60 cells. The data reveal Zn effects on NRF2 antioxidant and NFkappaB signaling. High-throughput approaches such as these are critical for the acquisition of temporally resolved, multicondition, large multiomic data sets such as those necessary to assess complex clinical and biological concerns. Ultimately, this type of approach will provide an expanded understanding of challenging scientific questions across many fields.

Here, we describe a force-clamp rheometry method to characterize the biomechanical properties of protein-based hydrogels. This method uses an analog proportional-integral-derivative (PID) system to apply controlled-force protocols on cylindrical protein-based hydrogel samples, which are tethered between a linear voice-coil motor and a force transducer. During operation, the PID system adjusts the extension of the hydrogel sample to follow a predefined force protocol by minimizing the difference between the measured and set-point forces. This unique approach to protein-based hydrogels enables the tethering of extremely low-volume hydrogel samples (< 5 µL) with different protein concentrations. Under force-ramp protocols, where the applied stress increases and decreases linearly with time, the system enables the study of the elasticity and hysteresis behaviors associated with the (un)folding of proteins and the measurement of standard elastic and viscoelastic parameters. Under constant-force, where the force pulse has a step-like shape, the elastic response, due to the change in force, is decoupled from the viscoelastic response, which comes from protein domain unfolding and refolding. Due to its low-volume sample and versatility in applying various mechanical perturbations, force-clamp rheometry is optimized to investigate the mechanical response of proteins under force using a bulk approach.

A convenient methodology for the synthesis of mono- and di-halogenated benzo[ b ]thiophenes is described herein, which utilizes copper(II) sulfate pentahydrate and various sodium halides in the presence of substituted 2-alkynylthioanisoles. The proposed method is facile, uses ethanol as a green solvent, and results in uniquely substituted benzo[ b ]thiophene structures with isolated yields up to 96%. The most useful component of this methodology is the selective introduction of bromine atoms at every available position (2-7) around the benzo[ b ]thiophene ring, while keeping position 3 occupied by a specific halogen atom such as Cl, Br or I. Aromatic halogens are useful reactive handles; therefore, the selective introduction of halogens at specific positions would be valuable in the targeted synthesis of bioactive molecules and complex organic materials via metal-catalyzed cross coupling reactions. This work is a novel approach towards the synthesis of dihalo substituted benzo[ b ]thiophene core structures, which provides a superior alternative to the current methods discussed herein.

Using tBLASTn and BLASTp searches, we queried recently sequenced arthropod genomes and expressed sequence tags (ESTs) using a database of known arthropod cecropins, defensins, and attacins. We identified and synthesized 6 potential AMPs and screened them for antimicrobial activity. Using radial diffusion assays and microtiter antimicrobial assays, we assessed the in vitro antimicrobial effects of these peptides against several human pathogens including Gram-positive and Gram-negative bacteria and fungi. We also conducted hemolysis assays to examine the cytotoxicity of these peptides to mammalian cells. Four of the six peptides identified showed antimicrobial effects in these assays. We also created truncated versions of these four peptides to assay their antimicrobial activity. Two cecropins derived from the monarch butterfly genome (Danaus plexippus), DAN1 and DAN2, showed minimum inhibitory concentrations (MICs) in the range of 2-16 μg/ml when screened against Gram-negative bacteria. HOLO1 and LOUDEF1, two defensin-like peptides derived from red flour beetle (Tribolium castaneum) and human body louse (Pediculus humanus humanus), respectively, exhibited MICs in the range of 13-25 μg/ml against Gram-positive bacteria. Furthermore, HOLO1 showed an MIC less than 5 μg/ml against the fungal species Candida albicans. These peptides exhibited no hemolytic activity at concentrations up to 200 μg/ml. The truncated peptides derived from DAN2 and HOLO1 showed very little antimicrobial activity. Our experiments show that the peptides DAN1, DAN2, HOLO1, and LOUDEF1 showed potent antimicrobial activity in vitro against common human pathogens, did not lyse mammalian red blood cells, and indicates their potential as templates for novel therapeutic agents against microbial infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Bovine enzootic haematuria (BEH) is a debilitating disease of cattle caused by chronic ingestion of bracken fern. Control of BEH is difficult when bracken fern is abundant and fodder resources are limited. To fill a significant knowledge gap on modifiable risk factors for BEH, we conducted a case-control study to identify cattle management practices associated with BEH in the Bhutanese cattle population. A case-control study involving 16 of the 20 districts of Bhutan was carried out between March 2012 and June 2014. In Bhutan sodium acid phosphate and hexamine (SAP&H) is used to treat BEH-affected cattle. All cattle greater than three years of age and treated with SAP&H in 2011 were identified from treatment records held by animal health offices. Households with at least one SAP&H-treated cattle were defined as probable cases. Probable case households were visited and re-classified as confirmed case households if the BEH status of cattle was confirmed following clinical examination and urinalysis. Two control households were selected from the same village as the case household. Households were eligible to be controls if: (1) householders reported that none of their cattle had shown red urine during the previous five years, and (2) haematuria was absent in a randomly selected animal from the herd following clinical examination. Details of cattle management practices were elicited from case and control householders using a questionnaire. A conditional logistic regression model was used to quantify the association between exposures of interest and household BEH status. A total of 183 cases and 345 controls were eligible for analysis. After adjusting for known confounders, the odds of free-grazing for two and three months in the spring were 3.81 (95% CI 1.27-11.7) and 2.28 (95% CI 1.15-4.53) times greater, respectively, in case households compared to controls. The odds of using fresh fern and dry fern as bedding in the warmer months were 2.05 (95% CI 1.03-4.10) and 2.08 (95% CI 0.88-4.90) times greater, respectively, in cases compared to controls. This study identified two husbandry practices that could be modified to reduce the risk of BEH in Bhutanese cattle. Avoiding the use of bracken fern as bedding is desirable, however, if fern is the only available material, it should be harvested during the colder months of the year. Improving access to alternative fodder crops will reduce the need for householders to rely on free-grazing as the main source of metabolisable energy for cattle during the spring., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Asian pika species are morphologically ∼similar and have overlapping ranges. This leads to uncertainty and species misidentification in the field. Phylogenetic analyses of such misidentified samples leads to taxonomic ambiguity. The ecology of many pika species remains understudied, particularly in the Himalaya, where sympatric species could be separated by elevation and/or substrate. We sampled, measured, and acquired genetic data from pikas in the Sikkim Himalaya. Our analyses revealed a cryptic lineage, Ochotona sikimaria, previously reported as a subspecies of O. thibetana. The results support the elevation of this lineage to the species level, as it is genetically divergent from O. thibetana, as well as sister species, O. cansus (endemic to central China) and O. curzoniae (endemic to the Tibetan plateau). The Sikkim lineage diverged from its sister species' about 1.7-0.8myrago, coincident with uplift events in the Himalaya. Our results add to the recent spate of cryptic diversity identified from the eastern Himalaya and highlight the need for further study within the Ochotonidae., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Highly pathogenic avian influenza A(H5N1), clade 2.3.2.1a, with an H9-like polymerase basic protein 1 gene, isolated in Bhutan in 2012, replicated faster in vitro than its H5N1 parental genotype and was transmitted more efficiently in a chicken model. These properties likely help limit/eradicate outbreaks, combined with strict control measures.

We describe the population demographics, rudimentary measures of reproductive performance, the prevalence of major disease conditions and reason-specific proportional mortality for cattle owned by villagers in 16 dzongkhags (districts) in Bhutan based on the findings of a cross-sectional study carried out between March 2012 and May 2014. The animal health issues that were of concern for livestock owners are also identified. Study households were selected using a stratified, two-stage cluster design. Districts (dzongkhags) formed the strata. Villages within dzongkhags were the first sampling stage and households within villages the second sampling stage. All cattle within each selected household comprised the study population. Questionnaires were collected from 409 households with 1480 standing cattle. A total of 71.0% (95% CI 67.9%-74.1%) of the standing cattle population were female and the median age of cattle was 5 years (Q25 3 years; Q75 7 years). Exotic breeds of cattle (Jerseys, Brown Swiss, and their crosses) comprised 41.4% of the cattle population with local breeds making up the remainder. Although exotic breeds of cattle had a significantly lower age at first calving (median 4 years) compared to local breeds (median 4.8 years) there was no significant difference in the number of calving events per cow for the two breed groups. Diarrhoea was the most prevalent disease condition with 2.8 (95% CI 1.5-4.6) cases per 100 animals followed by bovine enzootic haematuria with 1.9 (95% CI 1.0-3.3) cases per 100 animals. The most frequently cited cause of death was misadventure (proportional mortality 26.2%, 95% CI 15.7%-39.2%) followed by old age (17.8%, 95% CI 9.5%-29.4%). A lack of access to adequate fodder and pasture was the animal health issue that was cited by interviewees most frequently. We provide no evidence that exotic breeds of cattle have superior reproductive performance compared with local breeds. The major cattle health concern cited by interviewees, lack of access to fodder, is likely to contribute to suboptimal productivity and the relatively high incidence of bovine enzootic haematuria in this population. We propose that a prospective cohort study of Bhutanese cattle is warranted to more fully elucidate factors affecting the productivity and longevity of cattle in Bhutan., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Following the March 2013 outbreak of novel avian influenza A(H7N9) virus in humans and the subsequent isolation of the virus from chickens, ducks and pigeons in the People's Republic of China, concerns were raised that the H7N9 virus would spread beyond China through the poultry value chain linking to a number of bordering countries. For this reason, a rapid emergency surveillance exercise took place in Bhutan between May and July 2013 with the objective of determining whether influenza A(H7N9) virus was silently circulating in domestic poultryandwild birds in Bhutan.Atotal of 1716 oropharyngeal,tracheal and cloacal swabs together with faecal droppings were collected from poultry, wild birds and feral pigeons throughout the country; these samples included 150 that had been previously collected for surveillance of influenza A(H5N1) virus. Overall, 733 of the samples were tested. A QIAamp Viral RNA Mini K it was used to extract viral RNA from a mix of oropharyngeal, tracheal and cloacal swabs and faecal droppings. The matrix gene of avian influenza type A virus was detected using a specific real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay, and positive samples were further tested in RT-PCR for simultaneous detection of the H7 and N9 genes. Among the 733 samples tested, 46 (26 prospective, 20 retrospective) were confirmed positive for influenza A, a prevalence of 6.3% (95% CI: 4.6 to 8.3). The influenza A-positive samples were from areas in the south of Bhutan that had experienced previous outbreaks of highly pathogenic influenza A(H5N1). None of the samples tested positive for H7N9 strains, providing evidence that influenza A(H7N9) virus was not present in the sampled population. A risk-based approach for surveillance of influenza A(H7N9) and H5N1 is recommended in Bhutan, based on the epidemiology of the disease in China and other countries in South and Southeast Asia.

Functional brain networks (FBNs) are gaining increasing attention in computational neuroscience due to their ability to reveal dynamic interdependencies between brain regions. The dynamics of such networks during cognitive activity between stimulus and response using multi-channel electroencephalogram (EEG), recorded from 16 healthy human participants are explored in this research. Successive EEG segments of 500[Formula: see text]ms duration starting from the onset of cognitive stimulation have been used to analyze and understand the cognitive dynamics. The approach employs a combination of signal processing techniques, nonlinear statistical measures and graph-theoretical analysis. The efficacy of this approach in detecting and tracking cognitive load induced changes in EEG data is clearly demonstrated using graph metrics. It is revealed that most cognitive activity occurs within approximately 500[Formula: see text]ms of the stimulus presentation in addition to temporal variability in the FBNs. It is shown that mutual information (MI), a nonlinear measure, produces good correlations between the EEG channels thus enabling the construction of FBNs which are sensitive to cognitive load induced changes in EEG. Analyses of the dynamics of FBNs and the visualization approach reveal hard to detect subtle changes in cognitive function and hence may lead to a better understanding of cognitive processing in the brain. The techniques exploited have the potential to detect human cognitive dysfunction (impairments).

In Bhutan, Capture-Neuter-Vaccinate-Release (CNVR) programs have been implemented to manage the dog population and control rabies, but no detailed evaluation has been done to assess their coverage and impact. We compared estimates of the dog population using three analytical methods: Lincoln-Petersen index, the Chapman estimate, and the logit-normal mixed effects model, and a varying number of count periods at different times of the day to recommend a protocol for applying the mark-resight framework to estimate free-roaming dog population abundance. We assessed the coverage of the CNVR program by estimating the proportion of dogs that were ear-notched and visually scored the health and skin condition of free-roaming dogs in Gelephu and Phuentsholing towns in south Bhutan, bordering India, in September-October 2012. The estimated free-roaming dog population in Gelephu using the Lincoln-Petersen index and Chapman estimates ranged from 612 to 672 and 614 to 671, respectively, while the logit-normal mixed effects model estimate based on the combined two count events was 641 (95% CI: 603-682). In Phuentsholing the Lincoln-Petersen index and Chapman estimates ranged from 525 to 583 and 524 to 582, respectively, while the logit-normal mixed effects model estimate based on the combined four count events was 555 (95% CI: 526-587). The total number of dogs counted was significantly associated with the time of day (AM versus PM; P=0.007), with a 17% improvement in dog sightings during the morning counting events. We recommend to conduct a morning marking followed by one count event the next morning and estimate population size by applying the Lincoln-Peterson corrected Chapman method or conduct two morning count events and apply the logit-normal mixed model to estimate population size. The estimated proportion of vaccinated free-roaming dogs was 56% (95% CI: 52-61%) and 58% (95% CI: 53-62%) in Gelephu and Phuentsholing, respectively. Given coverage in many neighbourhoods was below the recommended threshold of 70%, we recommend conducting an annual "mass dog vaccination only" campaign in southern Bhutan to create an immune buffer in this high rabies-risk area. The male-to-female dog ratio was 1.34:1 in Gelephu and 1.27:1 in Pheuntsholing. Population size estimates using mark-resight surveys has provided useful baseline data for understanding the population dynamics of dogs at the study sites. Mark-resight surveys provide useful information for designing and managing the logistics of dog vaccination or CNVR programs, assessing vaccination coverage, and for evaluating the impact of neutering programs on the size and structure of dog populations over time., (Copyright © 2015. Published by Elsevier B.V.)

The worldwide increase of multidrug resistance in both community- and health-care associated bacterial infections has impaired the current antimicrobial therapy, warranting the search for other alternatives. We aimed to find the in vitro antibacterial activity of ethanolic extracts of 16 different traditionally used medicinal plants of Nepal against 13 clinical and 2 reference bacterial species using microbroth dilution method. The evaluated plants species were found to exert a range of in vitro growth inhibitory action against the tested bacterial species, and Cynodon dactylon was found to exhibit moderate inhibitory action against 13 bacterial species including methicillin-resistant Staphylococcus aureus, imipenem-resistant Pseudomonas aeruginosa, multidrug-resistant Salmonella typhi, and S. typhimurium. The minimum inhibitory concentration (MIC) values of tested ethanolic extracts were found from 31 to >25,000 μg/mL. Notably, ethanolic extracts of Cinnamomum camphora, Curculigo orchioides, and Curcuma longa exhibited the highest antibacterial activity against S. pyogenes with a MIC of 49, 49, and 195 μg/mL, respectively; whereas chloroform fraction of Cynodon dactylon exhibited best antibacterial activity against S. aureus with a MIC of 31 μg/mL. Among all, C. dactylon, C. camphora, C. orchioides, and C. longa plant extracts displayed a potential antibacterial activity of MIC < 100 μg/mL.

Edwardsiella ictaluri causes enteric septicemia in fish. Recently, we reported construction of E. ictaluri mutants with single and double gene deletions in tricarboxylic acid cycle (TCA) and one-carbon (C-1) metabolism. Here, we report the tissue persistence, virulence, and vaccine efficacy of TCA cycle (EiΔsdhC, EiΔfrdA, and EiΔmdh), C-1 metabolism (EiΔgcvP and EiΔglyA), and combination mutants (EiΔfrdAΔsdhC, EiΔgcvPΔsdhC, EiΔmdhΔsdhC, and EiΔgcvPΔglyA) in channel catfish. The tissue persistence study showed that EiΔsdhC, EiΔfrdA, EiΔfrdAΔsdhC, and EiΔgcvPΔsdhC were able to invade catfish and persist until 11 days post-infection. Vaccination of catfish fingerlings with all nine mutants provided significant (P<0.05) protection against subsequent challenge with the virulent parental strain. Vaccinated catfish fingerlings had 100% survival when subsequently challenged by immersion with wild-type E. ictaluri except for EiΔgcvPΔglyA and EiΔgcvP. Mutant EiΔgcvPΔsdhC was found to be very good at protecting catfish fry, as evidenced by 10-fold higher survival compared to non-vaccinated fish., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: The objective of our current study was to look for the EEG correlates that can reveal the engaged state of the brain while undertaking cognitive tasks. Specifically, we aimed to identify EEG features that could detect audio distraction during simulated driving., Approach: Time varying autoregressive (TVAR) analysis using Kalman smoother was carried out on short time epochs of EEG data collected from participants as they undertook two simulated driving tasks. TVAR coefficients were then used to construct all pole model enabling the identification of EEG features that could differentiate normal driving from audio distracted driving., Main Results: Pole analysis of the TVAR model led to the visualization of event related synchronization/desynchronization (ERS/ERD) patterns in the form of pole displacements in pole plots of the temporal EEG channels in the z plane enabling the differentiation of the two driving conditions. ERS in the EEG data has been demonstrated during audio distraction as an associated phenomenon., Significance: Visualizing the ERD/ERS phenomenon in terms of pole displacement is a novel approach. Although ERS/ERD has previously been demonstrated as reliable when applied to motor related tasks, it is believed to be the first time that it has been applied to investigate human cognitive phenomena such as attention and distraction. Results confirmed that distracted/non-distracted driving states can be identified using this approach supporting its applicability to cognition research.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of channel catfish (ESC). The disease causes considerable economic losses in the commercial catfish industry in the United States. Although antibiotics are used as feed additive, vaccination is a better alternative for prevention of the disease. Here we report the development and characterization of novel live attenuated E. ictaluri mutants. To accomplish this, several tricarboxylic acid cycle (sdhC, mdh, and frdA) and one-carbon metabolism genes (gcvP and glyA) were deleted in wild type E. ictaluri strain 93-146 by allelic exchange. Following bioluminescence tagging of the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, ΔgcvP, and ΔglyA mutants, their dissemination, attenuation, and vaccine efficacy were determined in catfish fingerlings by in vivo imaging technology. Immunogenicity of each mutant was also determined in catfish fingerlings. Results indicated that all of the E. ictaluri mutants were attenuated significantly in catfish compared to the parent strain as evidenced by 2,265-fold average reduction in bioluminescence signal from all the mutants at 144 h post-infection. Catfish immunized with the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, and ΔglyA mutants had 100% relative percent survival (RPS), while E. ictaluri ΔgcvP vaccinated catfish had 31.23% RPS after re-challenge with the wild type E. ictaluri.

A microwave-assisted heating method allows for the convenient and reproducible synthesis of defined Au-Rh core-shell metallic nanoparticles with tuneable shell thicknesses. Nanoparticles with shells as thin as two Rh monolayers can be prepared, which are effective in vapour-phase hydrogenation catalysis at room temperature without the need for pre-treatment. Particles with Rh shells consisting of two or four Rh overlayers show similar catalytic properties and are both significantly more highly active than pure Rh nanoparticles, per mol of Rh employed.

An extensive comparative study of the effects of microwave versus conventional heating on the nucleation and growth of near-monodisperse Rh, Pd, and Pt nanoparticles has revealed distinct and preferential effects of the microwave heating method. A one-pot synthetic method has been investigated, which combines nucleation and growth in a single reaction via precise control over the precursor addition rate. Using this method, microwave-assisted heating enables the convenient preparation of polymer-capped nanoparticles with improved monodispersity, morphological control, and higher crystallinity, compared with samples heated conventionally under otherwise identical conditions. Extensive studies of Rh nanoparticle formation reveal fundamental differences during the nucleation phase that is directly dependent on the heating method; microwave irradiation was found to provide more uniform seeds for the subsequent growth of larger nanostructures of desired size and surface structure. Nanoparticle growth kinetics are also markedly different under microwave heating. While conventional heating generally yields particles with mixed morphologies, microwave synthesis consistently provides a majority of tetrahedral particles at intermediate sizes (5-7 nm) or larger cubes (8+ nm) upon further growth. High-resolution transmission electron microscopy indicates that Rh seeds and larger nanoparticles obtained from microwave-assisted synthesis are more highly crystalline and faceted versus their conventionally prepared counterparts. Microwave-prepared Rh nanoparticles also show approximately twice the catalytic activity of similar-sized conventionally prepared particles, as demonstrated in the vapor-phase hydrogenation of cyclohexene. Ligand exchange reactions to replace polymer capping agents with molecular stabilizing agents are also easily facilitated under microwave heating, due to the excitation of polar organic moieties; the ligand exchange proceeds with excellent retention of nanoparticle size and structure.