Development of covalent chemogenetic K2P channel activators.

K2P potassium channels regulate excitability by affecting cellular resting membrane potential in the brain, cardiovascular system, immune cells, and sensory organs. Despite their important roles in anesthesia, arrhythmia, pain, hypertension, sleep, and migraine, the ability to control K2P function remains limited. Here, we describe a chemogenetic strategy termed CATKLAMP (covalent activation of TREK family K+ channels to clamp membrane potential) that leverages the discovery of a K2P modulator pocket site that reacts with electrophile-bearing derivatives of a TREK subfamily small-molecule activator, ML335, to activate the channel irreversibly. We show that CATKLAMP can be used to probe fundamental aspects of K2P function, as a switch to silence neuronal firing, and is applicable to all TREK subfamily members. Together, our findings exemplify a means to alter K2P channel activity that should facilitate molecular and systems level studies of K2P function and enable the search for new K2P modulators.

Correlation Between Visual Acuity and Foveal Threshold by Automated Perimetry With Size V Stimulus.

BACKGROUND: Visual acuity has been shown to correlate with foveal threshold as determined by automated perimetry. Although automated perimetry with size V stimulus is commonly used in neuro-ophthalmology practice, the relationship between the visual acuity and the foveal threshold with this larger stimulus is not well known. METHODS: Retrospective study of patients who had undergone neuro-ophthalmology evaluation and visual field testing with automated perimetry using size V stimulus. Healthy controls were also recruited. Using visual acuity and foveal threshold, Pearson correlation coefficients were calculated, and basic foveal threshold statistics were stratified by visual acuity. Prediction intervals for visual acuities by various foveal threshold were also calculated. RESULTS: A total of 106 unique eyes were included. The final Pearson correlation coefficient between visual acuities was -0.795 for the right eye and -0.578 for the left eye, with a pooled correlation coefficient of -0.751 (P < 0.001). A foveal threshold of at least 34 dB was present in 94.4% of eyes with 20/20 visual acuity, and a foveal threshold of greater than 35 dB was not observed in eyes with visual acuity of 20/40 or worse. CONCLUSIONS: Foveal threshold as determined by automated perimetry using size V stimulus has moderate-to-strong correlation with visual acuity in patients undergoing neuro-ophthalmology evaluation.

Development of covalent chemogenetic K2P channel activators.

K2P potassium channels regulate excitability by affecting cellular resting membrane potential in the brain, cardiovascular system, immune cells, and sensory organs. Despite their important roles in anesthesia, arrhythmia, pain, hypertension, sleep, and migraine, the ability to control K2P function remains limited. Here, we describe a chemogenetic strategy termed CATKLAMP (Covalent Activation of TREK family K+ channels to cLAmp Membrane Potential) that leverages the discovery of a site in the K2P modulator pocket that reacts with electrophile-bearing derivatives of a TREK subfamily small molecule activator, ML335, to activate the channel irreversibly. We show that the CATKLAMP strategy can be used to probe fundamental aspects of K2P function, as a switch to silence neuronal firing, and is applicable to all TREK subfamily members. Together, our findings exemplify a new means to alter K2P channel activity that should facilitate studies both molecular and systems level studies of K2P function and enable the search for new K2P modulators.

Bioorthogonal, Fluorogenic Targeting of Voltage-Sensitive Fluorophores for Visualizing Membrane Potential Dynamics in Cellular Organelles.

Electrical potential differences across lipid bilayers play foundational roles in cellular physiology. Plasma membrane voltage is the most widely studied; however, the bilayers of organelles like mitochondria, lysosomes, nuclei, and the endoplasmic reticulum (ER) also provide opportunities for ionic compartmentalization and the generation of transmembrane potentials. Unlike plasma membranes, organellar bilayers, cloistered within the cell, remain recalcitrant to traditional approaches like patch-clamp electrophysiology. To address the challenge of monitoring changes in organelle membrane potential, we describe the design, synthesis, and application of the LUnAR RhoVR (Ligation Unquenched for Activation and Redistribution Rhodamine-based Voltage Reporter) for optically monitoring membrane potential changes in the ER of living cells. We pair a tetrazine-quenched RhoVR for voltage sensing with a transcyclooctene (TCO)-conjugated ceramide (Cer-TCO) for targeting to the ER. Bright fluorescence is observed only at the coincidence of the LUnAR RhoVR and TCO in the ER, minimizing non-specific, off-target fluorescence. We show that the product of the LUnAR RhoVR and Cer-TCO is voltage-sensitive and that the LUnAR RhoVR can be targeted to an intact ER in living cells. Using the LUnAR RhoVR, we use two-color, ER-localized, fast voltage imaging coupled with cytosolic Ca2+ imaging to validate the electroneutrality of Ca2+ release from internal stores. Finally, we use the LUnAR RhoVR to directly visualize functional coupling between the plasma-ER membranes in patch clamped cell lines, providing the first direct evidence of the sign of the ER potential response to plasma membrane potential changes. We envision that the LUnAR RhoVR, along with other existing organelle-targeting TCO probes, could be applied widely for exploring organelle physiology.

Robust Institutional Support and Collaboration Between Summer Training Programs in Cancer and Biomedicine Drive the Pivot to a Virtual Format in Response to the COVID Pandemic.

Summer internships serve important roles in training the next generation of biomedical researchers and healthcare providers through laboratory and clinical experiences that excite trainees about these fields and help them make informed decisions about career paths. The SARS-CoV-2 (COVID) pandemic and associated physical distancing restrictions precluded implementation of traditional in-person summer curricula and led to the cancellation of many internships across the USA. COVID-related disruptions also created opportunities for trainees to engage in remote research, become proficient in online learning platforms, and explore multidisciplinary topics. These skills are highly relevant to trainees as virtual interfaces occupy an increasingly mainstream role in their professional paths. The response to the COVID pandemic required real-time adaptations at all levels for major biomedical institutions including the University of Maryland Baltimore (UMB). Pivoting summer programs to a virtual format as part of this response provided a "teachable moment" to expose trainees to the innovation and resilience that are essential components of the biomedical profession. UMB summer programs, which span diverse biomedical disciplines from cancer research to diabetes, consolidated resources and identified mentors with online research projects to develop a robust virtual curriculum. Herein, data from a cancer-focused internship illustrate the collaborative adaptations to established components and creation of new learning modules in the transition to, and implementation of, online training. Outcomes are presented in the context of the COVID pandemic and significant societal issues that arose in the summer of 2020. The utility of virtual components and their impact on future programs is discussed.

Antimicrobial-resistant Salmonella is detected more frequently in feed milling equipment than in raw feed components or processed animal feed.

Food for human and animal consumption can provide a vehicle for the transfer of pathogenic and antimicrobial-resistant bacteria into the food chain. We investigated the antimicrobial susceptibility of 453 Salmonella isolates collected from raw feed components, equipment and finished feed from 17 commercial feed mills in Australia between 2012 and 2021. Previous studies have found Salmonella prevalence and the diversity of Salmonella serotypes are greatest in the raw feed components. We, therefore, hypothesised that we would find a greater proportion of antimicrobial-resistant Salmonella isolates in the raw feed components compared to other sample types. We found that of 453 isolates tested, 356 (0.80) were susceptible to all antimicrobials tested, 49 (0.11) were nonsusceptible to streptomycin only and 48 (0.11) were resistant to two or more antimicrobials. Of the 48 antimicrobial-resistant isolates, 44 were found in feed milling equipment, two in raw feed components and two in finished feed. Statistical analysis, using a logistic regression with random effects model, found that the population-adjusted mean probability of detecting antimicrobial-resistant Salmonella isolates from feed milling equipment of 0.39, was larger than the probability of detecting resistant isolates in raw feed components 0.01, (P < 0.001) and in finished feed, 0.11, (P = 0.006). This propensity for antimicrobial-resistant bacteria to colonise feed milling equipment has not been previously reported. Further studies are required to understand the ecology of antimicrobial-resistant Salmonella in the feed milling environment.

Oral health-related self-efficacy and fatalism in a regional South Australian Aboriginal population.

OBJECTIVES: To assess the psychometric properties, including face, content, criterion and known-groups validity and reliability, of scales to measure oral health-related self-efficacy and fatalism in a regional Aboriginal adult population in Australia. METHODS: Four hundred Aboriginal adults (aged 18-82 years, 67% female) completed a self-report questionnaire including items pertaining to oral health-related self-efficacy and fatalism. Structural validity was determined in exploratory factor analysis (EFA) with principal components analysis for each scale. Criterion validity was assessed between the instruments and theoretically related variables. Known-groups validity was investigated by comparing the scores in different population groups according to age, sex, education and employment. Reliability of the scales was assessed through internal consistency. RESULTS: The EFA confirmed a single factor structure for self-efficacy and fatalism scales, with Cronbach's alphas of 0.93 and 0.89 respectively. The two scales were not correlated. Oral health-related self-efficacy was associated with toothbrush ownership and brushing the previous day supporting criterion validity. Oral health-related fatalism was associated with previous extractions and perceived need for extractions also supporting criterion validity. Both measures were associated with social impact of oral health as measured by the OHIP-14, supporting their criterion validity. Mixed findings were observed in terms of known-groups validity. CONCLUSIONS: There was initial evidence that measures of oral health-related self-efficacy and fatalism displayed adequate psychometric properties in this Aboriginal community. These constructs could have implications for approaches for improving oral health among Aboriginal people.

Reimagining Rural: Shifting Paradigms About Health and Well-Being in the Rural United States.

Rural health disparities have attracted increased national attention, compelling an expanded focus on rural health research. In this article, we deconstruct the definitions and narratives of "rural" communities and suggest that a paradigm shift is needed that centers the complexity and strength of rural places. We discuss the relevance of health equity frameworks, implementation science, and community-engaged approaches to promote rural well-being. Focusing on rural in its own right will lead to intervention innovations and reinvention with implications beyond rural areas. We conclude with suggestions for research and practice to inspire renewed interest in partnering with rural communities to promote health equity.

Fipronil use and associated effects on hematological and biochemical parameters of blue land crab (Cardisoma guanhumi Latreille): Ecological implication.

Fipronil is used to control pests to improve farm yield, however, indiscriminate use of fipronil has been reported to endanger crabs leading to their extinction. Therefore, this study investigated the impact of fipronil on several hematological and biochemical parameters of blue land crabs. We exposed blue land crabs to either fipronil or to a control treatment; fipronil reduced the protein content of the crab and also led to hematological and oxidative damages to the crabs' oxy-hemocyanin. Based on our results, there is need for guided use of agrochemicals such as fipronil to avoid/reduce their adverse effects on economically important species such as crabs.

Self-rated oral and general health among Aboriginal adults in regional South Australia.

BACKGROUND: In Australia, Aboriginal adults experience higher levels of poor oral and general health than the non-Aboriginal population. This study compared self-rated oral and general health among Aboriginal adults in regional South Australia with participants in the National Survey of Adult Oral Health (NSAOH). METHODS: Data were obtained from the Indigenous Oral Health Literacy Project (IOHLP) based in South Australia. Three sub-populations from the NSAOH were utilised for comparison: National Aboriginal, National non-Aboriginal and South Australian Regional Non-Aboriginal adults. All data were standardised by age group and sex, utilising Census data. RESULTS: Just over 70% of South Australian Regional Aboriginal participants gave a rating of 'excellent, very good or good' for general health, more than 17% lower than each of the other groups. Just over 50% rated their oral health highly, 20% fewer than the proportion for each other group. Stratifying by key socio-demographic factors did not account for all differences. CONCLUSIONS: Proportionally fewer South Australian Regional Aboriginal adults had high ratings of oral and general health than the Aboriginal and non-Aboriginal adults from the national survey, indicating that national-level data might underestimate the proportion of regional Aboriginal Australians with poor oral health.

Voltage Imaging in Drosophila Using a Hybrid Chemical-Genetic Rhodamine Voltage Reporter.

We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila.

Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes.

Fluorescent probes that change their spectral properties upon binding to small biomolecules, ions, or changes in the membrane potential (Vm) are invaluable tools to study cellular signaling pathways. Here, we introduce a novel technique for simultaneous recording of multiple probes at millisecond time resolution: frequency- and spectrally-tuned multiplexing (FASTM). Different from present multiplexing approaches, FASTM uses phase-sensitive signal detection, which renders various combinations of common probes for Vm and ions accessible for multiplexing. Using kinetic stopped-flow fluorimetry, we show that FASTM allows simultaneous recording of rapid changes in Ca2+, pH, Na+, and Vm with high sensitivity and minimal crosstalk. FASTM is also suited for multiplexing using single-cell microscopy and genetically encoded FRET biosensors. Moreover, FASTM is compatible with optochemical tools to study signaling using light. Finally, we show that the exceptional time resolution of FASTM also allows resolving rapid chemical reactions. Altogether, FASTM opens new opportunities for interrogating cellular signaling.

Evidence on yoga for health: A bibliometric analysis of systematic reviews.

OBJECTIVE: To support the research agenda in yoga for health by comprehensively identifying systematic reviews of yoga for health outcomes and conducting a bibliometric analysis to describe their publication characteristics and topic coverage. METHODS: We searched 7 databases (MEDLINE/PubMed, Embase, PsycINFO, CINAHL, AMED, the Cochrane Database of Systematic Reviews, and PROSPERO) from their inception to November 2019 and 1 database (INDMED) from inception to January 2017. Two authors independently screened each record for inclusion and one author extracted publication characteristics and topics of included reviews. RESULTS: We retrieved 2710 records and included 322 systematic reviews. 157 reviews were exclusively on yoga, and 165 were on yoga as one of a larger class of interventions (e.g., exercise). Most reviews were published in 2012 or later (260/322; 81 %). First/corresponding authors were from 32 different countries; three-quarters were from the USA, Germany, China, Australia, the UK or Canada (240/322; 75 %). Reviews were most frequently published in speciality journals (161/322; 50 %) complementary medicine journals (66/322; 20 %) or systematic review journals (59/322; 18 %). Almost all were present in MEDLINE (296/322; 92 %). Reviews were most often funded by government or non-profits (134/322; 42 %), unfunded (74/322; 23 %), or not explicit about funding (111/322; 34 %). Common health topics were psychiatric/cognitive (n = 56), cancer (n = 39) and musculoskeletal conditions (n = 36). Multiple reviews covered similar topics, particularly depression/anxiety (n = 18), breast cancer (n = 21), and low back pain (n = 16). CONCLUSIONS: Further research should explore the overall quality of reporting and conduct of systematic reviews of yoga, the direction and certainty of specific conclusions, and duplication or gaps in review coverage of topics.

Structural Insights into the Mechanisms and Pharmacology of K2P Potassium Channels.

Leak currents, defined as voltage and time independent flows of ions across cell membranes, are central to cellular electrical excitability control. The K2P (KCNK) potassium channel class comprises an ion channel family that produces potassium leak currents that oppose excitation and stabilize the resting membrane potential in cells in the brain, cardiovascular system, immune system, and sensory organs. Due to their widespread tissue distribution, K2Ps contribute to many physiological and pathophysiological processes including anesthesia, pain, arrythmias, ischemia, hypertension, migraine, intraocular pressure regulation, and lung injury responses. Structural studies of six homomeric K2Ps have established the basic architecture of this channel family, revealed key moving parts involved in K2P function, uncovered the importance of asymmetric pinching and dilation motions in the K2P selectivity filter (SF) C-type gate, and defined two K2P structural classes based on the absence or presence of an intracellular gate. Further, a series of structures characterizing K2P:modulator interactions have revealed a striking polysite pharmacology housed within a relatively modestly sized (~70 kDa) channel. Binding sites for small molecules or lipids that control channel function are found at every layer of the channel structure, starting from its extracellular side through the portion that interacts with the membrane bilayer inner leaflet. This framework provides the basis for understanding how gating cues sensed by different channel parts control function and how small molecules and lipids modulate K2P activity. Such knowledge should catalyze development of new K2P modulators to probe function and treat a wide range of disorders.