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Health care research applied to couples and families has been hampered by the high cost of obtaining and hand reviewing medical charts. Fortunately, self-report of medical use has been shown to be a useful measure of medical utilization with individuals. However, no such research has been reported to see if the same is true for couples and families. Self-reported medical use for 130 clients seen at a marriage and family therapy clinic and their report of family members' medical use, was compared with medical records. Results showed that self-report, spouse reports, and parent reports of medical visits were significantly correlated with medical records. These results suggest that researchers and clinicians may reliably use self and family member's reports instead of reviews of medical charts.

The present study investigated the associations between family functioning, acculturation between parents and their adolescents, and adolescent adjustment problems. Chinese adolescents and their parents (N = 41) living in the United States and Canada participated in this study. Results showed that differences in acculturation between parents and adolescents were related to adolescent depression and that family functioning was related to both depression and delinquency. Both parental and adolescent acculturation levels and their differences were important in predicting adolescent outcomes. Implications for conducting family therapy and parent education with Chinese North American adolescents and their parents are discussed. Key Words: acculturation, adolescent depression, Chinese adolescents, delinquency, family functioning.

The ability of a commercial fisher to limit red drum, Sciaenops ocellatus (Linnaeus), bycatch was evaluated by comparing the catch of directed sets (net site chosen by the commercial fisher) vs randomly placed gillnets during 70 days of gillnetting ( n = 691 sets). Catch rate did not differ between directed sets and nets set randomly. Thus, fisher experience did not lead to reduced bycatch; however, this may have been influenced by overall low red drum bycatch (0.41 fish net−1 for 12 h sets and 0.59 fish net−1 for 24 h sets). Catch rates of southern flounder were much higher at 3.97 and 3.37 fish net−1 for 12 h and 24 h sets, respectively. Fishing 10 nets per day, the catch of red drum exceeded the daily bag limit of seven red drum on 23% of the days. Of the 326 red drum retrieved from gillnets, 32.5% were dead at the time of retrieval; but only 15 of the 106 dead red drum had to be discarded due to size limits, and six were discarded dead based on bag limits. Current fisheries management regulations (bag limit and window limit of 457–686 mm) are supported by these findings given the conditions (e.g. red drum densities) at which the study was performed. [ABSTRACT FROM AUTHOR]

Examines the potential of Farming Systems Research/Extension (FSR/E) and some concepts used in the agricultural development activities of small farmers in the U.S. Background, evolution and stages of FSR/E; Use of the community of Waimanalo as an example of a formerly rural, now peri-urban community in Hawaii; Incorporation of other tools from FSR/E to address the situation of farmers in peri-urban areas.

Presents the annual report of the Committee on Affiliated and Related Corporations of the American Bar Association Section of Taxation. Issuance of the proposed consolidated returns regulations; Function of the special task force; Comments of the members on the regulations proposed.

Preclinical data have confirmed that human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can remuscularize the injured or diseased heart, with several clinical trials now in planning or recruitment stages. However, because ventricular arrhythmias represent a complication following engraftment of intramyocardially injected PSC-CMs, it is necessary to provide treatment strategies to control or prevent engraftment arrhythmias (EAs). Here, we show in a porcine model of myocardial infarction and PSC-CM transplantation that EAs are mechanistically linked to cellular heterogeneity in the input PSC-CM and resultant graft. Specifically, we identify atrial and pacemaker-like cardiomyocytes as culprit arrhythmogenic subpopulations. Two unique surface marker signatures, signal regulatory protein α (SIRPA) + CD90 - CD200 + and SIRPA + CD90 - CD200 - , identify arrhythmogenic and non-arrhythmogenic cardiomyocytes, respectively. Our data suggest that modifications to current PSC-CM-production and/or PSC-CM-selection protocols could potentially prevent EAs. We further show that pharmacologic and interventional anti-arrhythmic strategies can control and potentially abolish these arrhythmias., (© 2024. The Author(s).)

Technologies capable of assessing cellular metabolites with high precision and temporal resolution are currently limited. Recent developments in the field of nanopore sensors allow the non-stochastic quantification of metabolites, where a nanopore is acting as an electrical transducer for selective substrate binding proteins (SBPs). Here we show that incorporation of the pore-forming toxin Cytolysin A (ClyA) into the plasma membrane of Chinese hamster ovary cells (CHO-K1) results in the appearance of single-channel conductance amenable to multiplexed automated patch-clamp (APC) electrophysiology. In CHO-K1 cells, SBPs modify the ionic current flowing though ClyA nanopores, thus demonstrating its potential for metabolite sensing of living cells. Moreover, we developed a graphical user interface for the analysis of the complex signals resulting from multiplexed APC recordings. This system lays the foundation to bridge the gap between recent advances in the nanopore field ( e.g. , proteomic and transcriptomic) and potential cellular applications.

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, which defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and 2 additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of recombinantly expressed voltage-gated ion channels, Mu8.1 displayed the highest potency against the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study showcases the potential of uncovering novel structures and bioactivities within the largely unexplored group of macro-conotoxins., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Hackney et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Acid-sensing ion channels (ASICs) are transmembrane sensors of extracellular acidosis and potential drug targets in several disease indications, including neuropathic pain and cancer metastasis. The K + -sparing diuretic amiloride is a moderate nonspecific inhibitor of ASICs and has been widely used as a probe for elucidating ASIC function. In this work, we screened a library of 6-substituted and 5,6-disubstituted amiloride analogs using a custom-developed automated patch clamp protocol and identified 6-iodoamiloride as a potent ASIC1 inhibitor. Follow-up IC 50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 94 (hASIC1 94 IC 50 = 88 nM, cf. amiloride 11 IC 50 = 1.7 μM). A similar improvement in activity was observed in ASIC3-mediated currents from rat dorsal root ganglion neurons (rDRG single-concentration 94 IC 50 = 230 nM, cf. 11 IC 50 = 2.7 μM). 6-Iodoamiloride represents the amiloride analog of choice for studying the effects of ASIC inhibition on cell physiology.

Expanding literature on well-being within veterinary medicine has been instrumental in recognizing the prevalence of psychological distress among students and practitioners and promoting awareness and advocacy for well-being within teaching institutions, professional associations, and the workplace. However, greater focus on distress has also been critiqued for overemphasizing illness and reactive interventions, and a call has been made for more balanced conversations about veterinary well-being, with focus on strengths and proactive interventions. This Best Practices article highlights a proactive, strengths-focused intervention, aimed at increasing fourth-year students' awareness of self-care while in training and in their transition to the profession. Authentic conversations about self-care is a required part of clinical training at Kansas State University College of Veterinary Medicine. All students attend a private meeting with a behavioral scientist, engaging in an authentic conversation about their experience of stress and coping strategies. Current practices in providing stress management interventions are restricted to pre-clinical training. Authentic conversations about self-care are presented here as an alternative to current practices, which the authors argue are not adequately addressing students' needs during clinical training and the transition to the profession. Potential implications of providing self-care interventions during the clinical year of training include greater student engagement, increasing awareness, and self-efficacy as students make efforts to maintain well-being both in training and in the professional realm.

The analgesic α -conotoxins Vc1.1, RgIA, and PeIA attenuate nociceptive transmission via activation of G protein-coupled GABA B receptors (GABA B Rs) to modulate N-type calcium channels in primary afferent neurons and recombinantly coexpressed human GABA B R and Cav2.2 channels in human embryonic kidney 293T cells. Here, we investigate the effects of analgesic α -conotoxins following the mutation of amino acid residues in the Venus flytrap (VFT) domains of the GABA B R subunits predicted through computational peptide docking and molecular dynamics simulations. Our docking calculations predicted that all three of the α -conotoxins form close contacts with VFT residues in both B1 and B2 subunits, comprising a novel GABA B R ligand-binding site. The effects of baclofen and α -conotoxins on the peak Ba 2+ current (I Ba ) amplitude were investigated on wild-type and 15 GABA B R mutants individually coexpressed with human Cav2.2 channels. Mutations at the interface of the VFT domains of both GABA B R subunits attenuated baclofen-sensitive I Ba inhibition by the analgesic α -conotoxins. In contrast, mutations located outside the putative peptide-binding site (D380A and R98A) did not. The key GABA B R residues involved in interactions with the α -conotoxins are K168 and R207 on the B2 subunit and S130, S153, R162, E200, F227, and E253 on the B1 subunit. The double mutant, S130A + S153A, abolished inhibition by both baclofen and the α -conotoxins. Depolarization-activated I Ba mediated by both wild-type and all GABA B R mutants were inhibited by the selective GABA B R antagonist CGP 55845. This study identifies specific residues of GABA B R involved in the binding of the analgesic α -conotoxins to the VFT domains of the GABA B R. SIGNIFICANCE STATEMENT: This study defines the binding site of the analgesic α -conotoxins Vc1.1, RgIA, and PeIA on the human GABA B receptor to activate Gi/o proteins and inhibit Cav2.2 channels. Computational docking and molecular dynamics simulations of GABA B R identified amino acids of the Venus flytrap (VFT) domains with which the α -conotoxins interact. GABA B R alanine mutants attenuated baclofen-sensitive Cav2.2 inhibition by the α -conotoxins. We identify an allosteric binding site at the interface of the VFT domains of the GABA B R subunits for the analgesic α -conotoxins., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Low voltage-activated calcium currents are mediated by T-type calcium channels Ca V 3.1, Ca V 3.2, and Ca V 3.3, which modulate a variety of physiological processes including sleep, cardiac pace-making, pain, and epilepsy. Ca V 3 isoforms' biophysical properties, overlapping expression, and lack of subtype-selective pharmacology hinder the determination of their specific physiological roles in health and disease. We have identified μ-theraphotoxin Pn3a as the first subtype-selective spider venom peptide inhibitor of Ca V 3.3, with >100-fold lower potency against the other T-type isoforms. Pn3a modifies Ca V 3.3 gating through a depolarizing shift in the voltage dependence of activation thus decreasing Ca V 3.3-mediated currents in the normal range of activation potentials. Paddle chimeras of K V 1.7 channels bearing voltage sensor sequences from all four Ca V 3.3 domains revealed preferential binding of Pn3a to the S3-S4 region of domain II (Ca V 3.3 DII ). This novel T-type channel pharmacological site was explored through computational docking simulations of Pn3a, site-directed mutagenesis, and full domain II swaps between Ca V 3 channels highlighting it as a subtype-specific pharmacophore. This research expands our understanding of T-type calcium channel pharmacology and supports the suitability of Pn3a as a molecular tool in the study of the physiological roles of Ca V 3.3 channels., Competing Interests: JM, JW, AH, RF, DA No competing interests declared, (© 2022, McArthur et al.)

Familial hemiplegic migraine (FHM) is a severe neurogenetic disorder for which three causal genes, CACNA1A , SCN1A , and ATP1A2 , have been implicated. However, more than 80% of referred diagnostic cases of hemiplegic migraine (HM) are negative for exonic mutations in these known FHM genes, suggesting the involvement of other genes. Using whole-exome sequencing data from 187 mutation-negative HM cases, we identified rare variants in the CACNA1I gene encoding the T-type calcium channel Cav3.3. Burden testing of CACNA1I variants showed a statistically significant increase in allelic burden in the HM case group compared to gnomAD (OR = 2.30, P = 0.00005) and the UK Biobank (OR = 2.32, P = 0.0004) databases. Dysfunction in T-type calcium channels, including Cav3.3, has been implicated in a range of neurological conditions, suggesting a potential role in HM. Using patch-clamp electrophysiology, we compared the biophysical properties of five Cav3.3 variants (p.R111G, p.M128L, p.D302G, p.R307H, and p.Q1158H) to wild-type (WT) channels expressed in HEK293T cells. We observed numerous functional alterations across the channels with Cav3.3-Q1158H showing the greatest differences compared to WT channels, including reduced current density, right-shifted voltage dependence of activation and inactivation, and slower current kinetics. Interestingly, we also found significant differences in the conductance properties exhibited by the Cav3.3-R307H and -Q1158H variants compared to WT channels under conditions of acidosis and alkalosis. In light of these data, we suggest that rare variants in CACNA1I may contribute to HM etiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Maksemous, Blayney, Sutherland, Smith, Lea, Tran, Ibrahim, McArthur, Haupt, Cader, Finol-Urdaneta, Adams and Griffiths.)

Background and Purpose: Hydroxychloroquine, chloroquine and azithromycin are three drugs that were proposed to treat coronavirus disease 2019 (COVID-19). While concern already existed around their proarrhythmic potential, there are little data regarding how altered physiological states encountered in patients such as febrile state, electrolyte imbalances or acidosis might change their risk profiles., Experimental Approach: Potency of human ether-à-go-go related gene (hERG) block was measured using high-throughput electrophysiology in the presence of variable environmental factors. These potencies informed simulations to predict population risk profiles. Effects on cardiac repolarisation were verified in human induced pluripotent stem cell-derived cardiomyocytes from multiple individuals., Key Results: Chloroquine and hydroxychloroquine blocked hERG with IC 50 of 1.47 ± 0.07 and 3.78 ± 0.17 μM, respectively, indicating proarrhythmic risk at concentrations effective against severe acute respiratory syndrome-coronovirus-2 (SARS-CoV-2) in vitro. Hypokalaemia and hypermagnesaemia increased potency of chloroquine and hydroxychloroquine, indicating increased proarrhythmic risk. Acidosis significantly reduced potency of all drugs, whereas increased temperature decreased potency of chloroquine and hydroxychloroquine against hERG but increased potency for azithromycin. In silico simulations demonstrated that proarrhythmic risk was increased by female sex, hypokalaemia and heart failure and identified specific genetic backgrounds associated with emergence of arrhythmia., Conclusion and Implications: Our study demonstrates how proarrhythmic risk can be exacerbated by metabolic changes and pre-existing disease. More broadly, the study acts as a blueprint for how high-throughput in vitro screening, combined with in silico simulations, can help guide both preclinical screening and clinical management of patients in relation to drugs with potential to prolong repolarisation., (© 2021 The British Pharmacological Society.)

This study evaluated associations between healthy activities of daily living, common stressors, and psychological well-being among 230 veterinary medical students at Kansas State University. Participants completed the Psychological Wellbeing Scale during the fall semester of 2019. Additionally, students provided information about specific stressors, healthy activities of daily living, and relevant demographic information. Similar to previous studies, participants in this study reported being concerns about heavy workloads, being behind in studies, inefficient study, and academic performance. On average, the students in this study ate fewer than three meals per day, slept less than 7 hours per night, exercised only twice per week, and spent an average of 83 minutes per day on social media platforms. A higher number of daily meals, more days of exercise, and more frequent contact with one's support system, particularly significant others and family members, predicted students' increased psychological well-being. In contrast, lower psychological well-being scores were associated with comparing oneself to others and financial distress. This study identifies potential activities that students can engage in to improve psychological well-being. The discussion section provides specific suggestions for intervention.

The King Baboon spider, Pelinobius muticus , is a burrowing African tarantula. Its impressive size and appealing coloration are tempered by reports describing severe localized pain, swelling, itchiness, and muscle cramping after accidental envenomation. Hyperalgesia is the most prominent symptom after bites from P. muticus , but the molecular basis by which the venom induces pain is unknown. Proteotranscriptomic analysis of P. muticus venom uncovered a cysteine-rich peptide, δ/κ-theraphotoxin-Pm1a (δ/κ-TRTX-Pm1a), that elicited nocifensive behavior when injected into mice. In small dorsal root ganglion neurons, synthetic δ/κ-TRTX-Pm1a (sPm1a) induced hyperexcitability by enhancing tetrodotoxin-resistant sodium currents, impairing repolarization and lowering the threshold of action potential firing, consistent with the severe pain associated with envenomation. The molecular mechanism of nociceptor sensitization by sPm1a involves multimodal actions over several ion channel targets, including Na V 1.8, K V 2.1, and tetrodotoxin-sensitive Na V channels. The promiscuous targeting of peptides like δ/κ-TRTX-Pm1a may be an evolutionary adaptation in pain-inducing defensive venoms., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Background and Purpose: Activation of GIRK channels via G protein-coupled GABA B receptors has been shown to attenuate nociceptive transmission. The analgesic α-conotoxin Vc1.1 activates GABA B receptors resulting in inhibition of Ca v 2.2 and Ca v 2.3 channels in mammalian primary afferent neurons. Here, we investigated the effects of analgesic α-conotoxins on recombinant and native GIRK-mediated K + currents and on neuronal excitability., Experimental Approach: The effects of analgesic α-conotoxins, Vc1.1, RgIA, and PeIA, were investigated on inwardly-rectifying K + currents in HEK293T cells recombinantly co-expressing either heteromeric human GIRK1/2 or homomeric GIRK2 subunits, with GABA B receptors. The effects of α-conotoxin Vc1.1 and baclofen were studied on GIRK-mediated K + currents and the passive and active electrical properties of adult mouse dorsal root ganglion neurons., Key Results: Analgesic α-conotoxins Vc1.1, RgIA, and PeIA potentiate inwardly-rectifying K + currents in HEK293T cells recombinantly expressing human GIRK1/2 channels and GABA B receptors. GABA B receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen occurs via a pertussis toxin-sensitive G protein and is inhibited by the selective GABA B receptor antagonist CGP 55845. In adult mouse dorsal root ganglion neurons, GABA B receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen hyperpolarizes the cell membrane potential and reduces excitability., Conclusions and Implications: This is the first report of GIRK channel potentiation via allosteric α-conotoxin Vc1.1-GABA B receptor agonism, leading to decreased neuronal excitability. Such action potentially contributes to the analgesic effects of Vc1.1 and baclofen observed in vivo., (© 2021 The British Pharmacological Society.)

Friedreich ataxia (FRDA) is an autosomal recessive disease characterized by degeneration of dorsal root ganglia (DRG) sensory neurons, which is due to low levels of the mitochondrial protein Frataxin. To explore cell replacement therapies as a possible approach to treat FRDA, we examined transplantation of sensory neural progenitors derived from human embryonic stem cells (hESC) and FRDA induced pluripotent stem cells (iPSC) into adult rodent DRG regions. Our data showed survival and differentiation of hESC and FRDA iPSC-derived progenitors in the DRG 2 and 8 weeks post-transplantation, respectively. Donor cells expressed neuronal markers, including sensory and glial markers, demonstrating differentiation to these lineages. These results are novel and a highly significant first step in showing the possibility of using stem cells as a cell replacement therapy to treat DRG neurodegeneration in FRDA as well as other peripheral neuropathies., (© 2021 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Venomous animals hunt using bioactive peptides, but relatively little is known about venom small molecules and the resulting complex hunting behaviors. Here, we explored the specialized metabolites from the venom of the worm-hunting cone snail, Conus imperialis Using the model polychaete worm Platynereis dumerilii , we demonstrate that C. imperialis venom contains small molecules that mimic natural polychaete mating pheromones, evoking the mating phenotype in worms. The specialized metabolites from different cone snails are species-specific and structurally diverse, suggesting that the cones may adopt many different prey-hunting strategies enabled by small molecules. Predators sometimes attract prey using the prey's own pheromones, in a strategy known as aggressive mimicry. Instead, C. imperialis uses metabolically stable mimics of those pheromones, indicating that, in biological mimicry, even the molecules themselves may be disguised, providing a twist on fake news in chemical ecology., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Despite potently inhibiting the nociceptive voltage-gated sodium (Na v ) channel, Na v 1.7, µ -theraphotoxin Pn3a is antinociceptive only upon co-administration with sub-therapeutic opioid agonists, or by itself at doses >3,000-fold greater than its Na v 1.7 IC 50 by a yet undefined mechanism. Na v channels are structurally related to voltage-gated calcium (Ca v ) channels, Ca v 1 and Ca v 2. These channels mediate the high voltage-activated (HVA) calcium currents ( I Ca ) that orchestrate synaptic transmission in nociceptive dorsal root ganglion (DRG) neurons and are fine-tuned by opioid receptor (OR) activity. Using whole-cell patch clamp recording, we found that Pn3a (10 µM) inhibits ∼55% of rat DRG neuron HVA- I Ca and 60-80% of Ca v 1.2, Ca v 1.3, Ca v 2.1, and Ca v 2.2 mediated currents in HEK293 cells, with no inhibition of Ca v 2.3. As a major DRG I Ca component, Ca v 2.2 inhibition by Pn3a ( IC 50 = 3.71 ± 0.21 µM) arises from an 18 mV hyperpolarizing shift in the voltage dependence of inactivation. We observed that co-application of Pn3a and µ-OR agonist DAMGO results in enhanced HVA- I Ca inhibition in DRG neurons whereas co-application of Pn3a with the OR antagonist naloxone does not, underscoring HVA channels as shared targets of Pn3a and opioids. We provide evidence that Pn3a inhibits native and recombinant HVA Ca v s at previously reportedly antinociceptive concentrations in animal pain models. We show additive modulation of DRG HVA- I Ca by sequential application of low Pn3a doses and sub-therapeutic opioids ligands. We propose Pn3a's antinociceptive effects result, at least in part, from direct inhibition of HVA- I Ca at high Pn3a doses, or through additive inhibition by low Pn3a and mild OR activation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 McArthur, Munasinghe, Finol-Urdaneta, Adams and Christie.)

Sensory perception is fundamental to everyday life, yet understanding of human sensory physiology at the molecular level is hindered due to constraints on tissue availability. Emerging strategies to study and characterize peripheral neuropathies in vitro involve the use of human pluripotent stem cells (hPSCs) differentiated into dorsal root ganglion (DRG) sensory neurons. However, neuronal functionality and maturity are limited and underexplored. A recent and promising approach for directing hPSC differentiation towards functionally mature neurons involves the exogenous expression of Neurogenin-2 (NGN2). The optimized protocol described here generates sensory neurons from hPSC-derived neural crest (NC) progenitors through virally induced NGN2 expression. NC cells were derived from hPSCs via a small molecule inhibitor approach and enriched for migrating NC cells (66% SOX10+ cells). At the protein and transcript level, the resulting NGN2 induced sensory neurons ( NGN2 iSNs) express sensory neuron markers such as BRN3A (82% BRN3A+ cells), ISLET1 (91% ISLET1+ cells), TRKA, TRKB, and TRKC. Importantly, NGN2 iSNs repetitively fire action potentials (APs) supported by voltage-gated sodium, potassium, and calcium conductances. In-depth analysis of the molecular basis of NGN2 iSN excitability revealed functional expression of ion channels associated with the excitability of primary afferent neurons, such as Nav1.7, Nav1.8, Kv1.2, Kv2.1, BK, Cav2.1, Cav2.2, Cav3.2, ASICs and HCN among other ion channels, for which we provide functional and transcriptional evidence. Our characterization of stem cell-derived sensory neurons sheds light on the molecular basis of human sensory physiology and highlights the suitability of using hPSC-derived sensory neurons for modeling human DRG development and their potential in the study of human peripheral neuropathies and drug therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Hulme, McArthur, Maksour, Miellet, Ooi, Adams, Finol-Urdaneta and Dottori.)

Urotoxin (α-KTx 6), a peptide from venom of the Australian scorpion Urodacus yaschenkoi, is the most potent inhibitor of Kv1.2 described to date (IC 50  = 160 pM). The native peptide also inhibits Kv1.1, Kv1.3 and KCa3.1 with nanomolar affinity but its low abundance in venom precluded further studies of its actions. Here we produced recombinant Urotoxin (rUro) and characterized the molecular determinants of Kv1 channel inhibition. The 3D structure of rUro determined using NMR spectroscopy revealed a canonical cysteine-stabilised α/β (CSα/β) fold. Functional assessment of rUro using patch-clamp electrophysiology revealed the importance of C-terminal amidation for potency against Kv1.1-1.3 and Kv1.5. Neutralization of the putative pore-blocking K25 residue in rUro by mutation to Ala resulted in a major decrease in rUro potency against all Kv channels tested, without perturbing the toxin's structure. Reciprocal mutations in the pore of Uro-sensitive Kv1.2 and Uro-resistant Kv1.5 channels revealed a direct interaction between Urotoxin and the Kv channel pore. Our experimental work supports postulating a mechanism of action in which occlusion of the permeation pathway by the K25 residue in Urotoxin is the basis of its Kv1 inhibitory activity. Docking analysis was consistent with occlusion of the pore by K25 and the requirement of a small, non-charged amino acid in the Kv1 channel vestibule to facilitate toxin-channel interactions. Finally, computational studies revealed key interactions between the amidated C-terminus of Urotoxin and a conserved Asp residue in the turret of Kv1 channels, offering a potential rationale for potency differences between native and recombinant Urotoxin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Toxins from marine animals provide molecular tools for the study of many ion channels, including mammalian voltage-gated potassium channels of the Kv1 family. Selectivity profiling and molecular investigation of these toxins have contributed to the development of novel drug leads with therapeutic potential for the treatment of ion channel-related diseases or channelopathies. Here, we review specific peptide and small-molecule marine toxins modulating Kv1 channels and thus cover recent findings of bioactives found in the venoms of marine Gastropod (cone snails), Cnidarian (sea anemones), and small compounds from cyanobacteria. Furthermore, we discuss pivotal advancements at exploiting the interaction of κM-conotoxin RIIIJ and heteromeric Kv1.1/1.2 channels as prevalent neuronal Kv complex. RIIIJ's exquisite Kv1 subtype selectivity underpins a novel and facile functional classification of large-diameter dorsal root ganglion neurons. The vast potential of marine toxins warrants further collaborative efforts and high-throughput approaches aimed at the discovery and profiling of Kv1-targeted bioactives, which will greatly accelerate the development of a thorough molecular toolbox and much-needed therapeutics.

The intravenous anaesthetic ketamine, has been demonstrated to inhibit nicotinic acetylcholine receptor (nAChR)-mediated currents in dissociated rat intracardiac ganglion (ICG) neurons (Weber et al., 2005). This effect would be predicted to depress synaptic transmission in the ICG and would account for the inhibitory action of ketamine on vagal transmission to the heart (Inoue and König, 1988). This investigation was designed to examine the activity of ketamine on (i) postsynaptic responses to vagal nerve stimulation, (ii) the membrane potential, and (iii) membrane current responses evoked by exogenous application of ACh and nicotine in ICG neurons in situ. Intracellular recordings were made using sharp intracellular microelectrodes in a whole mount ICG preparation. Preganglionic nerve stimulation and recordings in current- and voltage-clamp modes were used to assess the action of ketamine on ganglionic transmission and nAChR-mediated responses. Ketamine attenuated the postsynaptic responses evoked by nerve stimulation. This reduction was significant at clinically relevant concentrations at high frequencies. The excitatory membrane potential and current responses to focal application of ACh and nicotine were inhibited in a concentration-dependent manner by ketamine. In contrast, ketamine had no effect on either the directly-evoked action potential or excitatory responses evoked by focal application of γ-aminobutyric acid (GABA). Taken together, ketamine inhibits synaptic transmission and nicotine- and ACh-evoked currents in adult rat ICG. Ketamine inhibition of synaptic transmission and nAChR-mediated responses in the ICG contributes significantly to its attenuation of the bradycardia observed in response to vagal stimulation in the mammalian heart., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

µ-Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide µ-conotoxins were first recognized as potent components of the venoms of fish-hunting cone snails that selectively inhibit voltage-gated skeletal muscle sodium channels, thus preventing muscle contraction. Intriguingly, computer simulations predicted that PIIIA binds to prokaryotic channel NavAb with much higher affinity than to fish (and other vertebrates) skeletal muscle sodium channel (Nav 1.4). Here, using whole-cell voltage clamp, we demonstrate that PIIIA inhibits NavBac mediated currents even more potently than predicted. From concentration-response data, with [PIIIA] varying more than 6 orders of magnitude (10 -12 to 10 -5 M), we estimated an IC 50 = ~5 pM, maximal block of 0.95 and a Hill coefficient of 0.81 for the inhibition of peak currents. Inhibition was stronger at depolarized holding potentials and was modulated by the frequency and duration of the stimulation pulses. An important feature of the PIIIA action was acceleration of macroscopic inactivation. Docking of PIIIA in a NaChBac (NavBh) model revealed two interconvertible binding modes. In one mode, PIIIA sterically and electrostatically blocks the permeation pathway. In a second mode, apparent stabilization of the inactivated state was achieved by PIIIA binding between P2 helices and trans-membrane S5s from adjacent channel subunits, partially occluding the outer pore. Together, our experimental and computational results suggest that, besides blocking the channel-mediated currents by directly occluding the conducting pathway, PIIIA may also change the relative populations of conducting (activated) and non-conducting (inactivated) states.

Background and Purpose: T-type calcium (Ca v 3) and transient receptor potential vanilloid-1 (TRPV1) channels play central roles in the control of excitability in the peripheral nervous system and are regarded as potential therapeutic pain targets. Modulators that either activate or inhibit TRPV1-mediated currents display analgesic properties in various pain models despite opposing effects on their connate target, TRPV1. We explored the effects of TRPV1-active compounds on Ca v 3-mediated currents., Experimental Approach: Whole-cell patch clamp recordings were used to examine the effects of TRPV1-active compounds on rat dorsal root ganglion low voltage-activated calcium currents and recombinant Ca v 3 isoforms in expression systems., Key Results: The classical TRPV1 agonist capsaicin as well as TRPV1 antagonists A-889425, BCTC, and capsazepine directly inhibited Ca v 3 channels. These compounds altered the voltage-dependence of activation and inactivation of Ca v 3 channels and delayed their recovery from inactivation, leading to a concomitant decrease in T-type current availability. The TRPV1 antagonist capsazepine potently inhibited Ca v 3.1 and 3.2 channels (K D  < 120 nM), as demonstrated by its slow off rate. In contrast, neither the TRPV1 agonists, Palvanil and resiniferatoxin, nor the TRPV1 antagonist AMG9810 modulated Ca v 3-mediated currents., Conclusions and Implications: Analgesic TRPV1-active compounds inhibit Ca v 3 currents in native and heterologous systems. Hence, their analgesic effects may not be exclusively attributed to their actions on TRPV1, which has important implications in the current understanding of nociceptive pathways. Importantly, our results highlight the need for attention in the experimental design used to address the analgesic properties of Ca v 3 channel inhibitors., (© 2019 The British Pharmacological Society.)

Batrachotoxin (BTX), an alkaloid from skin secretions of dendrobatid frogs, causes paralysis and death by facilitating activation and inhibiting deactivation of eukaryotic voltage-gated sodium (Nav) channels, which underlie action potentials in nerve, muscle, and heart. A full understanding of the mechanism by which BTX modifies eukaryotic Nav gating awaits determination of high-resolution structures of functional toxin-channel complexes. Here, we investigate the action of BTX on the homotetrameric prokaryotic Nav channels NaChBac and NavSp1. By combining mutational analysis and whole-cell patch clamp with molecular and kinetic modeling, we show that BTX hinders deactivation and facilitates activation in a use-dependent fashion. Our molecular model shows the horseshoe-shaped BTX molecule bound within the open pore, forming hydrophobic H-bonds and cation-π contacts with the pore-lining helices, leaving space for partially dehydrated sodium ions to permeate through the hydrophilic inner surface of the horseshoe. We infer that bulky BTX, bound at the level of the gating-hinge residues, prevents the S6 rearrangements that are necessary for closure of the activation gate. Our results reveal general similarities to, and differences from, BTX actions on eukaryotic Nav channels, whose major subunit is a single polypeptide formed by four concatenated, homologous, nonidentical domains that form a pseudosymmetric pore. Our determination of the mechanism by which BTX activates homotetrameric voltage-gated channels reveals further similarities between eukaryotic and prokaryotic Nav channels and emphasizes the tractability of bacterial Nav channels as models of voltage-dependent ion channel gating. The results contribute toward a deeper, atomic-level understanding of use-dependent natural and synthetic Nav channel agonists and antagonists, despite their overlapping binding motifs on the channel proteins., (© 2019 Finol-Urdaneta et al.)

μ-Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of μ-conotoxin GIIIC was determined using 2D NMR spectroscopy and simulated annealing calculations. Despite high sequence similarity, GIIIC adopts a three-dimensional structure that differs from the previously observed conformation of μ-conotoxins GIIIA and GIIIB due to the presence of a bulky, non-polar leucine residue at position 18. The side chain of L18 is oriented towards the core of the molecule and consequently the N-terminus is re-modeled and located closer to L18. The functional characterization of GIIIC defines it as a canonical μ-conotoxin that displays substantial selectivity towards skeletal muscle sodium channels (Na V ), albeit with ~2.5-fold lower potency than GIIIA. GIIIC exhibited a lower potency of inhibition of Na V 1.4 channels, but the same Na V selectivity profile when compared to GIIIA. These observations suggest that single amino acid differences that significantly affect the structure of the peptide do in fact alter its functional properties. Our work highlights the importance of structural factors, beyond the disulfide pattern and electrostatic interactions, in the understanding of the functional properties of bioactive peptides. The latter thus needs to be considered when designing analogues for further applications.

Cone snails are a diverse group of predatory marine invertebrates that deploy remarkably complex venoms to rapidly paralyse worm, mollusc or fish prey. ω-Conotoxins are neurotoxic peptides from cone snail venoms that inhibit Ca v 2.2 voltage-gated calcium channel, demonstrating potential for pain management via intrathecal (IT) administration. Here, we isolated and characterized two novel ω-conotoxins, MoVIA and MoVIB from Conus moncuri, the first to be identified in vermivorous (worm-hunting) cone snails. MoVIA and MoVIB potently inhibited human Ca v 2.2 in fluorimetric assays and rat Ca v 2.2 in patch clamp studies, and both potently displaced radiolabeled ω-conotoxin GVIA ( 125 I-GVIA) from human SH-SY5Y cells and fish brain membranes (IC 50 2-9 pM). Intriguingly, an arginine at position 13 in MoVIA and MoVIB replaced the functionally critical tyrosine found in piscivorous ω-conotoxins. To investigate its role, we synthesized MoVIB-[R13Y] and MVIIA-[Y13R]. Interestingly, MVIIA-[Y13R] completely lost Ca v 2.2 activity and MoVIB-[R13Y] had reduced activity, indicating that Arg at position 13 was preferred in these vermivorous ω-conotoxins whereas tyrosine 13 is preferred in piscivorous ω-conotoxins. MoVIB reversed pain behavior in a rat neuropathic pain model, confirming that vermivorous cone snails are a new source of analgesic ω-conotoxins. Given vermivorous cone snails are ancestral to piscivorous species, our findings support the repurposing of defensive venom peptides in the evolution of piscivorous Conidae.

Background and Purpose: Voltage-gated calcium channels are involved in nociception in the CNS and in the periphery. N-type (Ca v 2.2) and T-type (Ca v 3.1, Ca v 3.2 and Ca v 3.3) voltage-gated calcium channels are particularly important in studying and treating pain and epilepsy., Experimental Approach: In this study, whole-cell patch clamp electrophysiology was used to assess the potency and mechanism of action of a novel ortho-phenoxylanilide derivative, MONIRO-1, against a panel of voltage-gated calcium channels including Ca v 1.2, Ca v 1.3, Ca v 2.1, Ca v 2.2, Ca v 2.3, Ca v 3.1, Ca v 3.2 and Ca v 3.3., Key Results: MONIRO-1 was 5- to 20-fold more potent at inhibiting human T-type calcium channels, hCa v 3.1, hCa v 3.2 and hCa v 3.3 (IC 50 : 3.3 ± 0.3, 1.7 ± 0.1 and 7.2 ± 0.3 μM, respectively) than N-type calcium channel, hCa v 2.2 (IC 50 : 34.0 ± 3.6 μM). It interacted with L-type calcium channels Ca v 1.2 and Ca v 1.3 with significantly lower potency (IC 50  > 100 μM) and did not inhibit hCa v 2.1 or hCa v 2.3 channels at concentrations as high as 100 μM. State- and use-dependent inhibition of hCa v 2.2 channels was observed, whereas stronger inhibition occurred at high stimulation frequencies for hCa v 3.1 channels suggesting a different mode of action between these two channels., Conclusions and Implications: Selectivity, potency, reversibility and multi-modal effects distinguish MONIRO-1 from other low MW inhibitors acting on Ca v channels involved in pain and/or epilepsy pathways. High-frequency firing increased the affinity for MONIRO-1 for both hCa v 2.2 and hCa v 3.1 channels. Such Ca v channel modulators have potential clinical use in the treatment of epilepsies, neuropathic pain and other nociceptive pathophysiologies., Linked Articles: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc., (© 2017 The British Pharmacological Society.)

Conotoxins (conopeptides) are a diverse group of peptides isolated from the venom of marine cone snails. Conus peptides modulate pain by interacting with voltage-gated ion channels and G protein-coupled receptors (GPCRs). Opiate drugs targeting GPCRs have long been used, nonetheless, many undesirable side effects associated with opiates have been observed including addiction. Consequently, alternative avenues to pain management are a largely unmet need. It has been shown that various voltage-gated calcium channels (VGCCs) respond to GPCR modulation. Thus, regulation of VGCCs by GPCRs has become a valuable alternative in the management of pain. In this review, we focus on analgesic conotoxins that exert their effects via GPCR-mediated inhibition of ion channels involved in nociception and pain transmission. Specifically, α-conotoxin Vc1.1 activation of GABA B receptors and inhibition of voltage-gated calcium channels as a novel mechanism for reducing the excitability of dorsal root ganglion neurons is described. Vc1.1 and other α-conotoxins have been shown to be analgesic in different animal models of chronic pain. This review will outline the functional effects of conopeptide modulation of GPCRs and how their signalling is translated to downstream components of the pain pathways. Where available we present the proposed signalling mechanisms that couples metabotropic receptor activation to their downstream effectors to produce analgesia. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.', (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: To determine the effectiveness of various monitoring strategies on weight loss, body composition, blood markers, exercise, and psychosocial indices in adults with overweight and obesity following a 12-month weight loss program., Methods: Two hundred fifty adults with BMI ≥ 27 were randomized to brief, monthly, individual consults, daily self-monitoring of weight, self-monitoring of diet using MyFitnessPal, self-monitoring of hunger, or control over 12 months. All groups received diet and exercise advice, and 171 participants (68.4%) remained at 12 months., Results: No significant differences in weight, body composition, blood markers, exercise, or eating behavior were apparent between those in the four monitoring groups and the control condition at 12 months (all P ≥ 0.053). Weight differences between groups ranged from -1.1 kg (-3.8 to 1.6) to 2.2 kg (-1.0 to 5.3). However, brief support and hunger training groups reported significantly lower scores for depression (difference [95% CI]: -3.16 [-5.70 to -0.62] and -3.05 [-5.61 to -0.50], respectively) and anxiety (-1.84, [-3.67 to -0.02]) scores than control participants., Conclusions: Although adding a monitoring strategy to diet and exercise advice did not further increase weight loss, no adverse effects on eating behavior were observed, and some monitoring strategies may even benefit mental health., (© 2017 The Obesity Society.)

Human-animal relationships are increasingly incorporated into families as a normal part of family life. Despite this, relationships with animals are often viewed as inferior to human relationships. This becomes problematic during times of loss and grief when members of a grieving companion animal owner's support system do not understand the salience of the relationship with the animal. Veterinary and other helping professionals need basic information about the experience of companion animal loss in order to help support and normalize the experiences of grieving companion animal owners. The present study qualitatively describes human-animal relationships and the subsequent loss and coping experienced by owners of beloved companion animals. Comparison with human and other types of loss and factors unique to companion animal loss are discussed, and practical applications for veterinary and other helping professionals are provided.

Much has been discussed about the high prevalence of psychological distress among veterinary medical students. Studies investigating general samples of veterinary medical students indicate that, on average, depression and anxiety symptoms are present at higher rates than in comparison samples. However, little is known about veterinary medical students who seek counseling. This study intends to expand the literature on veterinary student well-being, as the first to examine a sample of veterinary medical students seeking counseling services. It offers an overview of student distress and help-seeking trends from a decade of counseling services provided in one College of Veterinary Medicine (CVM) in the US. The sample includes data from 279 participants. Results indicate a steady increase in students seeking counseling over the last decade. First-year students sought services at higher rates but second-year students experienced the greatest distress when compared to other cohorts. Students seeking counseling services experienced levels of overall distress, symptoms of anxiety and depression, and social role concerns that were, on average, above cut-off scores. Physical health was significantly associated with student distress, suggesting opportunities for intervention.

The present study evaluated third-year veterinary medical students' perceptions of a communication lab protocol. The protocol used clips of fourth-year veterinary medical students working with authentic clients. These clips supplemented course material. Clips showed examples of proficient communication as well as times of struggle for fourth-year students. Third-year students were asked to critique interactions during class. One hundred and eight third-year students provided feedback about the communication lab. While initial interest in communication proved low, interest in communication training at the end of the course increased substantially. The majority of students cited watching videos clips of authentic client interactions as being an important teaching tool.

Neuronal Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) calcium channels contribute to synaptic transmission and are modulated through G protein-coupled receptor pathways. The analgesic α-conotoxin Vc1.1 acts through γ-aminobutyric acid type B (GABAB) receptors (GABABRs) to inhibit Cav2.2 channels. We investigated GABABR-mediated modulation by Vc1.1, a cyclized form of Vc1.1 (c-Vc1.1), and the GABABR agonist baclofen of human Cav2.1 or Cav2.3 channels heterologously expressed in human embryonic kidney cells. 50 µM baclofen inhibited Cav2.1 and Cav2.3 channel Ba(2+) currents by ∼40%, whereas c-Vc1.1 did not affect Cav2.1 but potently inhibited Cav2.3, with a half-maximal inhibitory concentration of ∼300 pM. Depolarizing paired pulses revealed that ∼75% of the baclofen inhibition of Cav2.1 was voltage dependent and could be relieved by strong depolarization. In contrast, baclofen or Vc1.1 inhibition of Cav2.3 channels was solely mediated through voltage-independent pathways that could be disrupted by pertussis toxin, guanosine 5'-[β-thio]diphosphate trilithium salt, or the GABABR antagonist CGP55845. Overexpression of the kinase c-Src significantly increased inhibition of Cav2.3 by c-Vc1.1. Conversely, coexpression of a catalytically inactive double mutant form of c-Src or pretreatment with a phosphorylated pp60c-Src peptide abolished the effect of c-Vc1.1. Site-directed mutational analyses of Cav2.3 demonstrated that tyrosines 1761 and 1765 within exon 37 are critical for inhibition of Cav2.3 by c-Vc1.1 and are involved in baclofen inhibition of these channels. Remarkably, point mutations introducing specific c-Src phosphorylation sites into human Cav2.1 channels conferred c-Vc1.1 sensitivity. Our findings show that Vc1.1 inhibition of Cav2.3, which defines Cav2.3 channels as potential targets for analgesic α-conotoxins, is caused by specific c-Src phosphorylation sites in the C terminus.

Purpose: To describe and quantify a radiological phenomenon where the distal tibial plafond appears in valgus malalignment in intra-operative fluoroscopy owing to 15 degrees craniocaudal angulation of the X-ray beam., Methods: The lateral distal tibial angle (LDTA) of 14 male and 9 female skeletally mature patients was measured by a single reviewer using 2 types of anteroposterior radiographs, in which the X-ray beam was projected at 0 (orthogonal to the ankle) and then at 15 (in a craniocaudal direction) degrees. The LDTA was the angle between the long axis of the tibia and a line drawn across the most radiodense part of the tibial plafond. The paired t-test was used to compare the LDTA of the 2 measurements., Results: The mean LDTA on the 0-degree orthogonal radiographs was 89 (range, 87-92) degrees, whereas the mean LDTA on the 15-degree craniocaudal radiographs was 79 (range, 77-81) degrees. The mean difference was 10 degrees (range, 9-12; p<0.0001). In the 15-degree craniocaudal radiographs, the ankle joint appeared to have valgus malalignment., Conclusion: During intramedullary nailing of the tibia, the knee is usually flexed and the image intensifier may not swing over far enough. This can result in well-aligned reduction being incorrectly viewed as having valgus malalignment or a varus-malaligned fracture being incorrectly viewed as reduced.

Existing research consistently connects higher relationship satisfaction with improved psychological and physical functioning. Investigations focusing on relational satisfaction within veterinary medicine have been sparse. This study evaluated 240 veterinary medical students at Kansas State University. Results indicate that students within higher-functioning relationships are more likely to report fewer depressive symptoms, lower stress associated with balancing their school and home lives, less relationship conflict, better physical health, and improved ability to cope with academic expectations, while at the same time experiencing more stress from being behind in studies. Based on these findings, Colleges of Veterinary Medicine (CVMs) are encouraged to institute policies and programs which foster relationship-building for students.

Conotoxins have emerged as useful leads for the development of novel therapeutic analgesics. These peptides, isolated from marine molluscs of the genus Conus, have evolved exquisite selectivity for receptors and ion channels of excitable tissue. One such peptide, α-conotoxin Vc1.1, is a 16-mer possessing an interlocked disulfide framework. Despite its emergence as a potent analgesic lead, the molecular target and mechanism of action of Vc1.1 have not been elucidated to date. In this paper we describe the regioselective synthesis of dicarba analogues of Vc1.1 using olefin metathesis. The ability of these peptides to inhibit acetylcholine-evoked current at rat α9α10 and α3β4 nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes has been assessed in addition to their ability to inhibit high voltage-activated (HVA) calcium channel current in isolated rat DRG neurons. Their solution structures were determined by NMR spectroscopy. Significantly, we have found that regioselective replacement of the native cystine framework with a dicarba bridge can be used to selectively tune the cyclic peptide's innate biological activity for one receptor over another. The 2,8-dicarba Vc1.1 isomer retains activity at γ-aminobutyric acid (GABAB) G protein-coupled receptors, whereas the isomeric 3,16-dicarba Vc1.1 peptide retains activity at the α9α10 nAChR subtype. These singularly acting analogues will enable the elucidation of the biological target responsible for the peptide's potent analgesic activity.

Mental health needs of veterinary medical students have become the focus of concern in recent years. Literature to date is scarce, but indicates a large number of veterinary medical students experience clinical levels of anxiety and depression. The present study focused on the prevalence of anxiety and depression in a sample of veterinary medical students (N=142) across four-year cohorts. Findings indicate elevated scores of anxiety and depression across the four-year cohorts. Students in their second and third years had the highest anxiety and depression scores. Perceived physical health, unclear expectations, difficulty fitting in, heavy workload, and homesickness were most relevant in explaining anxiety and depression symptom prevalence. Implications for practice and future research are addressed based on these findings.