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Abstract Background Movement plays a key role in allowing animal species to adapt to sudden environmental shifts. Anthropogenic climate and land use change have accelerated the frequency of some of these extreme disturbances, including megafire. These megafires dramatically alter ecosystems and challenge the capacity of several species to adjust to a rapidly changing landscape. Ungulates and their movement behaviors play a central role in the ecosystem functions of fire-prone ecosystems around the world. Previous work has shown behavioral plasticity is an important mechanism underlying whether large ungulates are able to adjust to recent changes in their environments effectively. Ungulates may respond to the immediate effects of megafire by adjusting their movement and behavior, but how these responses persist or change over time following disturbance is poorly understood. Methods We examined how an ecologically dominant ungulate with strong site fidelity, Columbian black-tailed deer (Odocoileus hemionus columbianus), adjusted its movement and behavior in response to an altered landscape following a megafire. To do so, we collected GPS data from 21 individual female deer over the course of a year to compare changes in home range size over time and used resource selection functions (RSFs) and hidden Markov movement models (HMMs) to assess changes in behavior and habitat selection. Results We found compelling evidence of adaptive capacity across individual deer in response to megafire. Deer avoided exposed and severely burned areas that lack forage and could be riskier for predation immediately following megafire, but they later altered these behaviors to select areas that burned at higher severities, potentially to take advantage of enhanced forage. Conclusions These results suggest that despite their high site fidelity, deer can navigate altered landscapes to track rapid shifts in encounter risk with predators and resource availability. This successful adjustment of movement and behavior following extreme disturbance could help facilitate resilience at broader ecological scales.

Abstract Many wildlife species vary habitat selection across space, time, and behavior to maximize rewards and minimize risk. Multi‐scale research approaches that identify variation in wildlife habitat selection can highlight not only habitat preferences and risk tolerance but also movement strategies that afford coexistence or cause conflict with humans. Here, we examined how anthropogenic and natural features influenced coyote (Canis latrans) habitat selection in a mixed‐use, agricultural landscape in Mendocino County, California, USA. We used resource selection functions and hidden Markov models to test whether coyote selection for anthropogenic and natural features varied by time of day or by behavioral state (resting, foraging, and traveling). We found that coyotes avoided development, but, contrary to our expectations, coyotes selected for roads, agriculture, and areas with risk of human encounter and rifle use regardless of diel period or behavioral state. While traveling, coyotes increased selection for roads and avoided ruggedness, indicating that unpaved roads may enhance connectivity for coyotes in mixed‐use landscapes. Finally, we found that coyotes selected for mountain lion habitat when resting and at night, signifying that risk from natural predators was not a factor in habitat selection at coarse scales. Coyote habitat selection for places and times associated with human activity, without variation across scales, signals a potential for conflict if coyotes are perceived by people as a nuisance.

Abstract Gut microbiome dysbiosis is a major contributing factor to several pathological conditions. However, the mechanistic understanding of the communication between gut microbiota and extra‐intestinal organs remains largely elusive. Extracellular vesicles (EVs), secreted by almost every form of life, including bacteria, could play a critical role in this inter‐kingdom crosstalk and are the focus of present study. Here, we present a novel approach for isolating lipopolysaccharide (LPS)+ bacterial extracellular vesicles (bEVLPS) from complex biological samples, including faeces, plasma and the liver from lean and diet‐induced obese (DIO) mice. bEVLPS were extensively characterised using nanoparticle tracking analyses, immunogold labelling coupled with transmission electron microscopy, flow cytometry, super‐resolution microscopy and 16S sequencing. In liver tissues, the protein expressions of TLR4 and a few macrophage‐specific biomarkers were assessed by immunohistochemistry, and the gene expressions of inflammation‐related cytokines and their receptors (n = 89 genes) were measured using a PCR array. Faecal samples from DIO mice revealed a remarkably lower concentration of total EVs but a significantly higher percentage of LPS+ EVs. Interestingly, DIO faecal bEVLPS showed a higher abundance of Proteobacteria by 16S sequencing. Importantly, in DIO mice, a higher number of total EVs and bEVLPS consistently entered the hepatic portal vein and subsequently reached the liver, associated with increased expression of TLR4, macrophage markers (F4/80, CD86 and CD206), cytokines and receptors (Il1rn, Ccr1, Cxcl10, Il2rg and Ccr2). Furthermore, a portion of bEVLPS escaped liver and entered the peripheral circulation. In conclusion, bEV could be the key mediator orchestrating various well‐established biological effects induced by gut bacteria on distant organs.

To improve animal performance and modify growth by increasing lean tissue accretion, beef cattle production has relied on use of growth promoting technologies such as beta-adrenergic agonists. These synthetic catecholamines, combined with the variable inclusion of rumen degradable (RDP) and undegradable protein (RUP), improve feed efficiency and rate of gain in finishing beef cattle. However, research regarding the impact of beta-adrenergic agonists, protein level, and source on the ruminal microbiome is limited. The objective of this study was to determine the effect of different protein concentrations and beta-adrenergic agonist (ractopamine hydrochloride; RAC) on ruminal bacterial communities in finishing beef heifers. Heifers (n = 140) were ranked according to body weight and assigned to pens in a generalized complete block design with a 3 × 2 factorial arrangement of treatments of 6 different treatment combinations, containing 3 protein treatments (Control: 13.9% CP, 8.9% RDP, and 5.0% RUP; High RDP: 20.9% CP, 14.4% RDP, 6.5% RUP; or High RUP: 20.9% CP, 9.7% RDP, 11.2% RUP) and 2 RAC treatments (0 and 400 mg/day). Rumen samples were collected via orogastric tubing 7 days before harvest. DNA from rumen samples were sequenced to identify bacteria based on the V1-V3 hypervariable regions of the 16S rRNA gene. Reads from treatments were analyzed using the packages 'phyloseq' and 'dada2' within the R environment. Beta diversity was analyzed based on Bray-Curtis distances and was significantly different among protein and RAC treatments (P < 0.05). Alpha diversity metrics, such as Chao1 and Shannon diversity indices, were not significantly different (P > 0.05). Bacterial differences among treatments after analyses using PROC MIXED in SAS 9 were identified for the main effects of protein concentration (P < 0.05), rather than their interaction. These results suggest possible effects on microbial communities with different concentrations of protein but limited impact with RAC. However, both may potentially act synergistically to improve performance in finishing beef cattle.

Abstract Increasingly frequent megafires are dramatically altering landscapes and critical habitats around the world. Across the western United States, megafires have become an almost annual occurrence, but the implication of these fires for the conservation of native wildlife remains relatively unknown. Woodland savannas are among the world's most biodiverse ecosystems and provide important food and structural resources to a variety of wildlife, but they are threatened by megafires. Despite this, the great majority of fire impact studies have only been conducted in coniferous forests. Understanding the resistance and resilience of wildlife assemblages following these extreme perturbations can help inform future management interventions that limit biodiversity loss due to megafire. We assessed the resistance of a woodland savanna mammal community to the short‐term impacts of megafire using camera trap data collected before, during, and after the fire. Specifically, we utilized a 5‐year camera trap data set (2016–2020) from the Hopland Research and Extension Center to examine the impacts of the 2018 Mendocino Complex Fire, California's largest recorded wildfire at the time, on the distributions of eight observed mammal species. We used a multispecies occupancy model to quantify the effects of megafire on species' space use, to assess the impact on species size and diet groups, and to create robust estimates of fire's impacts on species diversity across space and time. Megafire had a negative effect on the detection of certain mammal species, but overall, most species showed high resistance to the disturbance and returned to detection and site use levels comparable to unburned sites by the end of the study period. Following megafire, species richness was higher in burned areas that retained higher canopy cover relative to unburned and burned sites with low canopy cover. Fire management that prevents large‐scale canopy loss is critical to providing refugia for vulnerable species immediately following fire in oak woodlands, and likely other mixed‐forest landscapes.

Beluga whales (Delphinapterus leucas) from Cook Inlet (CI), Alaska, are listed as “endangered” because of dramatic declines in abundance, with no indications of population recovery. Serum samples from this population are exceedingly rare. Longitudinal samples from aquarium (AQ) belugas can potentially provide health assessment reference ranges for free-ranging beluga, including reproductive and metabolic hormones. We analysed serum hormone concentrations from CI (n = 6, three females and three males) and Bristol Bay (Alaska; BB; n = 5, four males and one female), alongside AQ (n = 3, two females and one male) belugas, to conduct physiological comparisons of reproductive hormones (progesterone, testosterone and total oestrogens) and metabolic hormones (total thyroxine, triiodothyronine and cortisol) in beluga serum. Oestrogen and progesterone profiles from January through May from two AQ female beluga were typical of non-pregnant, cycling females. CI and BB sex steroid concentrations were within AQ hormone ranges, with the exception of elevated progesterone concentrations in four potentially pregnant females. Both CI and BB belugas had elevated metabolic hormones, which may indicate greater metabolic effort required in the wild environment or capture response. Because sample collection from CI belugas is rare, analysis of even the few samples that we analysed may contribute to the conservation of the small and declining population of genetically distinct CI beluga whales. It is important that each sample collected from free-range CI belugas provides the maximum biological information possible. Continued comparison of hormones in AQ and free-ranging beluga will enhance the interpretation of health data in both groups.

Abstract With rapid global change, the frequency and severity of extreme disturbance events are increasing worldwide. The ability of animal populations to survive these stochastic events depends on how individual animals respond to their altered environments, yet our understanding of the immediate and short‐term behavioral responses of animals to acute disturbances remains poor. We focused on animal behavioral responses to the environmental disturbance created by megafire. Specifically, we explored the effects of the 2018 Mendocino Complex Fire in northern California, USA, on the behavior and body condition of black‐tailed deer (Odocoileus hemionus columbianus). We predicted that deer would be displaced by the disturbance or experience high mortality post‐fire if they stayed in the burn area. We used data from GPS collars on 18 individual deer to quantify patterns of home range use, movement, and habitat selection before and after the fire. We assessed changes in body condition using images from a camera trap grid. The fire burned through half of the study area, facilitating a comparison between deer in burned and unburned areas. Despite a dramatic reduction in vegetation in burned areas, deer showed high site fidelity to pre‐fire home ranges, returning within hours of the fire. However, mean home range size doubled after the fire and corresponded to increased daily activity in a severely resource‐depleted environment. Within their home ranges, deer also selected strongly for patches of surviving vegetation and woodland habitat, as these areas provided forage and cover in an otherwise desolate landscape. Deer body condition significantly decreased after the fire, likely as a result of a reduction in forage within their home ranges, but all collared deer survived for the duration of the study. Understanding the ways in which large mammals respond to disturbances such as wildfire is increasingly important as the extent and severity of such events increases across the world. While many animals are adapted to disturbance regimes, species that exhibit high site fidelity or otherwise fixed behavioral strategies may struggle to cope with increased climate instability and associated extreme disturbance events.

Skeletal metastasis is the leading cause of morbidity and mortality in prostate cancer, with 80% of advanced prostate cancer patients developing bone metastases. Before metastasis, bone remodeling occurs, stimulating pre-metastatic niche formation and bone turnover, and platelets govern this process. Stem cell factor (SCF, Kit Ligand) is increased in advanced prostate cancer patient platelet releasates. Further, SCF and its receptor, CD117/c-kit, correlate with metastatic prostate cancer severity. We hypothesized that bone-derived SCF plays an important role in prostate cancer tumor communication with the bone inducing pre-metastatic niche formation. We generated two cell-specific SCF knockout mouse models deleting SCF in either mature osteoblasts or megakaryocytes and platelets. Using two syngeneic androgen-insensitive murine prostate cancer cell lines, RM1 (Ras and Myc co-activation) and mPC3 (Pten and Trp53 deletion), we examined the role of bone marrow-derived SCF in primary tumor growth and bone microenvironment alterations. Platelet-derived SCF was required for mPC3, but not RM1, tumor growth, while osteoblast-derived SCF played no role in tumor size in either cell line. While exogenous SCF induced proangiogenic protein secretion by RM1 and mPC3 prostate cancer cells, no significant changes in tumor angiogenesis were measured by immunohistochemistry. Like our previous studies, tumor-induced bone formation occurred in mice bearing RM1 or mPC3 neoplasms, demonstrated by bone histomorphometry. RM1 tumor-bearing osteoblast SCF knockout mice did not display tumor-induced bone formation. Bone stromal cell composition analysis by flow cytometry showed significant shifts in hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and osteoblast cell percentages in mice bearing RM1 or mPC3 tumors. There were no significant changes in the percentage of macrophages, osteoclasts, or osteocytes. Our study demonstrates that megakaryocyte/platelet-derived SCF regulates primary mPC3 tumor growth, while SCF originating from osteoblasts plays a role in bone marrow-derived progenitor cell composition and pre-metastatic niche formation. Further, we show that both the source of SCF and the genetic profile of prostate cancer determine the effects of SCF. Thus, targeting the SCF/CD117 signaling axis with tyrosine kinase inhibitors could affect primary prostate carcinomas or play a role in reducing bone metastasis dependent on the gene deletions or mutations driving the patients’ prostate cancer.

Abstract Angioimmunoblastic T‐cell lymphoma (AITL) is a rare but aggressive lymphoma that commonly presents with skin involvement, often morphologically similar to viral or drug‐induced morbilliform eruptions. Morbilliform rashes with systemic symptoms of unclear etiology mandate a thorough workup; empiric corticosteroid therapy is best avoided and may delay diagnosis of underlying malignancy such as AITL.

The complex ecosystem in which tumor cells reside and interact, termed the tumor microenvironment (TME), encompasses all cells and components associated with a neoplasm that are not transformed cells. Interactions between tumor cells and the TME are complex and fluid, with each facet coercing the other, largely, into promoting tumor progression. While the TME in humans is relatively well-described, a compilation and comparison of the TME in our canine counterparts has not yet been described. As is the case in humans, dog tumors exhibit greater heterogeneity than what is appreciated in laboratory animal models, although the current level of knowledge on similarities and differences in the TME between dogs and humans, and the practical implications of that information, require further investigation. This review summarizes some of the complexities of the human and mouse TME and interjects with what is known in the dog, relaying the information in the context of the temporo-spatial organization of the TME. To the authors' knowledge, the development of the TME over space and time has not been widely discussed, and a comprehensive review of the canine TME has not been done. The specific topics covered in this review include cellular invasion and interactions within the TME, metabolic derangements in the TME and vascular invasion, and the involvement of the TME in tumor spread and metastasis.

The δ-opioid receptor (δOR) holds great potential as a therapeutic target. Yet, clinical drug development, which has focused on δOR agonists that mimic the potent and selective tool compound SNC80 have largely failed. It has increasingly become apparent that the SNC80 scaffold carries with it potent and efficacious β-arrestin recruitment. Here, we screened a relatively small (5120 molecules) physical drug library to identify δOR agonists that underrecruit β-arrestin, as it has been suggested that compounds that efficaciously recruit β-arrestin are proconvulsant. The screen identified a hit compound and further characterization using cellular binding and signaling assays revealed that this molecule (R995045, compound 1) exhibited ten-fold selectivity over µ- and κ-opioid receptors. Compound 1 represents a novel chemotype at the δOR. A subsequent characterization of fourteen analogs of compound 1, however did not identify a more potent δOR agonist. Computational modeling and in vitro characterization of compound 1 in the presence of the endogenous agonist leu-enkephalin suggest compound 1 may also bind allosterically and negatively modulate the potency of Leu-enkephalin to inhibit cAMP, acting as a ‘NAM-agonist’ in this assay. The potential physiological utility of such a class of compounds will need to be assessed in future in vivo assays.

Between 2000 and 2005 several studies revealed that morphine is more potent and exhibits fewer side effects in beta-arrestin 2 knockout mice. These findings spurred efforts to develop opioids that signal primarily via G protein activation and do not, or only very weakly, recruit beta-arrestin. Development of such molecules targeting the mu opioid receptor initially outpaced those targeting the kappa, delta and nociceptin opioid receptors, with the G protein-biased mu opioid agonist oliceridine/TRV130 having completed phase III clinical trials with improved therapeutic window to treat moderate-to-severe acute pain. Recently however, there has been a sharp increase in the development of novel G protein-biased kappa agonists. It is hypothesized that G protein-biased kappa agonists can reduce pain and itch, but exhibit fewer side effects, such as anhedonia and psychosis, that have thus far limited the clinical development of unbiased kappa opioid agonists. Here we summarize recently discovered G protein-biased kappa agonists, comparing structures, degree of signal bias and preclinical effects. We specifically reviewed nalfurafine, 22-thiocyanatosalvinorin A (RB-64), mesyl-salvinorin B, 2-(4-(furan-2-ylmethyl)-5-((4-methyl-3-(trifluoromethyl)benzyl)thio)-4H-1,2,4-triazol-3-yl)pyridine (triazole 1.1), 3-(2-((cyclopropylmethyl)(phenethyl)amino)ethyl)phenol (HS666), N-n-butyl-N-phenylethyl-N-3-hydroxyphenylethyl-amine (compound 5/BPHA), 6-guanidinonaltrindole (6′GNTI), and collybolide. These agonists encompass a variety of chemical scaffolds and range in both their potency and efficacy in terms of G protein signaling and beta-arrestin recruitment. Thus unsurprisingly, the behavioral responses reported for these agonists are not uniform. Yet, it is our conclusion that the kappa opioid field will benefit tremendously from future studies that compare several biased agonists and correlate the degree of signaling bias to a particular pharmacological response.

Pre-clinical studies have demonstrated that tart cherries, rich in hydroxycinnamic acids and anthocyanins, protect against age-related and inflammation-induced bone loss. This study examined how daily consumption of Montmorency tart cherry juice (TC) alters biomarkers of bone metabolism in older women. Healthy women, aged 65–80 years (n = 27), were randomly assigned to consume ~240 mL (8 fl. oz.) of juice once (TC1X) or twice (TC2X) per day for 90 d. Dual-energy x-ray absorptiometry (DXA) scans were performed to determine bone density at baseline, and pre- and post-treatment serum biomarkers of bone formation and resorption, vitamin D, inflammation, and oxidative stress were assessed. Irrespective of osteoporosis risk, the bone resorption marker, tartrate resistant acid phosphatase type 5b, was significantly reduced with the TC2X dose compared to baseline, but not with the TC1X dose. In terms of indicators of bone formation and turnover, neither serum bone-specific alkaline phosphatase nor osteocalcin were altered. No changes in thiobarbituric acid reactive substances or high sensitivity C-reactive protein were observed in response to either TC1X or TC2X. We conclude that short-term supplementation with the higher dose of tart cherry juice decreased bone resorption from baseline without altering bone formation and turnover biomarkers in this cohort.

The inbred high drinking in the dark (iHDID1 and iHDID2) strains are two replicate lines bred from the parent HS/Npt (HS) line for achieving binge levels of blood ethanol concentration (≥80 mg/dL BEC) in a four-hour period. In this work, we sought to evaluate differences in baseline and ethanol-induced c-Fos activation between the HS, iHDID1, and iHDID2 genetic lines in brain regions known to process the aversive properties of ethanol. Methods: Male and female HS, iHDID1, and iHDID2 mice underwent an IP saline 2 3 g/kg ethanol injection. Brain sections were then stained for c-Fos expression in the basolateral/central amygdala (BLA/CeA), bed nucleus of the stria terminals (BNST), A2, locus coeruleus (LC), parabrachial nucleus (PBN), lateral/medial habenula (LHb/MHb), paraventricular nucleus of the thalamus (PVT), periaqueductal gray (PAG), Edinger–Westphal nuclei (EW), and rostromedial tegmental nucleus (RMTg). Results: The iHDID1 and iHDID2 lines showed similar and distinct patterns of regional c-Fos; however, in no region did the two both significantly differ from the HS line together. Conclusions: Our findings lend further support to the hypothesis the iHDID1 and the iHDID2 lines arrive at a similar behavior phenotype through divergent genetic mechanisms.

The transition from non-dependent alcohol use to alcohol dependence involves increased activity of the dorsal striatum. Interestingly, the dorsal striatum expresses a large number of inhibitory G-protein-coupled receptors (GPCRs), which when activated may inhibit alcohol-induced increased activity and can decrease alcohol consumption. Here, we explore the hypothesis that dorsal striatal Gi/o-protein activation is sufficient to reduce voluntary alcohol intake. Using a voluntary, limited-access, two-bottle choice, drink-in-the-dark model of alcohol (10%) consumption, we validated the importance of Gi/o signaling in this region by locally expressing neuron-specific, adeno-associated-virus encoded Gi/o-coupled muscarinic M4 designer receptors exclusively activated by designer drugs (DREADD) in the dorsal striatum and observed a decrease in alcohol intake upon DREADD activation. We validated our findings by activating Gi/o-coupled delta-opioid receptors (DORs), which are natively expressed in the dorsal striatum, using either a G-protein biased agonist or a β-arrestin-biased agonist. Local infusion of TAN-67, an in vitro-determined Gi/o-protein biased DOR agonist, decreased voluntary alcohol intake in wild-type and β-arrestin-2 knockout (KO) mice. SNC80, a β-arrestin-2 biased DOR agonist, increased alcohol intake in wild-type mice; however, SNC80 decreased alcohol intake in β-arrestin-2 KO mice, thus resulting in a behavioral outcome generally observed for Gi/o-biased agonists and suggesting that β-arrestin recruitment is required for SNC80-increased alcohol intake. Overall, these results suggest that activation Gi/o-coupled GPCRs expressed in the dorsal striatum, such as the DOR, by G-protein biased agonists may be a potential strategy to decrease voluntary alcohol consumption and β-arrestin recruitment is to be avoided.

Over a decade, declines in honey bee colonies have raised worldwide concerns. Several potentially contributing factors have been investigated, e.g. parasites, diseases, and pesticides. Neonicotinoid pesticides have received much attention due to their intensive use in crop protection, and their adverse effects on many levels of honey bee physiology led the European Union to ban these compounds. Due to their neuronal target, a receptor expressed throughout the insect nervous system, studies have focused mainly on neuroscience and behaviour. Through the Geometric Framework of nutrition, we investigated effects of the neonicotinoid thiamethoxam on survival, food consumption and sucrose sensitivity of honey bees (Apis mellifera). Thiamethoxam did not affect protein and carbohydrate intake, but decreased responses to high concentrations of sucrose. Interestingly, when bees ate fixed unbalanced diets, dietary protein facilitated better sucrose detection. Both thiamethoxam and dietary protein influenced survival. These findings suggest that, in the presence of a pesticide and unbalanced food, honey bee health may be severely challenged. Consequences for foraging efficiency and colony activity, cornerstones of honey bee health, are also discussed.

PURPOSE:The analysis of exosome/microvesicle (extracellular vesicles (EVs)) and the RNA packaged within them (exoRNA) has the potential to provide a non-invasive platform to detect and monitor disease related gene expression potentially in lieu of more invasive procedures such as biopsy. However, few studies have tested the diagnostic potential of EV analysis in humans. EXPERIMENTAL DESIGN:The ability of EV analysis to accurately reflect prostate tissue mRNA expression was examined by comparing urinary EV TMPRSS2:ERG exoRNA from pre-radical prostatectomy (RP) patients versus corresponding RP tissue in 21 patients. To examine the differential expression of TMPRSS2:ERG across patient groups a random urine sample was taken without prostate massage from a cohort of 207 men including prostate biopsy negative (Bx Neg, n = 39), prostate biopsy positive (Bx Pos, n = 47), post-radical prostatectomy (post-RP, n = 37), un-biopsied healthy age-matched men (No Bx, n = 44), and young male controls (Cont, n = 40). The use of EVs was also examined as a potential platform to non-invasively differentiate Bx Pos versus Bx Neg patients via the detection of known prostate cancer genes TMPRSS2:ERG, BIRC5, ERG, PCA3 and TMPRSS2. RESULTS:In this technical pilot study urinary EVs had a sensitivity: 81% (13/16), specificity: 80% (4/5) and an overall accuracy: 81% (17/21) for non-invasive detection of TMPRSS2:ERG versus RP tissue. The rate of TMPRSS2:ERG exoRNA detection was found to increase with age and the expression level correlated with Bx Pos status. Receiver operator characteristic analyses demonstrated that various cancer-related genes could differentiate Bx Pos from Bx Neg patients using exoRNA isolated from urinary EVs: BIRC5 (AUC 0.674 (CI:0.560-0.788), ERG (AUC 0.785 (CI:0.680-0.890), PCA3 (AUC 0.681 (CI:0.567-0.795), TMPRSS2:ERG (AUC 0.744 (CI:0.600-0.888), and TMPRSS2 (AUC 0.637 (CI:0.519-0.754). CONCLUSION:This pilot study suggests that urinary EVs have the potential to be used as a platform to non-invasively differentiate patients with prostate cancer with very good accuracy. Larger studies are needed to confirm the potential for clinical utility.

This essay describes the expectations and demands placed on one returning student to graduate school who is faced with an array of competing roles. Her reflection on the ups and downs of undergraduate school experiences offers key lessons for persons about to embark on a similar education choice and the lifestyle challenges that it brings.

The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems.

The transition to online learning due to COVID-19 changed the nature of participation in class. Typically quiet middle grades girls who had struggled to participate in their in-person classes were provided many different avenues to contribute to class online. While prior research has examined the reasons students are quiet and the ways teachers can support them, researchers have not yet studied the ways in which teachers can learn from the virtual environment to better do so. Thus, this research seeks to fill that gap by examining the pedagogical approaches utilized in a virtual middle grades environment that strengthen a quieter girl's confidence to speak up in class. Interviews and surveys with quieter girls and their teachers illuminated the ways in which the virtual environment allowed these students to actively participate in class. These quieter students benefitted from the use of chat and non-verbal participation tools, time in breakout rooms to discuss a topic before sharing out, and the increased space and time the online environment provided. Educators may draw from findings on the virtual environment to better support quieter students in the middle grades when returning to in-person learning.

The personal statement is a required component of the residency application, offering applicants the opportunity to showcase their personality as well as unique thoughts and experiences not explicitly stated elsewhere in their application. Although the applicant-generated nature of personal statements can gauge an applicant's professionalism, creativity, sound judgement, and tact, specific criteria for evaluating personal statements and comparing them to one another is lacking. Research on the value of personal statements in the application process is also lacking. The purpose of this research project is to quantify the perceived value of the personal statement to program directors and faculty members of U.S. dermatology programs involved in residency application review. Analysis of our survey responses determined that although the majority of participants consider the personal statement to be a necessary element of an application, it was rated least important compared to other components of the application. An applicant's Medical Student Performance Evaluation, clerkship grades, research projects and publications, board scores, and letters of recommendation were consistently rated as more important than the personal statement. These findings suggest the personal statement lacks the standardization needed for decision makers to confidently choose the best new dermatology residents for their program.

Simple Summary: Early detection of melanoma and differentiation from benign nevi can be challenging even for the most experienced dermatologists. To improve melanoma detection, artificial intelligence algorithms incorporating dermoscopy have been developed, but lack transparency and therefore have limited training value for healthcare providers. To address this, an automated approach utilizing imaging biomarker cues (IBCs), logical features extracted from images that mimic expert dermatologists' dermoscopic pattern recognition skills, was developed. This study excluded deep learning approaches to which IBCs are complementary or alternative. Ten participants assessed 78 dermoscopic images (39 melanomas and 39 nevi) first without IBCs and then with IBCs. Using IBCs significantly improved diagnostic accuracy: sensitivity increased significantly from 73.69% to 81.57% (p = 0.0051) and specificity increased from 60.50% to 67.25% (p = 0.059). These results indicate that incorporating IBCs can significantly enhance melanoma diagnosis, with potential implications for improved screening practices. Further research is needed to confirm these findings across a variety of healthcare providers. Incorporation of dermoscopy and artificial intelligence (AI) is improving healthcare professionals' ability to diagnose melanoma earlier, but these algorithms often suffer from a "black box" issue, where decision-making processes are not transparent, limiting their utility for training healthcare providers. To address this, an automated approach for generating melanoma imaging biomarker cues (IBCs), which mimics the screening cues used by expert dermoscopists, was developed. This study created a one-minute learning environment where dermatologists adopted a sensory cue integration algorithm to combine a single IBC with a risk score built on many IBCs, then immediately tested their performance in differentiating melanoma from benign nevi. Ten participants evaluated 78 dermoscopic images, comprised of 39 melanomas and 39 nevi, first without IBCs and then with IBCs. Participants classified each image as melanoma or nevus in both experimental conditions, enabling direct comparative analysis through paired data. With IBCs, average sensitivity improved significantly from 73.69% to 81.57% (p = 0.0051), and the average specificity improved from 60.50% to 67.25% (p = 0.059) for the diagnosis of melanoma. The index of discriminability (d′) increased significantly by 0.47 (p = 0.002). Therefore, the incorporation of IBCs can significantly improve physicians' sensitivity in melanoma diagnosis. While more research is needed to validate this approach across other healthcare providers, its use may positively impact melanoma screening practices. [ABSTRACT FROM AUTHOR]

Many wildlife species vary habitat selection across space, time, and behavior to maximize rewards and minimize risk. Multi‐scale research approaches that identify variation in wildlife habitat selection can highlight not only habitat preferences and risk tolerance but also movement strategies that afford coexistence or cause conflict with humans. Here, we examined how anthropogenic and natural features influenced coyote (Canis latrans) habitat selection in a mixed‐use, agricultural landscape in Mendocino County, California, USA. We used resource selection functions and hidden Markov models to test whether coyote selection for anthropogenic and natural features varied by time of day or by behavioral state (resting, foraging, and traveling). We found that coyotes avoided development, but, contrary to our expectations, coyotes selected for roads, agriculture, and areas with risk of human encounter and rifle use regardless of diel period or behavioral state. While traveling, coyotes increased selection for roads and avoided ruggedness, indicating that unpaved roads may enhance connectivity for coyotes in mixed‐use landscapes. Finally, we found that coyotes selected for mountain lion habitat when resting and at night, signifying that risk from natural predators was not a factor in habitat selection at coarse scales. Coyote habitat selection for places and times associated with human activity, without variation across scales, signals a potential for conflict if coyotes are perceived by people as a nuisance. [ABSTRACT FROM AUTHOR]

Gut microbiome dysbiosis is a major contributing factor to several pathological conditions. However, the mechanistic understanding of the communication between gut microbiota and extra‐intestinal organs remains largely elusive. Extracellular vesicles (EVs), secreted by almost every form of life, including bacteria, could play a critical role in this inter‐kingdom crosstalk and are the focus of present study. Here, we present a novel approach for isolating lipopolysaccharide (LPS)+ bacterial extracellular vesicles (bEVLPS) from complex biological samples, including faeces, plasma and the liver from lean and diet‐induced obese (DIO) mice. bEVLPS were extensively characterised using nanoparticle tracking analyses, immunogold labelling coupled with transmission electron microscopy, flow cytometry, super‐resolution microscopy and 16S sequencing. In liver tissues, the protein expressions of TLR4 and a few macrophage‐specific biomarkers were assessed by immunohistochemistry, and the gene expressions of inflammation‐related cytokines and their receptors (n = 89 genes) were measured using a PCR array. Faecal samples from DIO mice revealed a remarkably lower concentration of total EVs but a significantly higher percentage of LPS+ EVs. Interestingly, DIO faecal bEVLPS showed a higher abundance of Proteobacteria by 16S sequencing. Importantly, in DIO mice, a higher number of total EVs and bEVLPS consistently entered the hepatic portal vein and subsequently reached the liver, associated with increased expression of TLR4, macrophage markers (F4/80, CD86 and CD206), cytokines and receptors (Il1rn, Ccr1, Cxcl10, Il2rg and Ccr2). Furthermore, a portion of bEVLPS escaped liver and entered the peripheral circulation. In conclusion, bEV could be the key mediator orchestrating various well‐established biological effects induced by gut bacteria on distant organs. [ABSTRACT FROM AUTHOR]

Necro-politics by Achille Mbembe is a work that combines theory, politics, and race. It provides the reader with an opportunity to dive through a depth of knowledge of political, social, [...]