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With the increasing affordability of next-generation sequencing technologies, genotype-by-sequencing has become a cost-effective tool for ecologists and conservation biologists to describe a species' evolutionary history. For host–parasite interactions, genotype-by-sequencing can allow the simultaneous examination of host and parasite genomes and can yield insight into co-evolutionary processes. The eastern oyster, Crassostrea virginica, is among the most important aquacultured species in the United States. Natural and farmed oyster populations can be heavily impacted by ‘dermo’ disease caused by an alveolate protist, Perkinsus marinus. Here, we used restricted site-associated DNA sequencing (RADseq) to simultaneously examine spatial population genetic structure of host and parasite. We analysed 393 single-nucleotide polymorphisms (SNPs) for P. marinus and 52,100 SNPs for C. virginica from 36 individual oysters from the Gulf of Mexico (GOM) and mid-Atlantic coastline. All analyses revealed statistically significant genetic differentiation between the GOM and mid-Atlantic coast populations for both C. virginica and P. marinus, and genetic divergence between Chesapeake Bay and the outer coast of Virginia for C. virginica, but not for P. marinus. A co-phylogenetic analysis confirmed significant coupled evolutionary change between host and parasite across large spatial scales. The strong genetic divergence between marine basins raises the possibility that oysters from either basin would not be well adapted to parasite genotypes and phenotypes from the other, which would argue for caution with regard to both oyster and parasite transfers between the Atlantic and GOM regions. More broadly, our results demonstrate the potential of RADseq to describe spatial patterns of genetic divergence consistent with coupled evolution.

Abstract Introduction Transgender women in the United States experience high HIV incidence and suboptimal Pre‐exposure prophylaxis (PrEP) engagement. We sought to estimate PrEP initiation and discontinuation rates and characterize PrEP discontinuation experiences among a prospective cohort of transgender women. Methods Using a sequential, explanatory, mixed‐methods design, 1312 transgender women at risk for HIV acquisition were enrolled from March 2018 to August 2020 and followed through July 2022 (median follow‐up 24 months; interquartile range 15–36). Cox regression models assessed predictors of initiation and discontinuation. In‐depth interviews were conducted among 18 participants, including life history calendars to explore key events and experiences surrounding discontinuations. Qualitative and quantitative data were integrated to generate typologies of discontinuation, inform meta‐inferences and facilitate the interpretation of findings. Results 21.8% (n = 286) of participants reported taking PrEP at one or more study visits while under observation. We observed 139 PrEP initiations over 2127 person‐years (6.5 initiations/100 person‐years, 95% CI: 5.5–7.7). Predictors of initiation included identifying as Black and PrEP indication. The rate of initiation among those who were PrEP‐indicated was 9.6 initiations/100 person‐years (132/1372 person‐years; 95% CI: 8.1–11.4). We observed 138 PrEP discontinuations over 368 person‐years (37.5 discontinuations/100 person‐years, 95% CI: 31.7–44.3). Predictors of discontinuation included high school education or less and initiating PrEP for the first time while under observation. Four discontinuation typologies emerged: (1) seroconversion following discontinuation; (2) ongoing HIV acquisition risk following discontinuation; (3) reassessment of HIV/STI prevention strategy following discontinuation; and (4) dynamic PrEP use coinciding with changes in HIV acquisition risk. Conclusions PrEP initiation rates were low and discontinuation rates were high. Complex motivations to stop using PrEP did not consistently correspond with HIV acquisition risk reduction. Evidence‐based interventions to increase PrEP persistence among transgender women with ongoing acquisition risk and provide HIV prevention support for those who discontinue PrEP are necessary to reduce HIV incidence in this population.

Evidence suggests that estradiol-sensing preoptic area GABA neurons are involved in the preovulatory surge mechanism necessary for ovulation. In vivo CRISPR-Cas9 editing was used to achieve a 60–70% knockdown in estrogen receptor alpha (ESR1) expression by GABA neurons located within the regions of the rostral periventricular area of the third ventricle (RP3V) and medial preoptic nuclei (MPN) in adult female mice. Mice exhibited variable reproductive phenotypes with the only significant finding being mice with bilateral ESR1 deletion in RP3V GABA neurons having reduced cFos expression in gonadotropin-releasing hormone (GnRH) neurons at the time of the surge. One sub-population of RP3V GABA neurons expresses kisspeptin. Re-grouping ESR1-edited mice on the basis of their RP3V kisspeptin expression revealed a highly consistent phenotype; mice with a near-complete loss of kisspeptin immunoreactivity displayed constant estrus and failed to exhibit surge activation but retained pulsatile luteinizing hormone (LH) secretion. These observations demonstrate that ESR1-expressing GABA-kisspeptin neurons in the RP3V are essential for the murine preovulatory LH surge mechanism.

IntroductionPeople with HIV (PWH) are known to have underlying inflammation and immune activation despite virologic control. Substance use including opioid dependence is common in this population and is associated with increased morbidity and reduced lifespan. The primary objective of the present study termed opioid immunity study (OPIS), was to investigate the impact of chronic opioids in PWH.MethodsThe study recruited people with and without HIV who had opioid use disorder (OUD). Study participants (n=221) were categorized into four groups: HIV+OP+, n=34; HIV-OP+, n=66; HIV+OP-, n=55 and HIV-OP-, n=62 as controls. PWH were virally suppressed on ART and those with OUD were followed in a syringe exchange program with confirmation of OP use by urine drug screening. A composite cytokine score was developed for 20 plasma cytokines that are linked to inflammation. Cellular markers of immune activation (IA), exhaustion, and senescence were determined in CD4 and CD8 T cells. Regression models were constructed to examine the relationships of HIV status and opioid use, controlling for other confounding factors.ResultsHIV+OP+ participants exhibited highest inflammatory cytokines and cellular IA, followed by HIV-OP+ for inflammation and HIV+OP- for IA. Inflammation was found to be driven more by opioid use than HIV positivity while IA was driven more by HIV than opioid use. In people with OUD, expression of CD38 on CD28-CD57+ senescent-like T cells was elevated and correlated positively with inflammation.DiscussionGiven the association of inflammation with a multitude of adverse health outcomes, our findings merit further investigations to understand the mechanistic pathways involved.

Abstract Background Hospitalizations for severe injection drug use-related infections (SIRIs) are characterized by high costs, frequent patient-directed discharge, and high readmission rates. Beyond the health system impacts, these admissions can be traumatizing to people who inject drugs (PWID), who often receive inadequate treatment for their substance use disorders (SUD). The Jackson SIRI team was developed as an integrated infectious disease/SUD treatment intervention for patients hospitalized at a public safety-net hospital in Miami, Florida in 2020. We conducted a qualitative study to identify patient- and clinician-level perceived implementation barriers and facilitators to the SIRI team intervention. Methods Participants were patients with history of SIRIs (n = 7) and healthcare clinicians (n = 8) at one implementing hospital (Jackson Memorial Hospital). Semi-structured qualitative interviews were performed with a guide created using the Consolidated Framework for Implementation Research (CFIR). Interviews were transcribed, double coded, and categorized by study team members using CFIR constructs. Results Implementation barriers to the SIRI team intervention identified by participants included: (1) complexity of the SIRI team intervention; (2) lack of resources for PWID experiencing homelessness, financial insecurity, and uninsured status; (3) clinician-level stigma and lack of knowledge around addiction and medications for opioid use disorder (OUD); and (4) concerns about underinvestment in the intervention. Implementation facilitators of the intervention included: (1) a non-judgmental, harm reduction-oriented approach; (2) the team’s advocacy for PWID as a means of institutional culture change; (3) provision of close post-hospital follow-up that is often inaccessible for PWID; (4) strong communication with patients and their hospital physicians; and (5) addressing diverse needs such as housing, insurance, and psychological wellbeing. Conclusion Integration of infectious disease and SUD treatment is a promising approach to managing patients with SIRIs. Implementation success depends on institutional buy-in, holistic care beyond the medical domain, and an ethos rooted in harm reduction across multilevel (inner and outer) implementation contexts.

Estrogen secreted by the ovary controls how the brain drives pulsatile reproductive hormone secretion. The authors show that in mice, estrogen receptor alpha within a specific population of hypothalamic kisspeptin neurons is the principal pathway through which estrogen brings about this classic negative feedback pathway common to all mammals.

Abstract Background Dogs have a species‐specific susceptibility for developing mast cell tumours (MCTs). Mutations in the KIT proto‐oncogene (KIT) are known to contribute to the neoplastic biology of mast cells. In dogs, the most common KIT mutation is an internal tandem duplication (ITD) in exon 11 which has been considered a useful prognostic supplement to traditional histopathological tumour grading. Objective The aim of this retrospective study was to explore the importance of KIT exon 11 ITD mutation status and known clinical and pathological indices in predicting prognosis in a cohort of Australian dogs diagnosed with MCT. Methods Clinical parameters, survival data, and KIT mutation status were collected and assessed for 220 dogs with cutaneous or subcutaneous MCT (n = 189 and n = 31, respectively). Results In at least one of the multivariable models, tumour grade (cutaneous Kiupel low or high grade) or tumour subcutaneous location, multiple concurrent MCTs, metastasis at the time of surgery, and senior age were statistically significant in predicting the outcome (MCT‐related death and/or second MCT diagnosis) at 6‐ or 12‐month post‐tumour excision. KIT exon 11 ITD mutation status was not a significant predictor in any of the final multivariable models and was strongly correlated with high histological grade (p

Abstract Background Opioid use is escalating in North America and comes with a multitude of health consequences, including HIV and hepatitis C virus (HCV) outbreaks among persons who inject drugs (PWID). HIV pre-exposure prophylaxis (PrEP) and HCV treatment regimens have transformative potential to address these co-occurring epidemics. Evaluation of innovative multi-modal approaches, integrating harm reduction, opioid agonist therapy (OAT), PrEP, and HCV treatment is required. The aim of this study is to assess the effectiveness of an on-site integrated care model where delivery of PrEP and HCV treatment for PWID takes places at syringe service programs (SSP) and OAT programs compared with referring PWID to clinical services in the community through a patient navigation model and to examine how structural factors interact with HIV prevention adherence and HCV treatment outcomes. Methods The Miami-Montreal Hepatitis C and Pre-Exposure Prophylaxis trial (M2HepPrEP) is an open-label, multi-site, multi-center, randomized, controlled, superiority trial with two parallel treatment arms. A total of 500 persons who injected drugs in the prior 6 months and are eligible for PrEP will be recruited in OAT clinics and SSP in Miami, FL, and Montréal, Québec. Participants will be randomized to either on-site care, with adherence counseling, or referral to off-site clinics assisted by a patient navigator. PrEP will be offered to all participants and HCV treatment to those HCV-infected. Co-primary endpoints will be (1) adherence to pre-exposure prophylaxis medication at 6 months post-randomization and (2) HCV sustained virological response (SVR) 12 weeks post-treatment completion among participants who were randomized within the HCV stratum. Up to 100 participants will be invited to participate in a semi-structured interview regarding perceptions of adherence barriers and facilitators, after their 6-month assessment. A simulation model-based cost-effectiveness analysis will be performed to determine the comparative value of the strategies being evaluated. Discussion The results of this study have the potential to demonstrate the effectiveness and cost-effectiveness of offering PrEP and HCV treatment in healthcare venues frequently attended by PWID. Testing the intervention in two urban centers with high disease burden among PWID, but with different healthcare system dynamics, will increase generalizability of findings. Trial registration Clinicaltrials.gov NCT03981445 . Trial registry name: Integrated HIV Prevention and HCV Care for PWID (M2HepPrEP). Registration date: June 10, 201.

A rotary drum mower is a tractor-mounted harvester used for harvesting green fodder plants in agricultural fields. During transportation, it experiences significant dynamic road reaction forces that can cause deformation and functional failures. This study focuses on analysing the deformation behaviour of the machine during transportation to test the machine’s failure condition. To conduct the strength analysis, a total work cycle scenario reflecting actual load conditions and design challenges was created. Experimental strain-gauge-based stress analysis and advanced computer-aided engineering (CAE) simulation methods were employed. The study successfully conducted experimental stress analysis, 3D solid modelling, and validated finite element analysis (FEA). A comparison between experimental and simulation results showed an average relative difference of 24.25% with a maximum absolute difference of approximately 5 MPa. No functional failure issues were observed during physical experiments. The study also revealed that the mean dynamic loading value, when compared to the static linkage position, was calculated as 3.65 ± 0.40. Overall, this research provides a valuable approach for future studies on complex stress and deformation evaluations of agricultural machinery and equipment.

Abstract Pharmacokinetic drug interactions precipitated by botanical and other natural products (NPs) remain critically understudied. Investigating these complex interactions is fraught with difficulties due to the methodologic and technical challenges associated with the inherently complex chemistries and product variability of NPs. This knowledge gap is perpetuated by a continuing absence of a harmonized framework regarding the design of clinical pharmacokinetic studies of NPs and NP‐drug interactions. Accordingly, this Recommended Approach, the fourth in a series of Recommended Approaches released by the Center of Excellence for Natural Product Drug Interaction Research (NaPDI Center), provides recommendations for the design of clinical pharmacokinetic studies involving NPs. Building on prior Recommended Approaches and data generated from the NaPDI Center, such a framework is presented for the design of (1) phase 0 studies to assess the pharmacokinetics of an NP and (2) clinical pharmacokinetic NP‐drug interaction studies. Suggestions for NP sourcing, dosing, study design, participant selection, sampling periods, and data analysis are presented. With the intent to begin addressing the gap between regulatory agencies’ guidance documents about drug‐drug interactions and contemporary NPDI research, the objective of this Recommended Approach is to propose methods for the design of clinical pharmacokinetic studies of NPs and NP‐drug interactions.

Summary: The mechanism by which arcuate nucleus kisspeptin (ARNKISS) neurons co-expressing glutamate, neurokinin B, and dynorphin intermittently synchronize their activity to generate pulsatile hormone secretion remains unknown. An acute brain slice preparation maintaining synchronized ARNKISS neuron burst firing was used alongside in vivo GCaMP GRIN lens microendoscope and fiber photometry imaging coupled with intra-ARN microinfusion. Studies in intact and gonadectomized male mice revealed that ARNKISS neuron synchronizations result from near-random emergent network activity within the population and that this was critically dependent on local glutamate-AMPA signaling. Whereas neurokinin B operated to potentiate glutamate-generated synchronizations, dynorphin-kappa opioid tone within the network served as a gate for synchronization initiation. These observations force a departure from the existing “KNDy hypothesis” for ARNKISS neuron synchronization. A “glutamate two-transition” mechanism is proposed to underlie synchronizations in this key hypothalamic central pattern generator driving mammalian fertility.

This study focuses on the structural design analysis of a cura baglama, a traditional Turkish string instrument that does not have in place a regulated set of manufacturing standards to follow. The aim therefore is to introduce a structural deformation analysis for a sample cura baglama in three different string tensions via a numerical method-based engineering analysis technique. The three-dimensional solid model of a sample cura baglama was created using a 3D scanner and parametric 3D solid modelling software. Based on experimental frequency analysis, structural deformation analyses of the instrument were conducted using finite element method-based engineering simulation techniques. The simulation results revealed useful visual and numerical outputs related to the deformation behaviour of the instrument under pre-defined boundary conditions. A maximum deformation of 0.223 mm on the soundboard (at the D3 tune) and a maximum equivalent stress of 18.325 MPa on the bridge (at the D3 tune) were calculated. The outputs of this research contribute to further research into the usage of numerical method-based deformation simulation studies related to the standardisation, development, and preservation of such traditional string instruments.

Additive manufacturing (AM) is the name given to a family of manufacturing processes where materials are joined to make parts from 3D modelling data, generally in a layer-upon-layer manner. AM is rapidly increasing in industrial adoption for the manufacture of end-use parts, which is therefore pushing for the maturation of design, process, and production techniques. Machine learning (ML) is a branch of artificial intelligence concerned with training programs to self-improve and has applications in a wide range of areas, such as computer vision, prediction, and information retrieval. Many of the problems facing AM can be categorised into one or more of these application areas. Studies have shown ML techniques to be effective in improving AM design, process, and production but there are limited industrial case studies to support further development of these techniques.

Challenges caused by design complexities during the design stages of a product must be coordinated and overcome by the selection of a suitable manufacturing approach. Additive manufacturing (AM) is capable of fabricating complex shapes, yet there are limiting aspects to surface integrity, dimensional accuracy, and, in some instances, design restrictions. Therefore, the goal is essentially to establish the complex areas of a tool during the design stage to achieve the desired quality levels for the corresponding injection moulding tool insert. When adopting a manufacturing approach, it is essential to acknowledge limitations and restrictions. This paper presents the development of a feature-based manufacturability assessment system (FBMAS) to demonstrate the feasibility of integrating selective laser melting (SLM), a metal-based AM technology, with subtractive manufacturing for any given part. The areas on the tool inserts that hold the most geometrical complexities to manufacture are focused on the FBMAS and the design features that are critical for the FBMAS are defined. Furthermore, the structural approach used for developing the FBMAS graphical user interface is defined while explaining how it can be operated effectively and in a user-friendly approach. The systematic approach established is successful in capturing the benefits of SLM and subtractive methods of manufacturing, whilst defining design limitations of each manufacturing method. Finally, the FBMAS developed was validated and verified against the criteria set by experts in the field, and the system’s logic was proven to be accurate when tested. The decision recommendations proved to correlate with the determined recommendations of the field experts in evaluating the feature manufacturability of the tool inserts.

AbstractIntroduction Hospitalizations for severe injection-related infections (SIRI), such as endocarditis, osteomyelitis, and skin and soft tissue infections (SSTI) are increasingly common. People who inject drugs (PWID) experiencing SIRIs often receive inadequate substance use disorder (SUD) treatment and lack of access to harm reduction services. This translates into lengthy hospitalizations with high rates of patient-directed discharge, readmissions, and post-hospitalization mortality. The purpose of this study was to describe the development of an integrated “SIRI Team” and its initial barriers and facilitators to success.Materials and methods The Jackson SIRI Team was developed to improve both hospital and patient-level outcomes for individuals hospitalized with SIRIs at Jackson Memorial Hospital, a 1550-bed public hospital in Miami, Florida, United States. The SIRI Team provides integrated infectious disease and SUD treatment across the healthcare system starting from the inpatient setting and continuing for 90-days post-hospital discharge. The team uses a harm reduction approach, provides care coordination, focuses on access to medications for opioid use disorder (MOUD), and utilizes a variety of infection and addiction treatment modalities to suit each individual patient.Results Over the initial 8-months of the SIRI Team, 21 patients were treated with 20 surviving until discharge. Infections included osteomyelitis, endocarditis, bacteraemia/fungemia, SSTIs, and septic arthritis. All patients had OUD and 95% used stimulants. All patients were discharged on MOUD and 95% completed their prescribed antibiotic course. At 90-days post-discharge, 25% had been readmitted and 70% reported taking MOUD.Conclusions A model of integrated infectious disease and SUD care for the treatment of SIRIs has the potential to improve infection and addiction outcomes. Providing attentive, patient-centered care, using a harm reduction approach can facilitate engagement of this marginalized population with the healthcare system.KEY MESSAGESIntegrated infectious disease and addiction treatment is a novel approach to treating severe injection-related infections.Harm reduction should be applied to treating patients with severe injection-related infections with a goal of facilitating antibiotic completion, remission from substance use disorder, and reducing hospital readmissions.

BackgroundThe concentration and duration of antibodies (Ab) to SARS-CoV-2 infection predicts the severity of the disease and the clinical outcomes. Older people and those with HIV have impaired immune responses, worse outcomes after SARS-CoV-2 infection, and lower antibody responses after viral infection and vaccination. This study evaluated an Ab response to SARS-CoV-2 in people with HIV (PWH) and without HIV (HIV-) and its association with age.MethodsA total of 23 COVID+PWH and 21 COVID+HIV- participants were followed longitudinally for 6 months post-mild COVID-19. Immunoglobin G (IgG) and immunoglobin M (IgM) Ab responses were measured by an in-house developed ELISA. Time points and HIV status interaction were analyzed using Poisson generalized estimating equations, and correlations were analyzed using non-parametric tests.ResultsMedian age in PWH was 55 years with 28.6% women, while in the HIV- group was 36 years with 60.9% women. The mean time from COVID-19 diagnosis to study enrollment was 16 days for PWH and 11 days for HIV-. The mean CD4+ T-cell count/μl for PWH was 772.10 (±365.21). SARS-CoV-2 IgM and IgG were detected at all time points and Ab response levels did not differ by HIV status (p > 0.05). At entry, age showed a weak direct association with IgG responses (ρ = 0.44, p < 0.05) in HIV- but did not show any association in PWH. Similar associations between age, IgG, and HIV status emerged at day 14 (T1; ρ = 0.50, p < 0.05), 3 months (T3; ρ = 0.50, p < 0.05), and 6 months visit (T4; ρ = 0.78, p < 0.05) in the HIV- group.ConclusionThe Ab responses in the 6-month post-SARS-CoV-2 infection did not differ by HIV status, though a positive association was found between age and Ab response in older PWH. Results suggest that immune protection and vaccine responses are similar for PWH than for those without HIV infection.

The role of cytochrome P450 (CYP)2C9 and CYP2C19 genetic variation in risk for phenytoin‐induced cutaneous adverse drug events is not well understood independently of the human leukocyte antigen B (HLA‐B)*15:02 risk allele. In the multi‐ethnic resource for Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort, we identified 382 participants who filled a phenytoin prescription between 2005 and 2017. These participants included 21 people (5%) who self‐identified as Asian, 18 (5%) as black, 29 (8%) as white Hispanic, and 308 (81%) as white non‐Hispanic. We identified 264 (69%) CYP2C9*1/*1, 77 (20%) CYP2C9*1/*2, and 29 (8%) CYP2C9*1/*3. We also determined CYP2C19 genotypes, including 112 with the increased activity CYP2C19*17 allele. Using electronic clinical notes, we identified 32 participants (8%) with phenytoin‐induced cutaneous adverse events recorded within 100 days of first phenytoin dispensing. Adjusting for age, sex, daily dose, and race/ethnicity, participants with CYP2C9*1/*3 or CYP2C9*2/*2 genotypes were more likely to develop cutaneous adverse events compared with CYP2C9*1/*1 participants (odds ratio 4.47; 95% confidence interval 1.64–11.69; P

Warfarin is a narrow therapeutic index anticoagulant drug and its use is associated with infrequent but significant adverse bleeding events. The international normalized ratio (INR) is the most commonly used biomarker to monitor and titrate warfarin therapy. However, INR is derived from a functional assay, which determines clotting efficiency at the time of measurement and is susceptible to technical variability. Protein induced by vitamin K antagonist‐II (PIVKA‐II) has been suggested as a biomarker of long‐term vitamin K status, providing mechanistic insights about variation in the functional assay. However, the currently available antibody‐based PIVKA‐II assay does not inform on the position and number of des‐carboxylation sites in prothrombin. The assay presented in this paper provides simultaneous quantification of carboxy and des‐carboxy prothrombin that are essential for monitoring early changes in INR and, thus, serves as the superior tool for managing warfarin therapy. Additionally, this assay permits the quantification of total prothrombin level, which is affected by warfarin treatment. Prothrombin recovery from plasma was 95% and the liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) assay was linear (r2 = 0.98) with a dynamic range of 1–100 µg/mL. The assay interday precision was within 20%. A des‐carboxy peptide of prothrombin (GNLER) was negatively correlated with active prothrombin (Pearson r = 0.99, P

Excited charge carriers, such as photoelectrons, play an important role in fundamental and technological fields. Here the authors employ an ultrafast electron microscope to directly visualize the cyclotron oscillations and oblate-to-prolate shape change of a photoemitted electron gas from a laser-excited copper surface.

Abstract Background There is growing public health concern around the potential impact of the opioid crisis on efforts to eradicate HIV. This secondary analysis seeks to determine if those who report opioids as their primary problem drug compared to those who report other drugs and/or alcohol differ in engagement in HIV primary care among a sample of hospitalized people with HIV (PWH) who use drugs and/or alcohol, a traditionally marginalized and difficult to engage population key to ending the HIV epidemic. Setting and participants A total of 801 participants (67% male; 75% Black, non-Hispanic; mean age 44.2) with uncontrolled HIV and reported drug and/or alcohol use were recruited from 11 hospitals around the U.S. in cities with high HIV prevalence from 2012 to 2014 for a multisite clinical trial to improve HIV viral suppression. Methods A generalized linear model compared those who reported opioids as their primary problem drug to those who reported other problem drugs and/or alcohol on their previous engagement in HIV primary care, controlling for age, sex, race, education, income, any previous drug and/or alcohol treatment, length of time since diagnosis, and study site. Results A total of 95 (11.9%) participants reported opioids as their primary problem drug. In adjusted models, those who reported opioids were significantly less likely to have ever engaged in HIV primary care than those who reported no problem drug use (adjusted risk ratio, ARR = 0.84, 95% Confidence Interval, CI 0.73, 0.98), stimulants (ARR = 0.84, 95% CI 0.74, 0.95), and polydrug use but no alcohol (ARR = 0.79, 95% CI 0.68, 0.93). While not statistically significant, the trend in the estimates of the remaining drug and/or alcohol categories (alcohol, cannabis, polydrug use with alcohol, and [but excluding the estimate for] other), point to a similar phenomena—those who identify opioids as their primary problem drug are engaging in HIV primary care less. Conclusions These findings suggest that for hospitalized PWH who use drugs and/or alcohol, tailored and expanded efforts are especially needed to link those who report problem opioid use to HIV primary care. Trial registration This study was funded by National Institutes of Health (NIH) grant: U10-DA01372011 (Project HOPE—Hospital Visit as Opportunity for Prevention and Engagement for HIV-Infected Drug Users; Metsch); which is also a registered clinical trial under the Clinical Trials Network (CTN-0049). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

The cytochrome P450 2D6 (CYP2D6) gene locus is challenging to accurately genotype due to numerous single nucleotide variants and complex structural variation. Our goal was to determine whether the CYP2D6 genotype‐phenotype correlation is improved when diplotype assignments incorporate structural variation, identified by the bioinformatics tool Stargazer, with next‐generation sequencing data. Using CYP2D6 activity measured with substrates dextromethorphan and metoprolol, activity score explained 40% and 34% of variability in metabolite formation rates, respectively, when diplotype calls incorporated structural variation, increasing from 36% and 31%, respectively, when diplotypes did not incorporate structural variation. We also investigated whether the revised Clinical Pharmacogenetics Implementation Consortium (CPIC) recommendations for translating genotype to phenotype improve CYP2D6 activity predictions over the current system. Although the revised recommendations do not improve the correlation between activity score and CYP2D6 activity, perhaps because of low frequency of the CYP2D6*10 allele, the correlation with metabolizer phenotype group was significantly improved for both substrates. We also measured the function of seven rare coding variants: one (A449D) exhibited decreased (44%) and another (R474Q) increased (127%) activity compared with reference CYP2D6.1 protein. Allele‐specific analysis found that A449D is part of a novel CYP2D6*4 suballele, CYP2D6*4.028. The novel haplotype containing R474Q was designated CYP2D6*138 by PharmVar; another novel haplotype containing R365H was designated CYP2D6*139. Accuracy of CYP2D6 phenotype prediction is improved when the CYP2D6 gene locus is interrogated using next‐generation sequencing coupled with structural variation analysis. Additionally, revised CPIC genotype to phenotype translation recommendations provides an improvement in assigning CYP2D6 activity.

The opportunity to develop graduate attributes beyond disciplinary expertise is an essential component of any degree programme, and students should develop these during their time within higher education. Graduate attributes can be considered as high-level skills and qualities beyond technical knowledge, which enable higher education institutions to produce more employable graduates. However, there are significant challenges in developing such skills and qualities, where remote teaching and learning complicate this further. Online teaching does not wholly derail the opportunities for students to achieve existing graduate attributes—in contrast, it also creates opportunities for innovation and shapes graduates in preparation to be the next-generation workforce. In this paper, we aim to study and synthesise existing knowledge on the effect of online hybrid teaching on graduate attributes. A systematic literature survey was completed revealing that educational institutions continue using online or hybrid instructional modes, affecting graduate attributes positively and negatively. Therefore, to effectively develop graduate attributes, there is a requirement for fundamental changes in instructional strategies, teaching–learning behaviours, upgrading of facilities and curriculum adaptations. We review graduate attributes in the higher education literature, including the impacts resulting from remote teaching and learning. The paper also identifies the general challenges to developing graduate attributes, as well as more specific challenges as a result of hybrid and online teaching and learning.

The widespread use of nanomaterials in vaccines, therapeutics, and industrial applications creates an increasing demand for understanding their ingestion by living cells. Researchers in the field have called for a more robust understanding of physical/chemical particle–cell interactions and a means to determine the particles ingested per cell. Using superparamagnetic nanobeads, we measured the beads per cell and quantified the kinetics of the receptor-independent endocytosis of particles having seven surface chemistries. Poly(ethylene glycol) (PEG)-coated nanoparticles were ingested less effectively by cultured Chinese hamster ovary (CHO-K1) cells and more effectively by aminated nanoparticles than starch-coated particles. The cells ingested 2 to 4 × 105 of the most attractive particles. The interplay between Van der Waals and coulombic potentials was quantified on the basis of Derjaguin–Landau–Verwey–Overbeek (DLVO) theory modified to include hydration repulsion using physical parameters of the seven surface chemistries. Using dose–response curves for inhibitors of clathrin- or caveolae-dependent ingestion, we quantified how particle surface chemistry determines which endocytic pathway is used by the cell. Such characterization can be useful in predicting nanomaterial uptake in medical and toxicological applications and in the selection of particle surface chemistries for receptor-dependent endocytosis.

Summary: Liver disease is a major cause of premature death. Oxidative stress in the liver represents a key disease driver. Compounds, such as dimethyl fumarate (DMF), can activate the antioxidant response and are used clinically to treat disease. In this study, we tested the protective properties of DMF before or after paracetamol exposure. Following DMF administration, Nrf2 nuclear translocation was tracked at the single-cell level and target gene transactivation confirmed. Next, the protective properties of DMF were examined following paracetamol exposure. Transcriptomic and biochemical analysis revealed that DMF rescue was underpinned by reduced Nf-kB and TGF-β signaling and cell senescence. Following on from these studies, we employed a Zebrafish model to study paracetamol exposure in vivo. We combined a genetically modified Zebrafish model, expressing green fluorescent protein exclusively in the liver, with automated microscopy. Pre-treatment with DMF, prior to paracetamol exposure, led to reduced liver damage in Zebrafish demonstrating protective properties.

Background: Enhanced GABA activity in the brain and a hyperactive hypothalamic-pituitary-gonadal axis are associated with polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility. Women with PCOS exhibit elevated cerebrospinal fluid GABA levels and preclinical models of PCOS exhibit increased GABAergic input to GnRH neurons, the central regulators of reproduction. The arcuate nucleus (ARN) is postulated as the anatomical origin of elevated GABAergic innervation; however, the functional role of this circuit is undefined. Methods: We employed a combination of targeted optogenetic and chemogenetic approaches to assess the impact of acute and chronic ARN GABA neuron activation. Selective acute activation of ARN GABA neurons and their fiber projections was coupled with serial blood sampling for luteinizing hormone secretion in anesthetized male, female and prenatally androgenised (PNA) mice modelling PCOS. In addition, GnRH neuron responses to ARN GABA fiber stimulation were recorded in ex vivo brain slices. Chronic activation of ARN GABA neurons in healthy female mice was coupled with reproductive phenotyping for PCOS-like features. Findings: Acute stimulation of ARN GABA fibers adjacent to GnRH neurons resulted in a significant and long-lasting increase in LH secretion in male and female mice. The amplitude of this response was blunted in PNA mice, which also exhibited a blunted LH response to GnRH administration. Infrequent and variable GABAA-dependent changes in GnRH neuron firing were observed in brain slices. Chronic activation of ARN GABA neurons in healthy females impaired estrous cyclicity, decreased corpora lutea number and increased circulating testosterone levels. Interpretation: ARN GABA neurons can stimulate the hypothalamic-pituitary axis and chronic activation of ARN GABA neurons can mimic the reproductive deficits of PCOS in healthy females. Unexpectedly blunted HPG axis responses in PNA mice may reflect a history of high frequency GnRH/LH secretion and reduced LH stores, but also raise questions about impaired function within the ARN GABA population and the involvement of other circuits. Keywords: GnRH neurons, Luteinizing hormone, Mouse, Optogenetics, Chemogenetics, PCOS

Alaska Native and American Indian (AN/AI) people have unique pharmacogene variation that may affect warfarin disposition and therapeutic response. We performed targeted genotyping for cytochrome P450 (CYP)2C9, vitamin K epoxide oxidase reductase complex subunit 1 (VKORC1), CYP4F2, CYP4F11, and gamma‐glutamyl carboxylase (GGCX) variants in AN/AI people receiving warfarin. The primary outcome was stable warfarin dose, defined as one dose, and associated international normalized ratio within the target range, at least 6 months after starting therapy, with two matching doses at least 2 weeks apart. Genotype–phenotype relationships were assessed by multivariate regression analysis, adjusted for self‐reported heritage, age, gender, and concurrent statin use. VKORC1 genotype explained 34% of dose variability, with VKORC1 −1639G>A and 1173C>T associated with a 1.7 mg/day (P = 1.4e‐05) dose reduction. Additionally, CYP2C9 N218I was suggestively significant (P = 0.077), with heterozygotes requiring 1.1 mg/day less than reference individuals. Self‐reported heritage was significantly associated with dose, largely driven by differences in the diagnostic VKORC1 allele frequencies among AN/AI people.

Vitamin K (VK), in both its phylloquinone and menaquinone forms, has been hypothesized to undergo ω- and β-oxidation on its hydrophobic side chain in order to generate the observed urinary metabolites, K acid I and K acid II, which are excreted primarily as glucuronide conjugates. Synthetic standards of K acid I, K acid II, and a putative intermediate metabolite, menaquinone (MK)1 ω-COOH, were used to develop and optimize a new atmospheric pressure negative chemical ionization LC-MS/MS assay for the quantitation of these compounds in urine from untreated individuals and subjects treated with a high dose VK supplement. VK catabolites were extracted from urine, deconjugated, and converted to their methyl ester derivatives using previously reported methodology. The assay showed a high degree of sensitivity, with limits of detection below 10–50 fmol of metabolite per milliliter of urine, as well as an inter-assay precision of 8–12%. Metabolite standards provided unambiguous evidence for MK1 ω-COOH as a new human urinary metabolite of VK. This assay provides a minimally invasive, highly sensitive, and specific alternative for monitoring VK status in humans.

Background: Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder with a prevalence of 1:3000 births and a wide variety of clinical manifestations. Cutaneous neurofibromas (cNF) are among the most common visible manifestations of NF1 and present a major clinical burden for patients. NF1 patients with cNF often report decreased quality of life, emotional well-being and physical comfort. Developing effective medical therapies for cNF has been identified as a priority for the majority of adults with NF1. Methods: The study was an open, controlled and prospective proof-of-concept clinical trial. The topical treatment consisted of two steps: cNF microporation using a laser device followed by topical application of one drop of diclofenac 25 mg/mL on the surface of the cNF (T neurofibroma = treatment) or physiological saline (C neurofibroma = control) and reapplied twice daily for 3 days. Neurofibroma assessments included visual and dermatoscopy observations noting color and presence of necrosis, presence of flaccidity, measurements in two dimensions, photographs, and histopathology after excision. The primary efficacy variable was the presence of tissue necrosis. The primary safety variable was the occurrence of treatment-related adverse events. Results: Six patients were included in the study. The treatment resulted in transitory topical changes (healing of the microporation grid with formation of scintillating tissue layer, hyperemia and desquamation), with no statistically significant variation in the dimensions of the T and C neurofibromas in relation to pretreatment measurements. There was no necrosis in the T or C neurofibromas. In the histopathological analysis, there was no significant difference in the distribution of chronic (lymphocytic) inflammatory infiltrate in the papillary reticular dermis (subepithelial), type of infiltrate (diffuse, perivascular, or both), presence of fibrosis, and presence of atrophy among the T and C neurofibromas. No adverse events attributable to the use of diclofenac were reported during the treatment period. Conclusions: Treatment did not result in significant alterations in terms of presence of tissue necrosis, size, or histopathological features in the T neurofibromas or in comparison to the C neurofibromas. Topical diclofenac with laser microporation was well-tolerated, with no adverse events attributable to diclofenac reported. Whether these observations are due to minimal systemic and neurofibroma exposure remain to be explored in dosage studies with larger patient groups. Trial registration: ClinicalTrials.gov (NCT03090971) retrospectively registered March 27, 2017.

The necessity and functional significance of neurotransmitter co-transmission remains unclear. The glutamatergic ‘KNDy’ neurons co-express kisspeptin, neurokinin B (NKB), and dynorphin and exhibit a highly stereotyped synchronized behavior that reads out to the gonadotropin-releasing hormone (GnRH) neuron dendrons to drive episodic hormone secretion. Using expansion microscopy, we show that KNDy neurons make abundant close, non-synaptic appositions with the GnRH neuron dendron. Electrophysiology and confocal GCaMP6 imaging demonstrated that, despite all three neuropeptides being released from KNDy terminals, only kisspeptin was able to activate the GnRH neuron dendron. Mice with a selective deletion of kisspeptin from KNDy neurons failed to exhibit pulsatile hormone secretion but maintained synchronized episodic KNDy neuron behavior that is thought to depend on recurrent NKB and dynorphin transmission. This indicates that KNDy neurons drive episodic hormone secretion through highly redundant neuropeptide co-transmission orchestrated by differential post-synaptic neuropeptide receptor expression at the GnRH neuron dendron and KNDy neuron.

Increasing use of the botanical kratom to self-manage opioid withdrawal and pain has led to increased kratom-linked overdose deaths. Despite these serious safety concerns, rigorous fundamental pharmacokinetic knowledge of kratom in humans remains lacking. We assessed the pharmacokinetics of a single low dose (2 g) of a well-characterized kratom product administered orally to six healthy participants. Median concentration-time profiles for the kratom alkaloids examined were best described by a two-compartment model with central elimination. Pronounced pharmacokinetic differences between alkaloids with the 3S configuration (mitragynine, speciogynine, paynantheine) and alkaloids with the 3R configuration (mitraciliatine, speciociliatine, isopaynantheine) were attributed to differences in apparent intercompartmental distribution clearance, volumes of distribution, and clearance. Based on noncompartmental analysis of individual concentration-time profiles, the 3S alkaloids exhibited a shorter median time to maximum concentration (1–2 vs. 2.5–4.5 h), lower area under the plasma concentration-time curve (430–490 vs. 794–5120 nM × h), longer terminal half-life (24–45 vs. ~12–18 h), and higher apparent volume of distribution during the terminal phase (960–12,700 vs. ~46–130 L) compared to the 3R alkaloids. Follow-up mechanistic in vitro studies suggested differential hepatic/intestinal metabolism, plasma protein binding, blood-to-plasma partitioning, and/or distribution coefficients may explain the pharmacokinetic differences between the two alkaloid types. This first comprehensive pharmacokinetic characterization of kratom alkaloids in humans provides the foundation for further research to establish safety and effectiveness of this emerging botanical product.

Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR’s pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.

Preserving the ability to maintain an active lifestyle is a major concern in the reconstruction of the knee in young patients. for the healthy individual who desires to maintain a relatively active lifestyle, fresh osteochondral allografts may serve as an alternative to total joint reconstruction. The use of fresh allografts is primarily indicated in the patient suffering from a traumatic loss of articular seg- ments, who is too young or active for arthroplasty. In addition, fresh osteochon- dral allografts have a number of advantages over arthroplasty such as providing surgeons with a source of large grafts that can be ftted to replace osteochondral defects and cover the majority or entirety of articular surfaces without any donor site morbidity. In this case, a young, active patient lost a 7 x 8 cm portion of their distal femur, including a large portion of the articulating surface. Using a fresh os- teochondral allograft, harvested within 24 hours of donor death, a segment was ftted to match bony apposition, articular congruity, and congruity with the femoral notch and affxed with four partially threaded cancellous screws. Joint function was restored with the allograft in place, allowing the patient to delay the need for a total joint replacement.

Introduction: Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant genetic disorders. Some clinical manifestations are present at birth, while some develop during childhood, and others can occur at any age. Given the early age at which patients develop clinical features, diagnosis is often made during childhood. The most prevalent features of NF1 are café au lait spots, dermal and plexiform neurofibromas, and learning disability. A variety of skeletal problems may be seen in NF1, including scoliosis, short stature, and pseudoarthrosis. Reduced skeletal bone mass has been documented to be a common phenomenon in children and adults with NF1. Decreased serum 25-hydroxyvitamin D (vitamin D) levels have been noted in adults and children with NF1 and have been reported to be inversely correlated with the number of dermal neurofibromas in adults. However, the actual correlation of vitamin D level to bone density and dermal neurofibroma number in children with NF1 remains unclear. Objectives: The primary objective of this study was to evaluate vitamin D levels among children and adolescents with NF1. The secondary objective was to describe the levels of vitamin D among children and adolescents with NF1, to verify in which age group there is a higher frequency of vitamin D alterations, and to explore vitamin D level correlations between age, gender, sun exposure, number of neurofibromas, and number of plexiform neurofibromas. Methods: This was an observational, cross-sectional, hospital-based study. We obtained a convenience sample of individuals with confirmed diagnosis of NF1 from patients attending the Medical Genetics Service of the IPPMG-UFRJ and Santa Casa de Misericórdia of Rio de Janeiro over a 24-month period. We evaluated vitamin D levels in blood samples of patients with NF1 by a chemiluminescent immunoassay method, and we correlated the results with gender, age, number of neurofibromas, number of plexiform neurofibromas, and satisfactory sun exposure. Results: Of the 55 patients, 28 (50.9%) were female and 27 (49.1%) were male. Patient ages ranged from a minimum of 1.2 to a maximum of 19.6 years (mean age 10.95 years) and the median was 11.11 years. Median and mean body mass index (BMI; z score) were -0.09 (minimum value -1.63 and maximum of 4.62) and 0.16, respectively. The mean value of vitamin D was 30.82 ng/mL (±12.31) and the median was 29 ng/mL (minimum value of 10.40 ng/mL and maximum of 79.19 ng/mL). Conclusions: The levels of vitamin D did not differ according to gender, age group, or the presence or number of cutaneous neurofibromas. Among patients with adequate sun exposure, there was a higher incidence of sufficient serum vitamin D levels. Patients with cutaneous neurofibromas in the 0 to 11 age group had a greater tendency to vitamin D sufficiency in relation to patients aged 11 to 19 years.

The gonadotropin-releasing hormone (GnRH) neurons exhibit pulse and surge modes of activity to control fertility. They also exhibit an unusual bipolar morphology comprised of a classical soma-proximal dendritic zone and an elongated secretory process that can operate as both a dendrite and an axon, termed a ‘dendron’. We show using expansion microscopy that the highest density of synaptic inputs to a GnRH neuron exists at its distal dendron. In vivo, selective chemogenetic inhibition of the GnRH neuron distal dendron abolishes the luteinizing hormone (LH) surge and markedly dampens LH pulses. In contrast, inhibitory chemogenetic and optogenetic strategies targeting the GnRH neuron soma-proximal dendritic zone abolish the LH surge but have no effect upon LH pulsatility. These observations indicate that electrical activity at the soma-proximal dendrites of the GnRH neuron is only essential for the LH surge while the distal dendron represents an autonomous zone where synaptic integration drives pulsatile GnRH secretion.

mTORopathies are a heterogeneous group of neurological disorders characterized by malformations of cortical development (MCD), enhanced cellular mechanistic target of rapamycin (mTOR) signaling, and epilepsy that results from mutations in mTOR pathway regulatory genes. Homozygous mutations (del exon 9–13) in the pseudokinase STE20-related kinase adaptor alpha (STRAD-α; STRADA), an mTOR modulator, are associated with Pretzel Syndrome (PS), a neurodevelopmental disorder within the Old Order Mennonite Community characterized by megalencephaly, intellectual disability, and intractable epilepsy. To study the cellular mechanisms of STRADA loss, we generated CRISPR-edited Strada mouse N2a cells, a germline mouse Strada knockout (KO−/−) strain, and induced pluripotent stem cell (iPSC)-derived neurons from PS individuals harboring the STRADA founder mutation. Strada KO in vitro leads to enhanced mTOR signaling and iPSC-derived neurons from PS individuals exhibit enhanced cell size and mTOR signaling activation, as well as subtle alterations in electrical firing properties e.g., increased input resistance, a more depolarized resting membrane potential, and decreased threshold for action potential (AP) generation. Strada−/− mice exhibit high rates of perinatal mortality and out of more than 100 litters yielding both WT and heterozygous pups, only eight Strada−/− animals survived past P5. Strada−/− mice are hypotonic and tremulous. Histopathological examination (n = 5 mice) revealed normal gross brain organization and lamination but all had ventriculomegaly. Ectopic neurons were seen in all five Strada−/− brains within the subcortical white matter mirroring what is observed in human PS brain tissue. These distinct experimental platforms demonstrate that STRADA modulates mTOR signaling and is a key regulator of cell size, neuronal excitability, and cortical lamination.

Whereas recent clinical studies report metastatic melanoma survival rates high as 30-50%, many tumors remain nonresponsive or become resistant to current therapeutic strategies. Analyses of The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) data set suggests that a significant fraction of melanomas potentially harbor gain-of-function mutations in the gene that encodes for the ErbB4 receptor tyrosine kinase. In this work, a drug discovery strategy was developed that is based on the observation that the Q43L mutant of the naturally occurring ErbB4 agonist Neuregulin-2beta (NRG2β) functions as a partial agonist at ErbB4. NRG2β/Q43L stimulates tyrosine phosphorylation, fails to stimulate ErbB4-dependent cell proliferation, and inhibits agonist-induced ErbB4-dependent cell proliferation. Compounds that exhibit these characteristics likely function as ErbB4 partial agonists, and as such hold promise as therapies for ErbB4-dependent melanomas. Consequently, three highly sensitive and reproducible (Z' > 0.5) screening assays were developed and deployed for the identification of small-molecule ErbB4 partial agonists. Six compounds were identified that stimulate ErbB4 phosphorylation, fail to stimulate ErbB4-dependent cell proliferation, and appear to selectively inhibit ErbB4-dependent cell proliferation. Whereas further characterization is needed to evaluate the full therapeutic potential of these molecules, this drug discovery platform establishes reliable and scalable approaches for the discovery of ErbB4 inhibitors.

Mutations in DEPDC5 and NPRL3 subunits of GATOR1, a modulator of mechanistic target of rapamycin (mTOR), are linked to malformations of cortical development (MCD). Brain specimens from these individuals reveal abnormal cortical lamination, altered cell morphology, and hyperphosphorylation of ribosomal S6 protein (PS6), a marker for mTOR activation. While numerous studies have examined GATOR1 subunit function in non-neuronal cell lines, few have directly assessed loss of GATOR1 subunit function in neuronal cell types. We hypothesized that DEPDC5 or NPRL3 shRNA-mediated knockdown (DEPDC5/NPRL3 KD) leads to inappropriate functional activation of mTOR and mTOR-dependent alterations in neuronal morphology.Neuronal size was determined in human specimens harboring DEPDC5 or NPRL3 mutations resected for epilepsy treatment. DEPDC5/NPRL3 KD effects on cell size, filopodial extension, subcellular mTOR complex 1 (mTORC1) localization, and mTORC1 activation during nutrient deprivation were assayed in mouse neuroblastoma cells (N2aC) and mouse subventricular zone derived neural progenitor cells (mNPCs). mTORC1-dependent effects of DEPDC5/NPRL3 KD were determined using the mTOR inhibitor rapamycin. Changes in mTOR subcellular localization and mTORC1 pathway activation following DEPDC5/NPRL3 KD were determined by examining the proximity of mTOR to the lysosomal surface during amino acid starvation.Neurons exhibiting PS6 immunoreactivity (Ser 235/236) in human specimens were 1.5× larger than neurons in post-mortem control samples. DEPDC5/NPRL3 KD caused mTORC1, but not mTORC2, hyperactivation, soma enlargement, and increased filopodia in N2aC and mNPCs compared with wildtype cells. DEPDC5/NPRL3 KD led to inappropriate mTOR localization at the lysosome along with constitutive mTOR activation following amino acid deprivation. DEPDC5/NPRL3 KD effects on morphology and functional mTOR activation were reversed by rapamycin. mTOR-dependent effects of DEPDC5/NPRL3 KD on morphology and subcellular localization of mTOR in neurons suggests that loss-of-function in GATOR1 subunits may play a role in MCD formation during fetal brain development.

Abstract The roles of kisspeptin signaling outside the hypothalamus in the brain are unknown. We examined here the impact of Kiss1r-deletion on hippocampus-related behaviors of anxiety and spatial learning in adult male mice using two mouse models. In the first, global Kiss1r-null and control mice were gonadectomized (GDX KISS1R-KO). In the second, KISS1R signalling was rescued selectively in gonadotropin-releasing hormone neurons to generate Kiss1r-null mice with normal testosterone levels (intact KISS1R-KO). Intact KISS1R-KO rescue mice were found to spend twice as much time in the open arms of the elevated plus maze (EPM) compared to controls (P

The mechanisms responsible for stimulating biogeochemical activity in the hyporheic corridor (HC) are poorly understood. Here, the authors find that previously unrecognized thermodynamic mechanisms regulated by groundwater-river water mixing may strongly influence HC biogeochemical and microbial dynamics.

Education systems and institutions, often historically considered to be resolute, slow-moving entities transformed virtually overnight during the earlier stages of the COVID-19 pandemic, demonstrating nimbleness in adversity. This paper describes the first-hand experiences of teaching staff and students from a UK university which pivoted to emergency remote teaching for a core second-year module in engineering, focused on entrepreneurship. A range of methods are used including self-reflection, summative, formative, and focus-group student feedback. The paper provides an insight for readers who may be interested in the practical challenges associated with moving from an academic module typically delivered in a face-to-face learning environment accommodating a large student cohort (n = 177), to one that exists entirely in the digital domain. Our results show learning outcomes were fully met despite stark differences in quality of learning environments amongst students. Students reported benefits to remote learning because it offers a blended approach of both asynchronous content and synchronous sessions, with the latter enhancing engagement and providing structure to working weeks. Issues of presence emerged amongst group work: whilst it might be easier to confront some individuals for lack of contribution, it is also easier for those individuals to disengage. There was widespread support for the Microsoft Teams platform amongst students and staff but the former group reported this lacked a social environment in which relationships amongst team members could be nurtured informally, such as was experienced via social media.

Empirically quantifying tidally-influenced river discharge is typically laborious, expensive, and subject to more uncertainty than estimation of upstream river discharge. The tidal stage-discharge relationship is not monotonic nor necessarily single-valued, so conventional stage-based river rating curves fail in the tidal zone. Herein, we propose an expanded rating curve method incorporating stage-rate-of-change to estimate river discharge under tidal influences across progressive, mixed, and standing waves. This simple and inexpensive method requires (1) stage from a pressure transducer, (2) flow direction from a tilt current meter, and (3) a series of ADP surveys at different flow rates for model calibration. The method was validated using excerpts from 12 tidal USGS gauging stations during baseflow conditions. USGS gauging stations model discharge using a different more complex and expensive method. Comparison of new and previous models resulted in good R2 correlations (min 0.62, mean 0.87 with S.D. 0.10, max 0.97). The method for modeling tidally-influenced discharge during baseflow conditions was applied de novo to eight intertidal stations in the Mission and Aransas Rivers, Texas, USA. In these same rivers, the model was further expanded to identify and estimate tidally-influenced stormflow discharges. The Mission and Aransas examples illustrated the potential scientific and management utility of the applied tidal rating curve method for isolating transient tidal influences and quantifying baseflow and storm discharges to sensitive coastal waters.

We present a curriculum description, an initial student outcome investigation, and sample scientific results for a representative Course-Based Undergraduate Research Experience (CURE) that is part of the “Undergraduates Phenotyping Arabidopsis Knockouts” (unPAK) network. CUREs in the unPAK network characterize quantitative phenotypes of the model plant Arabidopsis from across environments to uncover connections between genotype and phenotype. Students in unPAK CUREs grow plants in a replicated block design and make quantitative measurements throughout the semester. This CURE enables students to answer plant science questions that draw from fields such as environmental science, genetics, ecology, and evolution. Findings indicate that this experience provides students with opportunities to make relevant scientific discoveries. Eighty percent of student datasets produced from the CURE met criteria for inclusion in the project database, indicative of student learning in data collection and analysis of quantitative plant traits. Student datasets uncovered novel effects of mutation on plant form. In addition, students’ science self-efficacy increased as a result of course participation, and faculty feedback on course implementation was positive. We present unPAK as a new network that supports CUREs and research experiences focused on collecting biological data made publicly available to the scientific community. The unPAK CUREs can be tailored to address instructor interests or pedagogical needs while involving students in research investigating quantitative plant phenotypes.

Objective: Puberty is a key developmental phenomenon highly sensitive to metabolic modulation. Worrying trends of changes in the timing of puberty have been reported in humans. These might be linked to the escalating prevalence of childhood obesity and could have deleterious impacts on later (cardio-metabolic) health, but their underlying mechanisms remain unsolved. The neuropeptide α-MSH, made by POMC neurons, plays a key role in energy homeostasis by mediating the actions of leptin and likely participates in the control of reproduction. However, its role in the metabolic regulation of puberty and interplay with kisspeptin, an essential puberty-regulating neuropeptide encoded by Kiss1, remain largely unknown. We aim here to unveil the potential contribution of central α-MSH signaling in the metabolic control of puberty by addressing its role in mediating the pubertal effects of leptin and its potential interaction with kisspeptin. Methods: Using wild type and genetically modified rodent models, we implemented pharmacological studies, expression analyses, electrophysiological recordings, and virogenetic approaches involving DREADD technology to selectively inhibit Kiss1 neurons, in order to interrogate the physiological role of a putative leptin→α-MSH→kisspeptin pathway in the metabolic control of puberty. Results: Stimulation of central α-MSH signaling robustly activated the reproductive axis in pubertal rats, whereas chronic inhibition of melanocortin receptors MC3/4R, delayed puberty, and prevented the permissive effect of leptin on puberty onset. Central blockade of MC3/4R or genetic elimination of kisspeptin receptors from POMC neurons did not affect kisspeptin effects. Conversely, congenital ablation of kisspeptin receptors or inducible, DREADD-mediated inhibition of arcuate nucleus (ARC) Kiss1 neurons resulted in markedly attenuated gonadotropic responses to MC3/4R activation. Furthermore, close appositions were observed between POMC fibers and ARC Kiss1 neurons while blockade of α-MSH signaling suppressed Kiss1 expression in the ARC of pubertal rats. Conclusions: Our physiological, virogenetic, and functional genomic studies document a novel α-MSH→kisspeptin→GnRH neuronal signaling pathway involved in transmitting the permissive effects of leptin on pubertal maturation, which is relevant for the metabolic (and, eventually, pharmacological) regulation of puberty onset. Keywords: α-MSH, Kisspeptin, Leptin, Metabolism, DREADDs, Puberty

Kisspeptin signaling at the gonadotropin-releasing hormone (GnRH) neuron is now relatively well characterized and established as being critical for the neural control of fertility. However, kisspeptin fibers and the kisspeptin receptor (KISS1R) are detected throughout the brain suggesting that kisspeptin is involved in regulating the activity of multiple neuronal circuits. We provide here a review of kisspeptin actions on neuronal populations throughout the brain including the magnocellular oxytocin and vasopressin neurons, and cells within the arcuate nucleus, hippocampus, and amygdala. The actions of kisspeptin in these brain regions are compared to its effects upon GnRH neurons. Two major themes arise from this analysis. First, it is apparent that kisspeptin signaling through KISS1R at the GnRH neuron is a unique, extremely potent form or neurotransmission whereas kisspeptin actions through KISS1R in other brain regions exhibit neuromodulatory actions typical of other neuropeptides. Second, it is becoming increasingly likely that kisspeptin acts as a neuromodulator not only through KISS1R but also through other RFamide receptors such as the neuropeptide FF receptors (NPFFRs). We suggest likely locations of kisspeptin signaling through NPFFRs but note that only limited tools are presently available for examining kisspeptin cross-signaling within the RFamide family of neuropeptides.

Groundwater-surface water mixing zones link critical ecosystem domains, but attendant microbe-biogeochemistry-hydrology interactions are poorly known. Here, the authors show that groundwater-surface water mixing stimulates respiration, alters carbon composition, and shifts the ecology from stochastic to deterministic.