An integrated multi-omics approach allowed ultra-rapid diagnosis of a deep intronic pathogenic variant in PDHX and precision treatment in a neonate critically ill with lactic acidosis.

The diagnosis of mitochondrial disorders is complex. Rapid whole genome sequencing is a first line test for critically ill neonates and infants allowing rapid diagnosis and treatment. Standard genomic technology and bioinformatic pipelines still have an incomplete diagnostic yield requiring complementary approaches. There are currently limited options for rapid additional tests to continue a diagnostic work-up after a negative rapid whole-genome sequencing result, reflecting a gap in clinical practice. Multi-modal integrative diagnostic approaches derived from systems biology including proteomics and transcriptomics show promise in suspected mitochondrial disorders. In this article, we report the case of a neonate who presented with severe lactic acidosis on the second day of life, for whom an initial report of ultra-rapid genome sequencing was negative. The patient was started on dichloroacetate as an emergency investigational new drug (eIND), with a sharp decline in lactic acid levels and clinical stabilization. A proteomics-based approach identified a complete absence of PDHX protein, leading to a re-review of the genome data for the PDHX gene in which a homozygous deep intronic pathogenic variant was identified. Subsequent testing in the following months confirmed the diagnosis with deficient pyruvate dehydrogenase enzyme activity, reduced protein levels of E3-binding protein, and confirmed by mRNA sequencing to lead to the inclusion of a cryptic exon and a premature stop codon. This case highlights the power of rapid proteomics in guiding genomic analysis. It also shows a promising role for dichloroacetate treatment in controlling lactic acidosis related to PDHX-related pyruvate dehydrogenase complex deficiency.

CNVoyant a machine learning framework for accurate and explainable copy number variant classification.

The precise classification of copy number variants (CNVs) presents a significant challenge in genomic medicine, primarily due to the complex nature of CNVs and their diverse impact on rare genetic diseases (RGDs). This complexity is compounded by the limitations of existing methods in accurately distinguishing between benign, uncertain, and pathogenic CNVs. Addressing this gap, we introduce CNVoyant, a machine learning-based multi-class framework designed to enhance the clinical significance classification of CNVs. Trained on a comprehensive dataset of 52,176 ClinVar entries across pathogenic, uncertain, and benign classifications, CNVoyant incorporates a broad spectrum of genomic features, including genome position, disease-gene annotations, dosage sensitivity, and conservation scores. Models to predict the clinical significance of copy number gains and losses were trained independently. Final models were selected after testing 29 machine learning architectures and 10,000 hyperparameter combinations each for deletions and duplications via fivefold cross-validation. We validate the performance of CNVoyant by leveraging a comprehensive set of 21,574 CNVs from the DECIPHER database, a highly regarded resource known for its extensive catalog of chromosomal imbalances linked to clinical outcomes. Compared to alternative approaches, CNVoyant shows marked improvements in precision-recall and ROC AUC metrics for binary pathogenic classifications while going one step further, offering multi-classification of clinical significance and corresponding SHAP explainability plots. Additionally, when provided germline CNV calls from real-world RGD cases with diagnostic CNV(s), CNVoyant correctly classified all diagnostic CNVs as having pathogenic significance with high confidence. This large-scale validation demonstrates CNVoyant's superior accuracy and underscores its potential to aid genomic researchers and clinical geneticists in interpreting the clinical implications of real CNVs.

Ultrastructural, metabolic and genetic characteristics of determinants facilitating the acquisition of macrolide resistance by Streptococcus pneumoniae.

AIMS: To investigate the molecular events associated with acquiring macrolide resistance genes [mefE/mel (Mega) or ermB] in Streptococcus pneumoniae (Spn) during nasopharyngeal colonization. METHODS AND RESULTS: Genomic analysis of 128 macrolide-resistant Spn isolates revealed recombination events in genes of the conjugation apparatus, or the competence system, in strains carrying Tn916-related elements. Studies using confocal and electron microscopy demonstrated that during the transfer of Tn916-related elements in nasopharyngeal cell biofilms, pneumococcal strains formed clusters facilitating their acquisition of resistance determinants at a high recombination frequency (rF). Remarkably, these aggregates comprise both encapsulated and nonencapsulated pneumococci that span extracellular and intracellular compartments. rF assessments showed similar rates regardless Mega was associated with large integrative and conjugative elements (ICEs) (>23 kb) or not (∼5.4 kb). The rF for Mega Class IV(c) insertion region (∼53 kb) was three orders of magnitude higher than the transformation of the capsule locus. Metabolomics studies of the microenvironment created by colonization of human nasopharyngeal cells revealed a link between the acquisition of ICEs and the pathways involving nicotinic acid and sucrose. CONCLUSIONS: Pneumococcal clusters, both extracellular and intracellular, facilitate macrolide resistance acquisition, and ICEs were acquired at a higher frequency than the capsule locus. Metabolic changes could serve as intervention targets.

NeoGx: Machine-Recommended Rapid Genome Sequencing for Neonates.

Background: Genetic disease is common in the Level IV Neonatal Intensive Care Unit (NICU), but neonatology providers are not always able to identify the need for genetic evaluation. We trained a machine learning (ML) algorithm to predict the need for genetic testing within the first 18 months of life using health record phenotypes. Methods: For a decade of NICU patients, we extracted Human Phenotype Ontology (HPO) terms from clinical text with Natural Language Processing tools. Considering multiple feature sets, classifier architectures, and hyperparameters, we selected a classifier and made predictions on a validation cohort of 2,241 Level IV NICU admits born 2020-2021. Results: Our classifier had ROC AUC of 0.87 and PR AUC of 0.73 when making predictions during the first week in the Level IV NICU. We simulated testing policies under which subjects begin testing at the time of first ML prediction, estimating diagnostic odyssey length both with and without the additional benefit of pursuing rGS at this time. Just by using ML to accelerate initial genetic testing (without changing the tests ordered), the median time to first genetic test dropped from 10 days to 1 day, and the number of diagnostic odysseys resolved within 14 days of NICU admission increased by a factor of 1.8. By additionally requiring rGS at the time of positive ML prediction, the number of diagnostic odysseys resolved within 14 days was 3.8 times higher than the baseline. Conclusions: ML predictions of genetic testing need, together with the application of the right rapid testing modality, can help providers accelerate genetics evaluation and bring about earlier and better outcomes for patients.

Efficacy of Eating Disorder Focused Family Therapy for Adolescents With Anorexia Nervosa: A Systematic Review and Meta-Analysis.

OBJECTIVE: To systematically review and evaluate the efficacy of eating disorder focused family therapy (FT-ED) in comparison to all other forms of psychotherapy for children and adolescents with anorexia nervosa. A secondary aim is to assess the relative efficacy of different variations of FT-ED (e.g., shorter vs. longer dose, parent-focused). METHODS: A search with relevant terms was systematically conducted on four databases. Twenty-three publications across 18 randomized controlled trials met inclusion criteria. Outcomes of interest included variables related to weight, eating psychopathology, and remission status. Study quality was assessed, and data were extracted by two independent researchers. RESULTS: Adolescents receiving FT-ED gained significantly more weight by the end of treatment in comparison to those receiving individual psychotherapy. FT-ED that was delivered just to parents or to parents and child separately offered preferable weight outcomes and rates of recovery at the end of treatment in comparison to conjoint FT-ED. No other outcomes tested in the meta-analysis were statistically significant at the end of treatment or follow-up. DISCUSSION: Currently available data suggest the use of FT-ED in its conjoint or separated/parent focused format is the best outpatient treatment option for adolescents with anorexia nervosa when immediate weight gain is paramount. The variability of outcome measurement, including the tools used and timepoints chosen, limit comparison among no more than a handful of studies. The field would benefit from the standardization of measurement and reporting guidelines for future clinical trials. TRIAL REGISTRATION: PROSPERO number: CRD42023396263.

OBJETIVO: Revisar y evaluar sistemáticamente la eficacia de la terapia familiar centrada en el trastorno de conducta alimentaria (TF­TCA; FT­ED por sus siglas en inglés) en comparación con todas las demás formas de psicoterapia para niños y adolescentes que padecen anorexia nerviosa. Un objetivo secundario es evaluar la eficacia relativa de diferentes variaciones de la TF­TCA (por ejemplo, dosis más corta vs. más larga, centrada en los padres). MÉTODOS: Se realizó una búsqueda sistemática con términos relevantes en cuatro bases de datos. Veintitrés publicaciones de 18 ensayos controlados aleatorios cumplieron con los criterios de inclusión. Los resultados de interés incluyeron variables relacionadas con el peso, la psicopatología alimentaria y el estado de remisión. La calidad del estudio fue evaluada y los datos fueron extraídos por dos investigadores independientes. RESULTADOS: Los adolescentes que recibieron TF­TCA ganaron significativamente más peso al final del tratamiento en comparación con aquellos que recibieron psicoterapia individual. La TF­TCA que se administró solo a los padres o a padres e hijos por separado ofreció mejores resultados en el peso y tasas de recuperación al final del tratamiento en comparación con la TF­TCA conjunta. Ningún otro resultado probado en el metaanálisis fue estadísticamente significativo al final del tratamiento o durante el seguimiento. DISCUSIÓN: Los datos disponibles actualmente sugieren que el uso de la TF­TCA en su formato conjunto o separado/centrado en los padres es la mejor opción de tratamiento ambulatorio para adolescentes que padecen anorexia nerviosa cuando la ganancia de peso inmediata es primordial. La variabilidad en la medición de los resultados, incluyendo las herramientas utilizadas y los puntos temporales elegidos, limita la comparación entre no más de un puñado de estudios. El campo se beneficiaría de la estandarización de la medición y las directrices de reporte para futuros ensayos clínicos.

Transient gas-induced differential refractive index effects in as-drawn hollow core optical fibers.

When a hollow core fiber is drawn, the core and cladding holes within the internal cane geometry are pressurized with an inert gas to enable precise control over the internal microstructure of the fiber and counteract surface tension forces. Primarily by considering the temperature drop as the fiber passes through the furnace and the geometrical transformation of the internal microstructure from preform-to-fiber, we recently established that the gas pressure within the final 'as-drawn' fiber is substantially below atmospheric pressure. We have also established that slight changes in the gas refractive index within the core and surrounding cladding holes induced by changes in gas pressure are sufficient to significantly affect both the modality and loss of the fiber. Here we demonstrate, through both simulations and experimental measurements, that the combination of these effects leads to transient changes in the fiber's attenuation when the fibers are opened to atmosphere post-fabrication. It is important to account for this phenomenon for accurate fiber characterization, particularly when long lengths of fiber are drawn where it could take many weeks for every part of the internal microstructure to reach atmospheric pressure.

ACAD9 treatment with bezafibrate and nicotinamide riboside temporarily stabilizes cardiomyopathy and lactic acidosis.

Pathogenic ACAD9 variants cause complex I deficiency. Patients presenting in infancy unresponsive to riboflavin have high mortality. A six-month-old infant presented with riboflavin unresponsive lactic acidosis and life-threatening cardiomyopathy. Treatment with high dose bezafibrate and nicotinamide riboside resulted in marked clinical improvement including reduced lactate and NT-pro-brain type natriuretic peptide levels, with stabilized echocardiographic measures. After a long stable period, the child succumbed from cardiac failure with infection at 10.5 months. Therapy was well tolerated. Peak bezafibrate levels exceeded its EC50. The clinical improvement with this treatment illustrates its potential, but weak PPAR agonist activity of bezafibrate limited its efficacy.

Effect of Peanut Butter Intake on Sleep Health in Firefighters: A Randomized Controlled Trial.

Sleep is often impaired in firefighters due to the psychologically and physiologically intense nature of their work and working shift schedules. Peanut butter is affordable and a substantial source of monounsaturated fatty acids, which may aid sleep health. Thus, this study sought to determine if a daily serving of peanut butter consumed before bedtime for seven weeks altered sleep quality and quantity among full-time firefighters. Forty firefighters (peanut butter group = 20; control group = 20) participated in this eight-week randomized controlled trial. All participants completed a subjective questionnaire on mood, focus, and alertness twice daily and wore an Actigraph wristwatch to measure sleep variables, including latency, efficiency, time in bed, time asleep, wake after sleep onset, number of awakenings, and time spent awake. After a baseline week, the peanut butter group consumed two tablespoons of peanut butter two hours prior to bedtime for seven weeks. Compared to the control group, the peanut butter group did not demonstrate significant changes (p > 0.05) in sleep measures or subjective feelings of mood, focus, or alertness after consuming peanut butter for seven weeks. Therefore, peanut butter as a source of peanuts did not alter sleep quality or quantity in this group of firefighters.

CNVoyant: A Highly Performant and Explainable Multi-Classifier Machine Learning Approach for Determining the Clinical Significance of Copy Number Variants.

The precise classification of copy number variants (CNVs) presents a significant challenge in genomic medicine, primarily due to the complex nature of CNVs and their diverse impact on genetic disorders. This complexity is compounded by the limitations of existing methods in accurately distinguishing between benign, uncertain, and pathogenic CNVs. Addressing this gap, we introduce CNVoyant, a machine learning-based multi-class framework designed to enhance the clinical significance classification of CNVs. Trained on a comprehensive dataset of 52,176 ClinVar entries across pathogenic, uncertain, and benign classifications, CNVoyant incorporates a broad spectrum of genomic features, including genome position, disease-gene annotations, dosage sensitivity, and conservation scores. Models to predict the clinical significance of copy number gains and losses were trained independently. Final models were selected after testing 29 machine learning architectures and 10,000 hyperparameter combinations each for deletions and duplications via 5-fold cross-validation. We validate the performance of the CNVoyant by leveraging a comprehensive set of 21,574 CNVs from the DECIPHER database, a highly regarded resource known for its extensive catalog of chromosomal imbalances linked to clinical outcomes. Compared to alternative approaches, CNVoyant shows marked improvements in precision-recall and ROC AUC metrics for binary pathogenic classifications while going one step further, offering multi-classification of clinical significance and corresponding SHAP explainability plots. This large-scale validation demonstrates CNVoyant's superior accuracy and underscores its potential to aid genomic researchers and clinical geneticists in interpreting the clinical implications of real CNVs.

A Swine Burn Model for Investigating the Healing Process in Multiple Depth Burn Wounds.

Burn wound healing is a complex and long process. Despite extensive experience, plastic surgeons and specialized teams in burn centers still face significant challenges. Among these challenges, the extent of the burned soft tissue can evolve in the early phase, creating a delicate balance between conservative treatments and necrosing tissue removal. Thermal burns are the most common type, and burn depth varies depending on multiple parameters, such as temperature and exposure time. Burn depth also varies in time, and the secondary aggravation of the "shadow zone" remains a poorly understood phenomenon. In response to these challenges, several innovative treatments have been studied, and more are in the early development phase. Nanoparticles in modern wound dressings and artificial skin are examples of these modern therapies still under evaluation. Taken together, both burn diagnosis and burn treatments need substantial advancements, and research teams need a reliable and relevant model to test new tools and therapies. Among animal models, swine are the most relevant because of their strong similarities in skin structure with humans. More specifically, Yucatan minipigs show interesting features such as melanin pigmentation and slow growth, allowing for studying high phototypes and long-term healing. This article aims to describe a reliable and reproducible protocol to study multi-depth burn wounds in Yucatan minipigs, enabling long-term follow-up and providing a relevant model for diagnosis and therapeutic studies.

Systems level analysis of time and stimuli specific signaling through PKA.

It is well known that eukaryotic cells create gradients of cAMP across space and time to regulate the cAMP dependent protein kinase (PKA) and, in turn, growth and metabolism. However, it is unclear how PKA responds to different concentrations of cAMP. Here, to address this question, we examine PKA signaling in Saccharomyces cerevisiae in different conditions, timepoints, and concentrations of the chemical inhibitor 1-NM-PP1, using phosphoproteomics. These experiments show that there are numerous proteins that are only phosphorylated when cAMP and PKA activity are at/near their maximum level, while other proteins are phosphorylated even when cAMP levels and PKA activity are low. The data also show that PKA drives cells into distinct growth states by acting on proteins with different thresholds for phosphorylation in different conditions. Analysis of the sequences surrounding the 118 PKA-dependent phosphosites suggests that the phosphorylation thresholds are set, at least in part, by the affinity of PKA for each site.

Multifunction fluorescence open source in vivo/in vitro imaging system (openIVIS).

The widespread availability and diversity of open-source microcontrollers paired with off-the-shelf electronics and 3D printed technology has led to the creation of a wide range of low-cost scientific instruments, including microscopes, spectrometers, sensors, data loggers, and other tools that can be used for research, education, and experimentation. These devices can be used to explore a wide range of scientific topics, from biology and chemistry to physics and engineering. In this study, we designed and built a multifunction fluorescent open source in vivo/in vitro imaging system (openIVIS) system that integrates a Raspberry Pi with commercial cameras and LEDs with 3D printed structures combined with an acrylic housing. Our openIVIS provides three excitation wavelengths of 460 nm, 520 nm, and 630 nm integrated with Python control software to enable fluorescent measurements across the full visible light spectrum. To demonstrate the potential applications of our system, we tested its performance against a diverse set of experiments including laboratory assays (measuring fluorescent dyes, using optical nanosensors, and DNA gel electrophoresis) to potentially fieldable applications (plant and mineral imaging). We also tested the potential use for a high school biology environment by imaging small animals and tracking their development over the course of ten days. Our system demonstrated its ability to measure a wide dynamic range fluorescent response from millimolar to picomolar concentrations in the same sample while measuring responses across visible wavelengths. These results demonstrate the power and flexibility of open-source hardware and software and how it can be integrated with customizable manufacturing to create low-cost scientific instruments with a wide range of applications. Our study provides a promising model for the development of low-cost instruments that can be used in both research and education.

Presenilin-1 mutation position influences amyloidosis, small vessel disease, and dementia with disease stage.

INTRODUCTION: Amyloidosis, including cerebral amyloid angiopathy, and markers of small vessel disease (SVD) vary across dominantly inherited Alzheimer's disease (DIAD) presenilin-1 (PSEN1) mutation carriers. We investigated how mutation position relative to codon 200 (pre-/postcodon 200) influences these pathologic features and dementia at different stages. METHODS: Individuals from families with known PSEN1 mutations (n = 393) underwent neuroimaging and clinical assessments. We cross-sectionally evaluated regional Pittsburgh compound B-positron emission tomography uptake, magnetic resonance imaging markers of SVD (diffusion tensor imaging-based white matter injury, white matter hyperintensity volumes, and microhemorrhages), and cognition. RESULTS: Postcodon 200 carriers had lower amyloid burden in all regions but worse markers of SVD and worse Clinical Dementia Rating® scores compared to precodon 200 carriers as a function of estimated years to symptom onset. Markers of SVD partially mediated the mutation position effects on clinical measures. DISCUSSION: We demonstrated the genotypic variability behind spatiotemporal amyloidosis, SVD, and clinical presentation in DIAD, which may inform patient prognosis and clinical trials. HIGHLIGHTS: Mutation position influences Aß burden, SVD, and dementia. PSEN1 pre-200 group had stronger associations between Aß burden and disease stage. PSEN1 post-200 group had stronger associations between SVD markers and disease stage. PSEN1 post-200 group had worse dementia score than pre-200 in late disease stage. Diffusion tensor imaging-based SVD markers mediated mutation position effects on dementia in the late stage.

Cell cycle plasticity underlies fractional resistance to palbociclib in ER+/HER2- breast tumor cells.

The CDK4/6 inhibitor palbociclib blocks cell cycle progression in Estrogen receptor-positive, human epidermal growth factor 2 receptor-negative (ER+/HER2-) breast tumor cells. Despite the drug's success in improving patient outcomes, a small percentage of tumor cells continues to divide in the presence of palbociclib-a phenomenon we refer to as fractional resistance. It is critical to understand the cellular mechanisms underlying fractional resistance because the precise percentage of resistant cells in patient tissue is a strong predictor of clinical outcomes. Here, we hypothesize that fractional resistance arises from cell-to-cell differences in core cell cycle regulators that allow a subset of cells to escape CDK4/6 inhibitor therapy. We used multiplex, single-cell imaging to identify fractionally resistant cells in both cultured and primary breast tumor samples resected from patients. Resistant cells showed premature accumulation of multiple G1 regulators including E2F1, retinoblastoma protein, and CDK2, as well as enhanced sensitivity to pharmacological inhibition of CDK2 activity. Using trajectory inference approaches, we show how plasticity among cell cycle regulators gives rise to alternate cell cycle "paths" that allow individual tumor cells to escape palbociclib treatment. Understanding drivers of cell cycle plasticity, and how to eliminate resistant cell cycle paths, could lead to improved cancer therapies targeting fractionally resistant cells to improve patient outcomes.

Thoracic and Lumbar Spine Injury: Evidence-Based Diagnosis, Management, and Outcomes.

BACKGROUND: Traumatic thoracolumbar spine injuries are associated with significant morbidity and mortality. Targeted for non-spine specialist trauma surgeons, this systematic scoping review aimed to examine literature for up-to-date evidence on presentation, management, and outcomes of thoracolumbar spine injuries in adult trauma patients. METHODS: This review was reported using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses checklist. We searched four bibliographic databases: PubMed, EMBASE, Web of Science, and the Cochrane Library. Eligible studies included experimental, observational, and evidence-synthesis articles evaluating patients with thoracic, lumbar, or thoracolumbar spine injury, published in English between January 1, 2010 and January 31, 2021. Studies which focused on animals, cadavers, cohorts with N <30, and pediatric cohorts (age <18 years old), as well as case studies, abstracts, and commentaries were excluded. RESULTS: A total of 2501 studies were screened, of which 326 unique studies were fully text reviewed and twelve aspects of injury management were identified and discussed: injury patterns, determination of injury status and imaging options, considerations in management, and patient quality of life. We found: (1) imaging is a necessary diagnostic tool, (2) no consensus exists for preferred injury characterization scoring systems, (3) operative management should be considered for unstable fractures, decompression, and deformity, and (4) certain patients experience significant burden following injury. DISCUSSION: In this systematic scoping review, we present the most up-to-date information regarding the management of traumatic thoracolumbar spine injuries. This allows non-specialist trauma surgeons to become more familiar with thoracolumbar spine injuries in trauma patients and provides a framework for their management.

Evaluating newly approved drugs in combination regimens for multidrug-resistant tuberculosis with fluoroquinolone resistance (endTB-Q): study protocol for a multi-country randomized controlled trial.

BACKGROUND: Treatment for fluoroquinolone-resistant multidrug-resistant/rifampicin-resistant tuberculosis (pre-XDR TB) often lasts longer than treatment for less resistant strains, yields worse efficacy results, and causes substantial toxicity. The newer anti-tuberculosis drugs, bedaquiline and delamanid, and repurposed drugs clofazimine and linezolid, show great promise for combination in shorter, less-toxic, and effective regimens. To date, there has been no randomized, internally and concurrently controlled trial of a shorter, all-oral regimen comprising these newer and repurposed drugs sufficiently powered to produce results for pre-XDR TB patients. METHODS: endTB-Q is a phase III, multi-country, randomized, controlled, parallel, open-label clinical trial evaluating the efficacy and safety of a treatment strategy for patients with pre-XDR TB. Study participants are randomized 2:1 to experimental or control arms, respectively. The experimental arm contains bedaquiline, linezolid, clofazimine, and delamanid. The control comprises the contemporaneous WHO standard of care for pre-XDR TB. Experimental arm duration is determined by a composite of smear microscopy and chest radiographic imaging at baseline and re-evaluated at 6 months using sputum culture results: participants with less extensive disease receive 6 months and participants with more extensive disease receive 9 months of treatment. Randomization is stratified by country and by participant extent-of-TB-disease phenotype defined according to screening/baseline characteristics. Study participation lasts up to 104 weeks post randomization. The primary objective is to assess whether the efficacy of experimental regimens at 73 weeks is non-inferior to that of the control. A sample size of 324 participants across 2 arms affords at least 80% power to show the non-inferiority, with a one-sided alpha of 0.025 and a non-inferiority margin of 12%, against the control in both modified intention-to-treat and per-protocol populations. DISCUSSION: This internally controlled study of shortened treatment for pre-XDR TB will provide urgently needed data and evidence for clinical and policy decision-making around the treatment of pre-XDR TB with a four-drug, all-oral, shortened regimen. TRIAL REGISTRATION: ClinicalTrials.Gov NCT03896685. Registered on 1 April 2018; the record was last updated for study protocol version 4.3 on 17 March 2023.

The Molecular Logic of Gtr1/2 and Pib2 Dependent TORC1 Regulation in Budding Yeast.

The Target of Rapamycin kinase Complex I (TORC1) regulates cell growth and metabolism in eukaryotes. Previous studies have shown that, in Saccharomyces cerevisiae, nitrogen and amino acid signals activate TORC1 via the highly conserved small GTPases, Gtr1/2, and the phosphatidylinositol 3-phosphate binding protein, Pib2. However, it was unclear if/how Gtr1/2 and Pib2 cooperate to control TORC1. Here we report that this dual regulator system pushes TORC1 into three distinct signaling states: (i) a Gtr1/2 on, Pib2 on, rapid growth state in nutrient replete conditions; (ii) a Gtr1/2 off, Pib2 on, adaptive/slow growth state in poor-quality growth medium; and (iii) a Gtr1/2 off, Pib2 off, quiescent state in starvation conditions. We suggest that other signaling pathways work in a similar way, to drive a multi-level response via a single kinase, but the behavior has been overlooked since most studies follow signaling to a single reporter protein.

Tamoxifen Response at Single-Cell Resolution in Estrogen Receptor-Positive Primary Human Breast Tumors.

PURPOSE: In estrogen receptor-positive (ER+)/HER2- breast cancer, multiple measures of intratumor heterogeneity are associated with a worse response to endocrine therapy. We sought to develop a novel experimental model to measure heterogeneity in response to tamoxifen treatment in primary breast tumors. EXPERIMENTAL DESIGN: To investigate heterogeneity in response to treatment, we developed an operating room-to-laboratory pipeline for the collection of live normal breast specimens and human tumors immediately after surgical resection for processing into single-cell workflows for experimentation and genomic analyses. Live primary cell suspensions were treated ex vivo with tamoxifen (10 µmol/L) or control media for 12 hours, and single-cell RNA libraries were generated using the 10X Genomics droplet-based kit. RESULTS: In total, we obtained and processed normal breast tissue from two women undergoing reduction mammoplasty and tumor tissue from 10 women with ER+/HER2- invasive breast carcinoma. We demonstrate differences in tamoxifen response by cell type and identify distinctly responsive and resistant subpopulations within the malignant cell compartment of human tumors. Tamoxifen resistance signatures from resistant subpopulations predict poor outcomes in two large cohorts of ER+ breast cancer patients and are enriched in endocrine therapy-resistant tumors. CONCLUSIONS: This novel ex vivo model system now provides the foundation to define responsive and resistant subpopulations within heterogeneous human tumors, which can be used to develop precise single cell-based predictors of response to therapy and to identify genes and pathways driving therapeutic resistance.

Homicide in the context of psychosis: analysis of prior service utilisation and age at onset of illness and violence.

BACKGROUND: Public stigma and fear are heightened in cases of extreme violence perpetrated by persons with serious mental illness (SMI). Prevention efforts require understanding of illness patterns and treatment needs prior to these events unfolding. AIMS: To examine mental health service utilisation by persons who committed homicide and entered into forensic care, to investigate the adequacy of mental healthcare preceding these offences. METHOD: Forensic patients across two mental health hospitals in Ontario with an admitting offence of homicide between 2011 and 2021 were identified (n = 112). Sociodemographic, clinical and offence-related variables were coded from the health record and reports prepared for the forensic tribunal. RESULTS: Most patients (75.7%) had mental health contacts preceding the homicide, with 28.4% having a psychiatric in-patient admission in the year prior. For those with service contacts in the year preceding, 50.9% had had only sporadic contact and 70.7% were non-adherent with prescribed medications. Victims were commonly known to the individual (35.7%) and were often family members in care-providing roles (55.4%). Examination of age at onset of illness and offending patterns suggested that most persons admitted to forensic care for homicide act in the context of illness and exhibit a low frequency of pre-homicide offending. CONCLUSIONS: Many individuals admitted to forensic care for homicide have had inadequate mental healthcare leading up to this point. Effective responses to reduce and manage risk should encompass services that proactively address illness-related (e.g. earlier access and better maintenance in care) and criminogenic (e.g. substance use treatment, employment and psychosocial supports) domains.

A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations.

The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as an approximation for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays identified substitutions which conferred strong nirmatrelvir resistance and others that compromised activity. On the other hand, N142A and the P132H mutation, carried by the Omicron variant, caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.