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Abstract Reference genomes of cattle and sheep have lacked contiguous assemblies of the sex-determining Y chromosome. Here, we assemble complete and gapless telomere to telomere (T2T) Y chromosomes for these species. We find that the pseudo-autosomal regions are similar in length, but the total chromosome size is substantially different, with the cattle Y more than twice the length of the sheep Y. The length disparity is accounted for by expanded ampliconic region in cattle. The genic amplification in cattle contrasts with pseudogenization in sheep suggesting opposite evolutionary mechanisms since their divergence 19MYA. The centromeres also differ dramatically despite the close relationship between these species at the overall genome sequence level. These Y chromosomes have been added to the current reference assemblies in GenBank opening new opportunities for the study of evolution and variation while supporting efforts to improve sustainability in these important livestock species that generally use sire-driven genetic improvement strategies.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are widely used in patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2 negative (HER2−) advanced/metastatic breast cancer (ABC/MBC) in first line (1L), but little is known about their real-world use and clinical outcomes long-term, in Canada. This study used Pentavere’s previously validated artificial intelligence (AI) to extract real-world data on the treatment patterns and outcomes of patients receiving CDK4/6i+endocrine therapy (ET) for HR+/HER2− ABC/MBC at Sinai Health in Toronto, Canada. Between 1 January 2016 and 1 July 2021, 48 patients were diagnosed with HR+/HER2− ABC/MBC and received CDK4/6i + ET. A total of 38 out of 48 patients received CDK4/6i + ET in 1L, of which 34 of the 38 (89.5%) received palbociclib + ET. In 2L, 12 of the 21 (57.1%) patients received CDK4/6i + ET, of which 58.3% received abemaciclib. In 3L, most patients received chemotherapy (10/12, 83.3%). For the patients receiving CDK4/6i in 1L, the median (95% CI) time to the next treatment was 42.3 (41.2, NA) months. The median (95% CI) time to chemotherapy was 46.5 (41.4, NA) months. The two-year overall survival (95% CI) was 97.4% (92.4, 100.0), and the median (range) follow-up was 28.7 (3.4–67.6) months. Despite the limitations inherent in real-world studies and a limited number of patients, these AI-extracted data complement previous studies, demonstrating the effectiveness of CDK4/6i + ET in the Canadian real-world 1L, with most patients receiving palbociclib as CDK4/6i in 1L.

Our core premise is that personalized variations of a ketogenic diet are likely to benefit pediatric patients with neuropsychiatric symptoms across multiple domains. Although pediatric epilepsy is currently a well-accepted indication for a strict ketogenic diet, there is a dearth of knowledge and therefore clinical guidelines upon which to recommend nutritional ketosis for pervasive pediatric conditions such as autism spectrum disorder and ADHD, even when comorbid epilepsy is present. However, there are published cohort studies and current clinical trials implementing medical ketogenic therapies for cognitive impairment, psychiatric comorbidities, motor disability, and even neuroinflammation. As holistic practitioners, it is imperative that we consider the health of a child in its entirety - and additionally offer the ketogenic diet as a therapeutic option when it may be synergistic in treating extra-neurologic diseases such as obesity. While there are uniquely pediatric potential adverse side effects such as linear growth deceleration and micronutrient deficiencies, previous trials in epilepsy and our center’s experience have already proven the ketogenic diet to be a low-risk intervention when optimized with appropriate patient monitoring and support.

Alberta grizzly bears are classified as a threatened species in the province of Alberta as of 2010, with human-caused mortality and habitat loss a primary threat. The people who live, work, and recreate within bear habitat play a crucial role in their conservation. While the public is often enthusiastic about grizzly bears, and opportunistically report their observations to government staff, these reports are not systematic or rigorously collected and lack key information. As such, we developed GrizzTracker as a community science program. Following several years of successful deployment, we analyzed community scientist data and evaluated the efficacy of the program through an online user survey. We found that the GrizzTracker app was useful as a data collection and public engagement tool, yielding information for applied management, and that community scientists were generally satisfied. We provide considerations for future program development, including considerations for human, social, technological, and financial capital investment related to design, development, and implementation of data collection protocols, the importance of clearly communicating outcomes, and opportunities for educational outreach. While there is continued trepidation by traditionally trained scientists to develop or engage in community science programs, and some noted areas of improvement for our program specifically, we think that GrizzTracker offers a success story in community science.

Ovine footrot is an infectious disease with important contributions from Dichelobacter nodosus and Fusobacterium necrophorum. Footrot is characterized by separation of the hoof from underlying tissue, and this causes severe lameness that negatively impacts animal wellbeing, growth, and profitability. Large economic losses result from lost production as well as treatment costs, and improved genetic tools to address footrot are a valuable long-term goal. Prior genetic studies had examined European wool sheep, but hair sheep breeds such as Katahdin and Blackbelly have been reported to have increased resistance to footrot, as well as to intestinal parasites. Thus, footrot condition scores were collected from 251 U.S. sheep including Katahdin, Blackbelly, and European-influenced crossbred sheep with direct and imputed genotypes at OvineHD array (>500,000 single nucleotide polymorphism) density. Genome-wide association was performed with a mixed model accounting for farm and principal components derived from animal genotypes, as well as a random term for the genomic relationship matrix. We identified three genome-wide significant associations, including SNPs in or near GBP6 and TCHH. We also identified 33 additional associated SNPs with genome-wide suggestive evidence, including a cluster of 6 SNPs in a peak near the genome-wide significance threshold located near the glutamine transporter gene SLC38A1. These findings suggest genetic susceptibility to footrot may be influenced by genes involved in divergent biological processes such as immune responses, nutrient availability, and hoof growth and integrity. This is the first genome-wide study to investigate susceptibility to footrot by including hair sheep and also the first study of any kind to identify multiple genome-wide significant associations with ovine footrot. These results provide a foundation for developing genetic tests for marker-assisted selection to improve resistance to ovine footrot once additional steps like fine mapping and validation are complete.

Measurement(s) Image Segmentation • hepatocellular carcinoma Technology Type(s) Neural Network • Multiphasic Computed Tomography of the Abdomen Sample Characteristic - Organism multiphasic CT of the abdomen Sample Characteristic - Location contiguous United States of America

Mesenchymal stromal cells (MSCs) demonstrate therapeutic benefits in multiple diseases, but the mechanisms remain unclear as infused MSCs do not persist in the body. Here, the authors show that MSC apoptosis is an important mechanistic element, as MSCs rendered genetically incapable of apoptosis lose their ability to ameliorate disease.

Abstract The neuropilin receptors and their secreted semaphorin ligands play key roles in brain circuit development by regulating numerous crucial neuronal processes, including the maturation of synapses and migration of GABAergic interneurons. Consistent with its developmental roles, the neuropilin 2 (Nrp2) locus contains polymorphisms in patients with autism spectrum disorder (ASD). Nrp2-deficient mice show autism-like behavioral deficits and propensity to develop seizures. In order to determine the pathophysiology in Nrp2 deficiency, we examined the hippocampal numbers of interneuron subtypes and inhibitory regulation of hippocampal CA1 pyramidal neurons in mice lacking one or both copies of Nrp2. Immunostaining for interneuron subtypes revealed that Nrp2 −/− mice have a reduced number of parvalbumin, somatostatin, and neuropeptide Y cells, mainly in CA1. Whole-cell recordings identified reduced firing and hyperpolarized shift in resting membrane potential in CA1 pyramidal neurons from Nrp2 +/− and Nrp2 −/− mice compared to age-matched wild-type controls indicating decrease in intrinsic excitability. Simultaneously, the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) are reduced in Nrp2-deficient mice. A convulsive dose of kainic acid evoked electrographic and behavioral seizures with significantly shorter latency, longer duration, and higher severity in Nrp2 −/− compared to Nrp2 +/+ animals. Finally, Nrp2 +/− and Nrp2 −/− but not Nrp2 +/+ , mice have impaired cognitive flexibility demonstrated by reward-based reversal learning, a task associated with hippocampal circuit function. Together these data demonstrate a broad reduction in interneuron subtypes and compromised inhibition in CA1 of Nrp2 −/− mice, which could contribute to the heightened seizure susceptibility and behavioral deficits consistent with an ASD/epilepsy phenotype.

Wildlife exclusion fencing has become a standard component of highway mitigation systems designed to reduce collisions with large mammals. Past work on the effectiveness of exclusion fencing has relied heavily on control–impact (i.e., space-for-time substitutions) and before–after study designs. These designs limit inference and may confound the effectiveness of mitigation with co-occurring process that also changes the rate of collisions. We used a replicated (n = 2 sites monitored for over 1000 km years combined) before-after-control-impact study design to assess fencing effectiveness along the Trans-Canada Highway in the Rocky Mountains of Canada. We found that collisions declined for common ungulates species (elk, mule deer, and white-tailed deer) by up to 96% but not for large carnivores. The weak response of carnivores is likely due to the combination of fence intrusions and low sample sizes. We calculated realized fencing effectiveness by applying the same change in collision rates observed at control (unfenced) sites as the expected change for adjacent fenced sections. Compared with the apparent fencing effectiveness (i.e., the difference in WVCs rates before and after fencing was installed), the realized estimates of fencing effectiveness declined by 6% at one site and increased by 10% at another site. When factoring in the cost of ungulate collisions to society, fencing provided a net economic gain within 1 year of construction. Over a 10-year period, fencing would provide a net economic gain of > $500,000 per km in reduced collisions. Our study highlights the benefits of long-term monitoring of road mitigation projects and provides evidence of fencing effectiveness for reducing wildlife–vehicle collisions involving large mammals.

Extracellular signaling proteins serve as neuronal growth cone guidance molecules during development and are well positioned to be involved in neuronal regeneration and recovery from injury. Semaphorins and their receptors, the plexins, are a family of conserved proteins involved in development that, in the nervous system, are axonal guidance cues mediating axon pathfinding and synapse formation. The Caenorhabditis elegans genome encodes for three semaphorins and two plexin receptors: the transmembrane semaphorins, SMP-1 and SMP-2, signal through their receptor, PLX-1, while the secreted semaphorin, MAB-20, signals through PLX-2. Here, we evaluate the locomotion behavior of knockout animals missing each of the semaphorins and plexins and the neuronal morphology of plexin knockout animals; we described the cellular expression pattern of the promoters of all plexins in the nervous system of C. elegans; and we evaluated their effect on the regrowth and reconnection of motoneuron neurites and the recovery of locomotion behavior following precise laser microsurgery. Regrowth and reconnection were more prevalent in the absence of each plexin, while recovery of locomotion surpassed regeneration in all genotypes.

Mesenchymal stromal cells (MSCs) have demonstrated therapeutic potential in inflammatory models of human disease. However, clinical translation has fallen short of expectations, with many trials failing to meet primary endpoints. Failure to fully understand their mechanisms of action is a key factor contributing to the lack of successful commercialisation. Indeed, it remains unclear how the long-ranging immunomodulatory effects of MSCs can be attributed to their secretome, when MSCs undergo apoptosis in the lung shortly after intravenous infusion. Their apoptotic fate suggests that efficacy is not based solely on their viable properties, but also on the immune response to dying MSCs. The secondary lymphoid organs (SLOs) orchestrate immune responses and play a key role in immune regulation. In this review, we will discuss how apoptotic cells can modify immune responses and highlight the importance of MSC-immune cell interactions in SLOs for therapeutic outcomes.

KCNT1 encodes the sodium-activated potassium channel KNa1.1, expressed preferentially in the frontal cortex, hippocampus, cerebellum, and brainstem. Pathogenic missense variants in KCNT1 are associated with intractable epilepsy, namely epilepsy of infancy with migrating focal seizures (EIMFS), and sleep-related hypermotor epilepsy (SHE). In vitro studies of pathogenic KCNT1 variants support predominantly a gain-of-function molecular mechanism, but how these variants behave in a neuron or ultimately drive formation of an epileptogenic circuit is an important and timely question. Using CRISPR/Cas9 gene editing, we introduced a gain-of-function variant into the endogenous mouse Kcnt1 gene. Compared to wild-type (WT) littermates, heterozygous and homozygous knock-in mice displayed greater seizure susceptibility to the chemoconvulsants kainate and pentylenetetrazole (PTZ), but not to flurothyl. Using acute slice electrophysiology in heterozygous and homozygous Kcnt1 knock-in and WT littermates, we demonstrated that CA1 hippocampal pyramidal neurons exhibit greater amplitude of miniature inhibitory postsynaptic currents in mutant mice with no difference in frequency, suggesting greater inhibitory tone associated with the Kcnt1 mutation. To address alterations in GABAergic signaling, we bred Kcnt1 knock-in mice to a parvalbumin-tdTomato reporter line, and found that parvalbumin-expressing (PV+) interneurons failed to fire repetitively with large amplitude current injections and were more prone to depolarization block. These alterations in firing can be recapitulated by direct application of the KNa1.1 channel activator loxapine in WT but are occluded in knock-in littermates, supporting a direct channel gain-of-function mechanism. Taken together, these results suggest that KNa1.1 gain-of-function dampens interneuron excitability to a greater extent than it impacts pyramidal neuron excitability, driving seizure susceptibility in a mouse model of KCNT1-associated epilepsy.

Abstract Cities worldwide are expanding in area and human population, posing multiple challenges to amphibian populations, including habitat loss from removal of wetlands and terrestrial upland habitat, habitat fragmentation due to roads and the built environment, and habitat degradation from pollutants, extensive human use and introduced species. We developed an eight‐step urban amphibian conservation framework based on established monitoring, analytical methods and community engagement to enable amphibian conservation in a large urban centre. The framework outlines a process used to conserve biodiversity in a complex landuse and decision‐making environment supported by a series of successive complementary modelling techniques to measure amphibian presence, priority habitat and functional connectivity. We applied the framework in Calgary, Alberta, Canada to illustrate its potential. Here, urbanization has reduced wetlands by 90% and ecological knowledge on amphibians was poor. We improved knowledge on amphibian diversity and distribution, identified core wetlands and movement pathways for amphibian species and identified barriers in the wetland network where construction or restoration measures could re‐establish amphibians or increase their densities. This knowledge was shared with ecologists and city planners for implementation through appropriate policies and plans. Our framework provides a series of stepwise products to improve an urban municipality's ability to restore or conserve priority habitat and movement pathways necessary for amphibian survival under pressure from multiple land uses. The framework provides a platform to identify city plans, policy and or programmes and to derive necessary information to support amphibian conservation.

With approximately 95 million Americans diagnosed with high cholesterol, and many searching for a nonmedicinal treatment, intermittent fasting as a method to improve health has become increasingly popular in the lay public. We conducted a clinical inquiry to determine whether intermittent fasting is superior to a low-calorie diet in improving lipids, searching the Cochrane, EBSCOhost, Embase, MEDLINE, and Scopus databases using the terms intermittent fasting, lipids, and calorie-restricted diet. Studies that included surgical weight loss or medicine-assisted weight loss were excluded. We identified 6 published studies, 5 of which were randomized controlled trials. In reviewing the selected studies, there did not appear to be a consistent difference in lipid change between restricted-calorie diet and intermittent fasting. Because of differences in study methods and in how intermittent fasting was defined, additional studies are needed.

Neuroimaging is among the most active research domains for the creation and management of open-access data repositories. Notably lacking from most data repositories are integrated capabilities for semantic representation. The Arkansas Imaging Enterprise System (ARIES) is a research data management system which features integrated capabilities to support semantic representations of multi-modal data from disparate sources (imaging, behavioral, or cognitive assessments), across common image-processing stages (preprocessing steps, segmentation schemes, analytic pipelines), as well as derived results (publishable findings). These unique capabilities ensure greater reproducibility of scientific findings across large-scale research projects. The current investigation was conducted with three collaborating teams who are using ARIES in a project focusing on neurodegeneration. Datasets included magnetic resonance imaging (MRI) data as well as non-imaging data obtained from a variety of assessments designed to measure neurocognitive functions (performance scores on neuropsychological tests). We integrate and manage these data with semantic representations based on axiomatically rich biomedical ontologies. These instantiate a knowledge graph that combines the data from the study cohorts into a shared semantic representation that explicitly accounts for relations among the entities that the data are about. This knowledge graph is stored in a triple-store database that supports reasoning over and querying these integrated data. Semantic integration of the non-imaging data using background information encoded in biomedical domain ontologies has served as a key feature-engineering step, allowing us to combine disparate data and apply analyses to explore associations, for instance, between hippocampal volumes and measures of cognitive functions derived from various assessment instruments.

The endocochlear potential (EP) generated by the stria vascularis (SV) is necessary for hair cell mechanotransduction in the mammalian cochlea. We sought to create a model of EP dysfunction for the purposes of transcriptional analysis and treatment testing. By administering a single dose of cisplatin, a commonly prescribed cancer treatment drug with ototoxic side effects, to the adult mouse, we acutely disrupt EP generation. By combining these data with single cell RNA-sequencing findings, we identify transcriptional changes induced by cisplatin exposure, and by extension transcriptional changes accompanying EP reduction, in the major cell types of the SV. We use these data to identify gene regulatory networks unique to cisplatin treated SV, as well as the differentially expressed and druggable gene targets within those networks. Our results reconstruct transcriptional responses that occur in gene expression on the cellular level while identifying possible targets for interventions not only in cisplatin ototoxicity but also in EP dysfunction.

The sample mean in statistics is a concept of great importance, with its properties being extensively utilized in other areas, such as computer science. This research centers on the concept of the sample mean and its characteristics in a cohort of computer engineering students undertaking a required course in statistics at a university in the Netherlands. The objective of this study was to analyze students' comprehension of the fundamental structure, properties, and applications of the sample mean. A digital mini-course on sample mean was developed and employed with 97 students, who had the option to apply the concepts at their own pace. The action-process-object-schema (APOS) theoretical framework was utilized to analyze the interview responses of seven participants. The majority of participants were able to demonstrate an understanding of the Process conception of the sample mean, with only a minority demonstrating an understanding of the Object conception. The lack of comprehension of the sample mean hindered students' capacity to effectively utilize the associated applications and properties. These findings imply that educators should strive to ensure that students have a sound comprehension of the sample mean so that they are better equipped to work with related applications. Suggestions of this study and further research ideas are indicated.

Abstract Objective We identified a novel de novo KCNT1 variant in a patient with early‐infantile epileptic encephalopathy (EIEE) and status dystonicus, a life‐threatening movement disorder. We determined the functional consequences of this variant on the encoded KNa1.1 channel to investigate the molecular mechanisms responsible for this disorder. Methods A retrospective case review of the proband is presented. We performed manual and automated electrophysiologic analyses of the KCNT1‐L437F variant expressed heterologously in Chinese hamster ovary (CHO) cells in the presence of channel activators/blockers. Results The KCNT1‐L437F variant, identified in a patient with refractory EIEE and status dystonicus, confers a gain‐of‐function channel phenotype characterized by instantaneous, voltage‐dependent activation. Channel openers do not further increase L437F channel function, suggesting maximal activation, whereas channel blockers similarly block wild‐type and variant channels. We further demonstrated that KCNT1 current can be measured on a high‐throughput automated electrophysiology platform with potential value for future screening of novel and repurposed pharmacotherapies. Interpretation A novel pathogenic variant in KCNT1 associated with early‐onset, medication‐refractory epilepsy and dystonia causes gain‐of‐function with rapid activation kinetics. Our findings extend the genotype–phenotype relationships of KCNT1 variants to include severe dystonia.

Abstract Background Patient reported outcome (PRO) information is crucial for establishing better patient-provider communication, improving shared decision-making between clinicians and patients, assessing patient responses to therapeutic interventions, and increasing satisfaction with care. We used the Brittle Bones Disease Consortium (BBDC) Contact Registry for People with OI, managed by the Rare Disease Clinical Research Network (RDCRN) to (1) to evaluate the construct validity of the Patient-Reported Outcome Measurement Information System® (PROMIS®) to record important components of the disease experience among individuals with OI; and (2) explore the feasibility of using a registry to recruit individuals with OI to report on health status. Our long-term goal is to enhance communication of health and disease management findings back to the OI community, especially those who do not have access to major OI clinical centers. Results We demonstrated the construct validity of PROMIS instruments in OI. Our results confirm that the scores from most domains differ significantly from the general US population: individuals with OI have worse symptom burden and functioning. We found no excessive floor or ceiling effects. Our study demonstrates that the BBDC Contact Registry can be used to recruit participants for online health status surveys. However, there are numerous challenges that must be addressed: lack of self-knowledge of OI type, under-representation of men, limited ethnic diversity, and imperfect questionnaire completion rates. Conclusion Our pilot study demonstrated the feasibility of using a contact registry to recruit respondents from the OI community and to obtain analyzable PROMIS data regarding disease experience. Because the results differ from the general population and avoid excessive floor and ceiling effects, PROMIS instruments can be used to assess response to therapeutic interventions in individuals with OI. Future directions will include (1) development and validation of an OI-specific patient-based classification system that aggregates persons with similar clinical characteristics and risks for complications to identify treatment needs; and (2) integrating these PRO tools into routine patient care and research studies.

Abstract The intersection of wildlife and people on roads raises two critical issues: the barrier and mortality effects of roads on wildlife and risks to people from animal‐vehicle collisions (AVCs). Road mitigation decisions are typically made at the discretion of transportation departments that are mandated to primarily address motorist safety. Therefore, prioritization of road sections for mitigation currently focuses on identification of spatial clusters of AVCs. We sought to understand if AVC clusters align with multispecies connectivity across roads to accurately identify multipurpose mitigation hotspots. We developed a decision‐support tool based on weighted priorities for mitigation planning across 7,900 km of roads over an 84,000‐km2 area of southern Alberta, Canada. To assess alignment, we built functional connectivity models for four focal species (prairie rattlesnake, grizzly bear, mule deer, and pronghorn) and a species‐neutral structural connectivity model. We integrated AVC risk and wildlife connectivity indices into Mitigation Priority Indices that varied the weighting of individual indices. Our results demonstrated poor spatial alignment between road sections of high motorist safety risk and those of high value for wildlife connectivity. Transportation planning would benefit from integrating motorist safety risk and wildlife management needs to prioritize mitigation neighborhoods along roadways.

Citizen science offers an excellent opportunity to engage the public in scientific data collection, educational opportunities, and applied management. However, the practicalities of developing and implementing citizen science programming are often more complex than considered. Some challenges to effective citizen science include scientists’ skepticism about the ability of public participants to rigorously collect quality data; a lack of clarity on or confidence in the utility of data; scientists’ hesitancy in engaging the public in projects; limited financial commitments; and challenges associated with the temporal and geographic scales of projects. To address these challenges, and provide a foundation upon which practitioners, scientists, and the public can credibly engage in citizen science, the Government of Alberta developed a set of citizen science principles. These principles offer a framework for planning, designing, implementing, and evaluating citizen science projects that extend beyond Alberta. Here, we present a case study using these principles to evaluate GrizzTracker, a citizen science program developed to help inform provincial species-at-risk recovery efforts. While we found that GrizzTracker applied each of the six principles in some way, including successful public engagement, strengthened relationships, and raising public awareness about northwest Alberta’s grizzly bears, we also identified a number of challenges. These included ongoing skepticism from the traditional scientific community about the utility of citizen science and governance challenges related to program leadership, staff capacity, and funding. By using the principles as a guideline, we provide policy recommendations for future citizen science efforts, including considerations for program design, implementation, and evaluation.

Abstract Airway smooth muscle (ASM) plays a major role in acute airway narrowing and reducing ASM thickness is expected to attenuate airway hyper‐responsiveness and disease burden. There are two therapeutic approaches to reduce ASM thickness: (a) a direct approach, targeting specific airways, best exemplified by bronchial thermoplasty (BT), which delivers radiofrequency energy to the airway via bronchoscope; and (b) a pharmacological approach, targeting airways more broadly. An example of the less well‐established pharmacological approach is the calcium‐channel blocker gallopamil which in a clinical trial effectively reduced ASM thickness; other agents may act similarly. In view of established anti‐proliferative properties of the macrolide antibiotic azithromycin, we examined its effects in naive mice and report a reduction in ASM thickness of 29% (p

Cell therapeutics — using cells as living drugs — have made advances in many areas of medicine. One of the most clinically studied cell-based therapy products is mesenchymal stromal cells (MSCs), which have shown promising results in promoting tissue regeneration and modulating inflammation. However, MSC therapy requires large numbers of cells, the generation of which is not feasible via conventional planar tissue culture methods. Scale-up manufacturing methods (e.g., propagation on microcarriers in stirred-tank bioreactors), however, are not specifically tailored for MSC expansion. These processes may, in principle, alter the cell secretome, a vital component underlying the immunosuppressive properties and clinical effectiveness of MSCs. This review outlines our current understanding of MSC properties and immunomodulatory function, expansion in commercial manufacturing systems, and gaps in our knowledge that need to be addressed for effective up-scaling commercialization of MSC therapy.

ABSTRACT: Objective: To describe a patient who presented with hypercalcemic crisis and severe hyperparathyroidism who had urgent surgery. She was found to have a huge mediastinal atypical parathyroid adenoma (APA). We discuss our case, the difficulty discriminating an APA from parathyroid cancer, and the tools that help differentiate these conditions.Methods: We present the clinical manifestations, laboratory results, radiologic and surgical findings, and management in a patient with hyperparathyroid crisis. Discussion follows.Results: We report a case of a 39-year-old woman who presented with symptoms of severe hypercalcemia. Her intact parathyroid hormone level was found to be markedly elevated, consistent with primary hyperparathyroidism. Imaging revealed an anterior mediastinal mass, suggestive of possible ectopic parathyroid tissue. Urgent surgery and subsequent pathologic study of the excised mass revealed a supernumerary 37.5-g parathyroid gland within the mediastinum surrounded by a rim of thymic tissue. Histologically, the adenoma did not meet the World Health Organization criteria for the diagnosis of parathyroid carcinoma; however, the adenoma had atypical pathologic features given an abnormal mitotic figure. Parafibromin staining was performed and largely negative in the center of the tumor. Germline analysis of the MEN1 and CDC73 genes revealed no pathogenic variants. Her postoperative course was complicated by hypocalcemia but took several days to develop.Conclusion: The distinction between APA and parathyroid carcinoma may be difficult, and parafibromin staining and genetic testing for CDC73 mutations should be considered, as the results have implications for patient follow-up as well as for patients' relatives.Abbreviations: APA a typical parathyroid adenoma PHPT primary hyperparathyroidism PTH parathyroid hormone

The γδ T cell pool includes abundant IL-17-producing cells that protect mucosal surfaces, but the signals that control γδ T cell specification are unclear. Here the authors identify a role for the transcription factor HEB, and antagonistic activity of Id3, in the development of these cells.

Objectives: This study aimed to find the optimal radiotherapy VMAT plans, that achieved high conformity and homogeneity to the planned target volume (PTV), and minimize the dose to nearby organs at risk including the non-PTV lung, heart and oesophagus for patients with centrally located non-small Cell Lung Cancer. Methods: A total of 18 patients who were treated for stage III centrally located non-small Cell Lung Cancer were selected retrospectively for this study. Identical CT datasets, 4D CT and structure dataset were used for radiotherapy planning based on single-planar VMAT (SP-VMAT), dual-planar VMAT (DP-VMAT) and Hybrid VMAT (H-VMAT). For SP-VMAT, one full arc and two half arcs were created on single-plane with couch at 0°. For DP-VMAT, one full arc was created with couch at 0°, and two half arcs with couch rotation of 330° or 30°. For H-VMAT, anterior-posterior opposing fixed beam and two half arcs were planned at couch at 0°. Dose constraints were adhered to the RTOG0617. Dose volumetric parameters were collected for statistical analysis. Results: There were no significant differences for the PTV, HI, CI between the SP-VMAT, DP-VMAT and H-VMAT. For the non-PTV lungs, Dmean, V20, V10, V5, D1500 and D1000 were significantly lower (2.05 Gy, 6.47%, 15.89%, 11.66% 4.17 Gy and 5.47 Gy respectively) in H-VMAT than that of SP-VMAT (all p < 0.001). For the oesophagus, Dmax, Dmean, V30 and V18.8 of H-VMAT were 0.08 Gy, 1.73 Gy, 5.54% and 7.17% lower than that of the SP-VMAT plan. For the heart, Dmean, V34, V28, V20 and V10 of DP-VMAT were lower than that of SP-VMAT by 1.45 Gy, 0.65%, 1.74%, 4.8% and 7.11% respectively. Conclusion: The proposed H-VMAT showed more favourable plan quality than the SP-VMAT for centrally located stage III NSCLC, in particular for non-PTV lungs and the oesophagus. It will benefit patients, especially those who planned for immunotherapy (Durvalumab) after standard chemo-irradiation. The proposed DP-VMAT plan showed significant dose reduction to the heart when compared to the H-VMAT plan.

Coronavirus disease 2019 (COVID-19), a condition associated with SARS-CoV-2, typically results in mild infection in infants and children. However, children with risk factors such as chronic lung disease and immunosuppression have higher risk of severe illness from COVID-19. We report a case of a 27-week-gestation extremely premature infant born to a mother with COVID-19 infection. The infant, initially treated for surfactant deficiency, developed worsening hypoxic respiratory failure on the fifth day of life requiring escalating ventilatory support, an elevated level of C-reactive protein, thrombocytopenia, and an elevated level of d-dimer. The infant was positive for SARS-CoV-2 by RT-PCR from Day 1 to Day 42 of his life. The infant responded to a seven-day course of dexamethasone with a gradually decreasing oxygen requirement and could be extubated to non-invasive ventilation by the end of the fifth week after birth. The infant is currently on home oxygen by nasal cannula. Prolonged shedding of the virus may be a unique feature of the disease in premature infants. Extreme prematurity, immature lungs, and an immunocompromised status may predispose these infants to severe respiratory failure and a prolonged clinical course. Instituting appropriate COVID-19 protocols to prevent the spread of the disease in the neonatal intensive care unit (NICU) is of utmost importance. Infection with SARS-CoV-2 may have implications in the management of extremely premature infants in the NICU.

We describe a case of a 24-year-old overweight woman who presented with hirsutism, secondary amenorrhea, clitoromegaly, and symptoms of diabetes mellitus (DM). While a diagnosis of polycystic ovary syndrome (PCOS) with its associated metabolic disturbances was initially considered, serum total testosterone, androstenedione, and 17-hydroxyprogesterone (17-OHP) measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) were significantly increased. As 17-OHP did not increase upon ACTH (Synacthen) stimulation and the urinary steroid profile (USP) was compatible with an ovarian source of 17-OHP excess rather than adrenal, non classical congenital adrenal hyperplasia (NCCAH) was unlikely and an androgen-secreting tumor was suspected. Transabdominal ultrasound revealed the presence of an enlarged right ovary with a polycystic ovary morphology and no discrete mass. Transvaginal ultrasound and [18F]− fluorodeoxyglucose positron emission tomography–computed tomography (FDG PET–CT) enabled the localization of a right ovarian tumor. Laparoscopic right salpingo-oophorectomy was performed and a histological diagnosis of steroid cell tumor, not otherwise specified (SCT–NOS) was made. Hyperandrogenism and menstrual disturbances resolved postoperatively. A literature review revealed that 17-OHP-secreting SCT–NOS may uncommonly show positive responses to ACTH stimulation similar to 21-hydroxylase deficiency. Alternatively, USP might be useful in localizing the source of 17-OHP to the ovaries. Its diagnostic performance should be evaluated in further studies.

As cities adopt mandates to protect, maintain and restore urban biodiversity, the need for urban ecology studies grows. Species-specific information on the effects of urbanization is often a limiting factor in designing and implementing effective biodiversity strategies. In suburban and exurban areas, amphibians play an important social-ecological role between people and their environment and contribute to ecosystem health. Amphibians are vulnerable to threats and imbalances in the aquatic and terrestrial environment due to a biphasic lifestyle, making them excellent indicators of local environmental health. We developed a citizen science program to systematically monitor amphibians in a large city in Alberta, Canada, where 90% of pre-settlement wetlands have been removed and human activities continue to degrade, alter, and/or fragment remaining amphibian habitats. We demonstrate successes and challenges of using publicly collected data in biodiversity monitoring. Through amphibian monitoring, we show how a citizen science program improved ecological knowledge, engaged the public in urban biodiversity monitoring and improved urban design and planning for biodiversity. We outline lessons learned to inform citizen science program design, including the importance of early engagement of decision makers, quality control assessment, assessing tensions in program design for data and public engagement goals, and incorporating conservation messaging into programming.

The emergence of nanoinformatics as a key component of nanotechnology and nanosafety assessment for the prediction of engineered nanomaterials (NMs) properties, interactions, and hazards, and for grouping and read-across to reduce reliance on animal testing, has put the spotlight firmly on the need for access to high-quality, curated datasets. To date, the focus has been around what constitutes data quality and completeness, on the development of minimum reporting standards, and on the FAIR (findable, accessible, interoperable, and reusable) data principles. However, moving from the theoretical realm to practical implementation requires human intervention, which will be facilitated by the definition of clear roles and responsibilities across the complete data lifecycle and a deeper appreciation of what metadata is, and how to capture and index it. Here, we demonstrate, using specific worked case studies, how to organise the nano-community efforts to define metadata schemas, by organising the data management cycle as a joint effort of all players (data creators, analysts, curators, managers, and customers) supervised by the newly defined role of data shepherd. We propose that once researchers understand their tasks and responsibilities, they will naturally apply the available tools. Two case studies are presented (modelling of particle agglomeration for dose metrics, and consensus for NM dissolution), along with a survey of the currently implemented metadata schema in existing nanosafety databases. We conclude by offering recommendations on the steps forward and the needed workflows for metadata capture to ensure FAIR nanosafety data.

SUMMARY Infection with Helicobacter pylori is a major risk factor for the development of gastric cancer, and infection with strains carrying the virulence factor CagA significantly increases this risk. To investigate the mechanisms by which CagA promotes carcinogenesis, we generated transgenic zebrafish expressing CagA ubiquitously or in the anterior intestine. Transgenic zebrafish expressing either the wild-type or a phosphorylation-resistant form of CagA exhibited significantly increased rates of intestinal epithelial cell proliferation and showed significant upregulation of the Wnt target genes cyclinD1, axin2 and the zebrafish c-myc ortholog myca. Coexpression of CagA with a loss-of-function allele encoding the β-catenin destruction complex protein Axin1 resulted in a further increase in intestinal proliferation. Coexpression of CagA with a null allele of the key β-catenin transcriptional cofactor Tcf4 restored intestinal proliferation to wild-type levels. These results provide in vivo evidence of Wnt pathway activation by CagA downstream of or in parallel to the β-catenin destruction complex and upstream of Tcf4. Long-term transgenic expression of wild-type CagA, but not the phosphorylation-resistant form, resulted in significant hyperplasia of the adult intestinal epithelium. We further utilized this model to demonstrate that oncogenic cooperation between CagA and a loss-of-function allele of p53 is sufficient to induce high rates of intestinal small cell carcinoma and adenocarcinoma, establishing the utility of our transgenic zebrafish model in the study of CagA-associated gastrointestinal cancers.

Investigators from Harvard University and UCLA have reported that despite evidence of structural abnormalities in the visual pathway of animal models and children with tuberous sclerosis complex (TSC), visual evoked potentials (VEPs) in 12-month old children with TSC compared to an age-matched control group are not significantly altered.

Investigators from Johns Hopkins University reported a cohort of 27 patients with incidentally-noted rolandic spikes (RS) on EEG.

Investigators from Wayne State University studied a cohort of children with Sturge-Weber syndrome (SWS) and epilepsy using both glucose-based positron emission tomography (FDG-PET) to evaluate metabolic activity and proton magnetic resonance spectroscopic imaging (MRSI) to evaluate glutamate turnover.

This article draws on data from a larger longitudinal qualitative case study which is tracking the progress of students over the course of their undergraduate degrees at a South African university. For this paper, we used background questionnaires and semi-structured interviews with 62 first-year students from working-class, township schools who were first registered for Extended Degree Programmes in 2009. The article draws on post-structuralist theory on learning and identity to describe and analyse the participants’ perspectives on how they negotiated their high school contexts. We analyse the subject positions in which participants invested, as well as how they negotiated their way through social networks and used resources. Our data illustrate the ways in which students had to carry the burden of negotiating their way through home, school and neighbourhood spaces that were generally not conducive to learning. Nevertheless, participants consciously positioned themselves as agents. They were resilient, motivated and took highly strategic adult decisions about their learning. We argue that a focus on how successful students negotiate their environments challenges the pathologising paradigm of “disadvantage” that characterises research and debates in higher education. It also offers an additional lens for admissions processes and for providing appropriate intervention strategies in the tertiary setting.

The ovine Callipyge mutation causes postnatal muscle hypertrophy localized to the pelvic limbs and torso, as well as body leanness. The mechanism underpinning enhanced muscle mass is unclear, as is the systemic impact of the mutation. Using muscle fibre typing immunohistochemistry, we confirmed muscle specific effects and demonstrated that affected muscles had greater prevalence and hypertrophy of type 2X fast twitch glycolytic fibres and decreased representation of types 1, 2C, 2A and/or 2AX fibres. To investigate potential systemic effects of the mutation, proton NMR spectra of plasma taken from lambs at 8 and 12 weeks of age were measured. Multivariate statistical analysis of plasma metabolite profiles demonstrated effects of development and genotype but not gender. Plasma from Callipyge lambs at 12 weeks of age, but not 8 weeks, was characterized by a metabolic profile consistent with contributions from the affected hypertrophic fast twitch glycolytic muscle fibres. Microarray analysis of the perirenal adipose tissue depot did not reveal a transcriptional effect of the mutation in this tissue. We conclude that there is an indirect systemic effect of the Callipyge mutation in skeletal muscle in the form of changes of blood metabolites, which may contribute to secondary phenotypes such as body leanness.

The gene encoding human spectrin Src homology domain binding protein 1, or Hssh3bpl, which is a marker of macropinocytic vesicles and a potential regulator of macropinocytosis, co-localizes to a YAC containing chromosome 10p sequences at loci D10S89 and D10S111 that are frequently deleted in prostate tumors. Expression of Hssh3bp1 was evaluated at the protein level in 17 paired normal and malignant prostate tumor samples using the monoclonal antibody 2G8 to Hssh3bpl. These experiments demonstrated that 4/6 tumors (67%) with 10p deletion failed to express Hssh3bp1 protein compared to 5/11 (46%) tumors with intact 10p. Thus, loss of Hssh3bp1 expression is concordant with allelic loss of adjacent 10p sequences in human prostate tumors. In addition, two prostate tumor cell lines contain an exon skipping mutation in the Hssh3bp1 gene that leads to the abnormal splicing of the mRNA and loss of a portion of Abl tyrosine kinase SH3 domain binding site in the protein. These data are consistent with a role for Hssh3bp1 as a candidate tumor suppressor gene inactivated during prostate tumorigenesis.

Like many complex dynamic systems, the brain exhibits scale-free dynamics that follow power law scaling. Broadband power spectral density (PSD) of brain electrical activity exhibits state-dependent power law scaling with a log frequency exponent that varies across frequency ranges. Widely divergent naturally occurring neural states, awake and slow wave sleep (SWS) periods, were used evaluate the nature of changes in scale-free indices. We demonstrate two analytic approaches to characterizing electrocorticographic (ECoG) data obtained during Awake and SWS states. A data driven approach was used, characterizing all available frequency ranges. Using an Equal Error State Discriminator (EESD), a single frequency range did not best characterize state across data from all six subjects, though the ability to distinguish awake and SWS states in individual subjects was excellent. Multisegment piecewise linear fits were used to characterize scale-free slopes across the entire frequency range (0.2-200 Hz). These scale-free slopes differed between Awake and SWS states across subjects, particularly at frequencies below 10 Hz and showed little difference at frequencies above 70 Hz. A Multivariate Maximum Likelihood Analysis (MMLA) method using the multisegment slope indices successfully categorized ECoG data in most subjects, though individual variation was seen. The ECoG spectrum is not well characterized by a single linear fit across a defined set of frequencies, but is best described by a set of discrete linear fits across the full range of available frequencies. With increasing computational tractability, the use of scale-free slope values to characterize EEG data will have practical value in clinical and research EEG studies.

Recent evidence suggests that the decline in resistance to viral infections with age occurs predominantly as a result of a gradual loss of naïve antigen-specific T cells. As such, restoration of the naïve T cell repertoire to levels seen in young healthy adults may improve defence against infection in the aged. We have previously shown that sex steroid ablation (SSA) rejuvenates the ageing thymus and increases thymic export of naïve T cells, but it remains unclear whether T cell responses are improved. Using mouse models of clinically relevant diseases, we now demonstrate that SSA increases the number of naïve T cells able to respond to antigen, thereby enhancing effector responses in aged mice. Specifically, aged mice exhibit a delay in clearing influenza A virus, which correlates with diminished specific cytotoxic activity. This is due to a decreased magnitude of response and not an intrinsic defect in effector T cell function. Upon SSA, aged mice exhibit increased T cell responsiveness that restores efficient viral clearance. We further demonstrate that SSA decreases the incidence of an inducible tumour in aged mice and can potentially increase their responsiveness to a low-dose human papillomavirus vaccine in clearing pre-formed tumours. As thymectomy abrogates the increase in T cell numbers and responsiveness following SSA, we propose that the T cell effects of SSA are dependent on thymic reactivation and subsequent replenishment of the peripheral T cell pool with newly emigrated naïve T cells. These findings have important implications for strategies to improve protection from infection and responsiveness to vaccination in the aged.

Electroporation (EP) of mammalian tissue is a technique that has been used successfully in the clinic for the delivery of genetic-based vaccines in the form of DNA plasmids. There is great interest in platforms which efficiently deliver RNA molecules such as messenger RNA and small interfering RNA (siRNA) to mammalian tissue. However, the in vivo delivery of RNA enhanced by EP has not been extensively characterized. This paper details the optimization of electrical parameters for a novel low-voltage EP method to deliver oligonucleotides (both DNA and RNA) to dermal tissue in vivo. Initially, the electrical parameters were optimized for dermal delivery of plasmid DNA encoding green fluorescent protein (GFP) using this novel surface dermal EP device. While all investigated parameters resulted in visible transfection, voltage parameters in the 10 V range elicited the most robust signal. The parameters optimized for DNA, were then assessed for translation of successful electrotransfer of siRNA into dermal tissue. Robust tagged-siRNA transfection in skin was detected. We then assessed whether these parameters translated to successful transfer of siRNA resulting in gene knockdown in vivo. Using a reporter gene construct encoding GFP and tagged siRNA targeting the GFP message, we show simultaneous transfection of the siRNA to the skin via EP and the concomitant knockdown of the reporter gene signal. The siRNA delivery was accomplished with no evidence of injection site inflammation or local tissue damage. The minimally invasive low-voltage EP method is thus capable of efficiently delivering both DNA and RNA molecules to dermal tissue in a tolerable manner.