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Abstract Background Mechanical ventilation (MV) is a life‐saving measure for patients in respiratory failure. However, prolonged MV results in significant diaphragm atrophy and contractile dysfunction, a condition referred to as ventilator‐induced diaphragm dysfunction (VIDD). While there are currently no clinically approved countermeasures to prevent VIDD, increased expression of heat shock protein 72 (HSP72) has been demonstrated to attenuate inactivity‐induced muscle wasting. HSP72 elicits cytoprotection via inhibition of NF‐κB and FoxO transcriptional activity, which contribute to VIDD. In addition, exercise‐induced prevention of VIDD is characterized by an increase in the concentration of HSP72 in the diaphragm. Therefore, we tested the hypothesis that increased HSP72 expression is required for the exercise‐induced prevention of VIDD. We also determined whether increasing the abundance of HSP72 in the diaphragm, independent of exercise, is sufficient to prevent VIDD. Methods Cause and effect was determined by inhibiting the endurance exercise‐induced increase in HSP72 in the diaphragm of exercise trained animals exposed to prolonged MV via administration of an antisense oligonucleotide targeting HSP72. Additional experiments were performed to determine if increasing HSP72 in the diaphragm via genetic (rAAV‐HSP72) or pharmacological (BGP‐15) overexpression is sufficient to prevent VIDD. Results Our results demonstrate that the exercise‐induced increase in HSP72 protein abundance is required for the protective effects of exercise against VIDD. Moreover, both rAAV‐HSP72 and BGP‐15‐induced overexpression of HSP72 were sufficient to prevent VIDD. In addition, modification of HSP72 in the diaphragm is inversely related to the expression of NF‐κB and FoxO target genes. Conclusions HSP72 overexpression in the diaphragm is an effective intervention to prevent MV‐induced oxidative stress and the transcriptional activity of NF‐κB and FoxO. Therefore, overexpression of HSP72 in the diaphragm is a potential therapeutic target to protect against VIDD.

Abstract Huntington’s disease (HD) is a progressive neurodegenerative disease. Involuntary movements, cognitive impairment and psychiatric disturbance are the major clinical manifestations, and gradual atrophy and selective neuronal loss in the striatum and cerebral cortex are the pathologic hallmarks. HD is caused by expanded CAG trinucleotide repeats at the N-terminus of IT15 that encodes the huntingtin (HTT) protein, though the molecular mechanisms through which the mutant HTT (mHTT) exerts toxic effects remain obscure. Members of the caspase family, including caspase-2 (Casp2), play an important role in HD pathogenesis. Genetic ablation of Casp2 ameliorates cognitive and motor deficits of HD mice, though the molecular targets of Casp2 are still unclear. It is well established that the microtubule-associated protein tau potentiates cognitive dysfunction in a variety of neurodegenerative disorders, including HD. Our recent study indicates that Casp2-catalyzed tau cleavage at aspartate 314 (tau 2N4R isoform numbering system) mediates synaptotoxicity, cognitive deficits and neurodegeneration in cellular and mouse models of frontotemporal dementia; further, levels of Δtau314, the soluble, N-terminal cleavage product, are elevated in individuals with mild cognitive impairment and Alzheimer’s disease, compared with cognitively normal individuals. Here, we identified the presence of Δtau314 proteins in the striatum (caudate nucleus) and prefrontal cortex (Brodmann’s area 8/9) of human subjects, and showed that in both structures, levels of Casp2 and Δtau314 proteins correlate well, and both proteins are higher in HD patients than non-HD individuals. Our findings advance our understanding of the contribution of Casp2-mediated Δtau314 production to HD pathogenesis.

Sphingolipids (SLs) have been implicated in numerous important cellular biologies; however, their study has been hindered by the complexities of SL metabolism. Furthermore, enzymes of SL metabolism represent a dynamic and interconnected network in which one metabolite can be transformed into other bioactive SLs through further metabolism, resulting in diverse cellular responses. Here we explore the effects of both lethal and sublethal doses of doxorubicin (Dox) in MCF-7 cells. The two concentrations of Dox resulted in the regulation of SLs, including accumulations in sphingosine, sphingosine-1-phosphate, dihydroceramide, and ceramide, as well as reduced levels of hexosylceramide. To further define the effects of Dox on SLs, metabolic flux experiments utilizing a d17 dihydrosphingosine probe were conducted. Results indicated the regulation of ceramidases and sphingomyelin synthase components specifically in response to the cytostatic dose. The results also unexpectedly demonstrated dose-dependent inhibition of dihydroceramide desaturase and glucosylceramide synthase in response to Dox. Taken together, this study uncovers novel targets in the SL network for the action of Dox, and the results reveal the significant complexity of SL response to even a single agent. This approach helps to define the role of specific SL enzymes, their metabolic products, and the resulting biologies in response to chemotherapeutics and other stimuli.

The Scholarship of Teaching and Learning (SoTL) is a rich forum where scholars from different fields and philosophical orientations find space to share their research on teaching and learning in higher education. Within this paper, we will share our individual and collective experiences of why we perceive phenomenology as a methodology well-suited for a broad range of SoTL purposes. Phenomenology is a research approach that focuses on describing the common meaning of the lived experience of several individuals about a particular phenomenon. We will discuss how phenomenology informed our own SoTL research projects, exploring the experiences of faculty and undergraduates in higher education. We will highlight the challenges and affordances that emerged from our use of this methodology. Phenomenology has motivated us to tell our stories of SoTL research and within those, to share the stories that faculty and students shared.

In actinide chemistry, a longstanding bonding model describes metal-ligand binding using 6d-orbitals, with the 5f-orbitals remaining non-bonding. Here the authors explore the inverse-trans-influence — a case where the model breaks down — finding that the f-orbitals play a crucial role in dictating a trans-ligand-directed geometry.

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, prolonged MV results in rapid diaphragmatic atrophy and contractile dysfunction, collectively termed ventilator-induced diaphragm dysfunction (VIDD). Recent evidence reveals that endurance exercise training, performed prior to MV, protects the diaphragm against VIDD. While the mechanism(s) responsible for this exercise-induced protection against VIDD remain unknown, increased diaphragm antioxidant expression may be required. To investigate the role that increased antioxidants play in this protection, we tested the hypothesis that elevated levels of the mitochondrial antioxidant enzyme superoxide dismutase 2 (SOD2) is required to achieve exercise-induced protection against VIDD. Cause and effect was investigated in two ways. First, we prevented the exercise-induced increase in diaphragmatic SOD2 via delivery of an antisense oligonucleotide targeted against SOD2 post-exercise. Second, using transgene overexpression of SOD2, we determined the effects of increased SOD2 in the diaphragm independent of exercise training. Results from these experiments revealed that prevention of the exercise-induced increases in diaphragmatic SOD2 results in a loss of exercise-mediated protection against MV-induced diaphragm atrophy and a partial loss of protection against MV-induced diaphragmatic contractile dysfunction. In contrast, transgenic overexpression of SOD2 in the diaphragm, independent of exercise, did not protect against MV-induced diaphragmatic atrophy and provided only partial protection against MV-induced diaphragmatic contractile dysfunction. Collectively, these results demonstrate that increased diaphragmatic levels of SOD2 are essential to achieve the full benefit of exercise-induced protection against VIDD. Keywords: Diaphragm, ROS, Antioxidants, Muscle atrophy, SOD2, Exercise training

The design and development of analgesics with mixed-opioid receptor interactions has been reported to decrease side effects, minimizing respiratory depression and reinforcing properties to generate safer analgesic therapeutics. We synthesized bis-cyclic guanidine heterocyclic peptidomimetics from reduced tripeptides. In vitro screening with radioligand competition binding assays demonstrated variable affinity for the mu-opioid receptor (MOR), delta-opioid receptor (DOR), and kappa-opioid receptor (KOR) across the series, with compound 1968-22 displaying good affinity for all three receptors. Central intracerebroventricular (i.c.v.) administration of 1968-22 produced dose-dependent, opioid receptor-mediated antinociception in the mouse 55 °C warm-water tail-withdrawal assay, and 1968-22 also produced significant antinociception up to 80 min after oral administration (10 mg/kg, p.o.). Compound 1968-22 was detected in the brain 5 min after intravenous administration and was shown to be stable in the blood for at least 30 min. Central administration of 1968-22 did not produce significant respiratory depression, locomotor effects or conditioned place preference or aversion. The data suggest these bis-cyclic guanidine heterocyclic peptidomimetics with multifunctional opioid receptor activity may hold potential as new analgesics with fewer liabilities of use.

Limited research exists on the effectiveness of product placement in secondary schools. We explored the impact of re-positioning sweet-baked goods, fruit, sugar-sweetened beverages (SSBs) and water on pupil’s lunchtime purchases in two secondary schools in North-East England. We employed a stepped-wedge design with two clusters and four time periods. The intervention(s) involved re-positioning selected food and drinks to increase and decrease accessibility of ‘healthier’ and ‘less healthy’ items, respectively. Unidentifiable smartcard data measured the change in number of pupil’s purchasing the above items. McNemar tests were undertaken on paired nominal data in Stata(v15). In School A, pupils purchasing fruit pots from control to intervention increased (n = 0 cf. n = 81; OR 0, 95% CI 0 to 0.04); post-intervention, this was not maintained. In School B, from control to intervention pupil’s purchasing sweet-baked goods decreased (n = 183 cf. n = 147; OR 1.2, 95% CI 1 to 1.6). This continued post-intervention (n = 161 cf. n = 122; OR 1.3, 95% CI 1.0 to 1.7) and was similar for SSBs (n = 180 cf. n = 79; OR 2.3, 95% CI 1.7 to 3.0). We found no evidence of other changes. There is some evidence that product placement may positively affect pupil’s food and drink purchases. However, there are additional aspects to consider, such as, product availability, engaging canteen staff and the individual school context.

Background: Fluoride exposure has the potential to disrupt thyroid functioning, though adequate iodine intake may mitigate this effect. This is the first population-based study to examine the impact of chronic low-level fluoride exposure on thyroid function, while considering iodine status. The objective of this study was to determine whether urinary iodine status modifies the effect of fluoride exposure on thyroid stimulating hormone (TSH) levels. Methods: This cross-sectional study utilized weighted population-based data from Cycle 3 (2012−2013) of the Canadian Health Measures Survey (CHMS). Information was collected via a home interview and a visit to a mobile examination centre. The weighted sample represented 6,914,124 adults in Canada aged 18–79 who were not taking any thyroid-related medication. Urinary fluoride concentrations were measured in spot samples using an ion selective electrode and adjusted for specific gravity (UFSG). Serum TSH levels provided a measure of thyroid function. Multivariable regression analyses examined the relationship between UFSG and TSH, controlling for covariates. Results: Approximately 17.8% of participants fell in the moderately-to-severely iodine deficient range. The mean (SD) age of the sample was 46.5 (15.6) years and the median UFSG concentration was 0.74 mg/L. Among iodine deficient adults, a 1 mg/L increase in UFSG was associated with a 0.35 mIU/L increase in TSH [95% CI: 0.06, 0.64; p = 0.01, one-tailed]. Conclusions: Adults living in Canada who have moderate-to-severe iodine deficiencies and higher levels of urinary fluoride may be at an increased risk for underactive thyroid gland activity. Keywords: Fluoride, Thyroid, Iodine status, Thyroid stimulating hormone

Abstract Background Dietary inequalities between population groups are common with older and more affluent individuals tending to have healthier diets. Differential responses to health interventions may exacerbate inequalities. Changing what foods are displayed at supermarket checkouts is one intervention that has the potential to change diets. The aim of this study was to assess whether differences in purchases of common checkout foods from supermarkets with different checkout food policies varied according to age group and social grade. Methods We analysed annual household purchase data for 2013–17 from nine leading UK supermarkets, split according to age of the main household shopper and household social grade. Checkout food policies were categorised as clear and consistent, vague or inconsistent, and none. Policies were heterogeneous but all included removal of confectionery and/or chocolate from checkouts. Mixed effects linear regression models were used to assess differences in purchases of common checkout foods (sugary confectionery, chocolate and potato crisps) by checkout food policy and whether these varied by age group or occupational social grade. Results Relative to supermarkets with no checkout food policy, 14% (95% CI: 4–22%) fewer purchases of common checkout foods per household per percentage market share were made in supermarkets with a clear and consistent policy. Adjusted mean numbers of purchases were higher in older age groups than the youngest, but there were no differences between the highest and other social grades. There were significant interactions between checkout food policy and both age group and social grade. In supermarkets with clear and consistent policies, 23% (6–36%), 20% (2–34%), and 23% (7–37%) fewer purchases were made in age groups 45–54, 55–64 and 65+ years respectively, compared to all groups combined. In supermarkets with clear and consistent policies, there were 21% (4–35%), 26% (9–39%) and 21% (3–35%) fewer purchases made by households in the highest two and lowest social grades respectively, compared to all groups combined. Conclusions Households with older main shoppers and those in the most and least affluent social grades may be most responsive to supermarket checkout food policies. As older and more affluent groups tend to have healthier diets overall, it is unlikely that supermarket checkout food policies contribute to greater dietary equity.

Summary: Caspase-11 sensing of intracellular lipopolysaccharide (LPS) plays critical roles during infections and sepsis. However, the key cell types that sense intracellular LPS and their contributions to the host responses at the organismal level are not completely clear. Here, we show that macrophage/monocyte-specific caspase-11 plays a dominant role in mediating the pathological manifestations of endotoxemia, including gasdermin D (GSDMD) activation, interleukin (IL)-1β, IL-18, and damage-associated molecular pattern (DAMP) release, tissue damage, and death. Surprisingly, caspase-11 expression in CD11c+ cells and intestinal epithelial cells (IECs) plays minor detrimental roles in LPS shock. In contrast, caspase-11 expression in neutrophils is dispensable for LPS-induced lethality. Importantly, caspase-11 sensing of intracellular LPS in LyzM+ myeloid cells and MRP8+ neutrophils, but not CD11c+ cells and IECs, is necessary for bacterial clearance and host survival during intracellular bacterial infection. Thus, we reveal hierarchical cell-type-specific roles of caspase-11 that govern the host-protective and host-detrimental functions of the cytosolic LPS surveillance.

Competition for resources shapes ecological and evolutionary relationships. Physiological capacities such as in locomotor performance can influence the fitness of individuals by increasing competitive success. Social hierarchy too can affect outcomes of competition by altering locomotor behaviour or because higher ranking individuals monopolize resources. Here, we tested the hypotheses that competitive success is determined by sprint performance or by social status. We show that sprint performance of individuals measured during escape responses (fast start) or in an accelerated sprint test did not correlate with realized sprint speed while competing for food within a social group of five fish; fast start and accelerated sprint speed were higher than realized speed. Social status within the group was the best predictor of competitive success, followed by realized speed. Social hierarchies in zebrafish are established within 7 days of their first encounter, and interestingly, there was a positive correlation between social status and realized speed 1 and 4 days after fish were placed in a group, but not after 7 days. These data indicate that physiological performance decreases in importance as social relationships are established. Also, maximal physiological capacities were not important for competitive success, but swimming speed changed with social context.

Immune dysfunction is an important factor driving mortality and adverse outcomes after trauma but remains poorly understood, especially at the cellular level. To deconvolute the trauma-induced immune response, we applied single-cell RNA sequencing to circulating and bone marrow mononuclear cells in injured mice and circulating mononuclear cells in trauma patients. In mice, the greatest changes in gene expression were seen in monocytes across both compartments. After systemic injury, the gene expression pattern of monocytes markedly deviated from steady state with corresponding changes in critical transcription factors, which can be traced back to myeloid progenitors. These changes were largely recapitulated in the human single-cell analysis. We generalized the major changes in human CD14+ monocytes into 6 signatures, which further defined 2 trauma patient subtypes (SG1 vs. SG2) identified in the whole-blood leukocyte transcriptome in the initial 12 hours after injury. Compared with SG2, SG1 patients exhibited delayed recovery, more severe organ dysfunction, and a higher incidence of infection and noninfectious complications. The 2 patient subtypes were also recapitulated in burn and sepsis patients, revealing a shared pattern of immune response across critical illness. Our data will be broadly useful to further explore the immune response to inflammatory diseases and critical illness.

Breast cancer is a very heterogeneous disease, and ~30% of breast cancer patients succumb to metastasis, highlighting the need to understand the mechanisms of breast cancer progression in order to identify new molecular targets for treatment. Sphingosine kinase 1 (SK1) has been shown to be upregulated in patients with breast cancer, and several studies have suggested its involvement in breast cancer progression and/or metastasis, mostly based on cell studies. In this work we evaluated the role of SK1 in breast cancer development and metastasis using a transgenic breast cancer model, mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT), that closely resembles the characteristics and evolution of human breast cancer. The results show that SK1 deficiency does not alter tumor latency or growth, but significantly increases the number of metastatic lung nodules and the average metastasis size in the lung of MMTV-PyMT mice. Additionally, analysis of Kaplan-Meier plotter of human disease shows that high SK1 mRNA expression can be associated with a better prognosis for breast cancer patients. These results suggest a metastasis-suppressing function for SK1 in the MMTV-PyMT model of breast cancer, and that its role in regulating human breast cancer progression and metastasis may be dependent on the breast cancer type.

Summary: Isogenic cells manifest distinct cellular fates for a single stress; however, the nongenetic mechanisms driving such fates remain poorly understood. Here, we implement a robust multi-channel live-cell imaging approach to uncover noncanonical factors governing cell fate. We show that in response to acute glucose removal (AGR), budding yeast undergoes distinct fates, becoming either quiescent or senescent. Senescent cells fail to resume mitotic cycles following glucose replenishment but remain responsive to nutrient stimuli. Whereas quiescent cells manifest starvation-induced adaptation, senescent cells display perturbed endomembrane trafficking and defective nucleus-vacuole junction (NVJ) expansion. Surprisingly, senescence occurs even in the absence of lipid droplets. Importantly, we identify the nutrient-sensing kinase Rim15 as a key biomarker predicting cell fates before AGR stress. We propose that isogenic yeast challenged with acute nutrient shortage contains determinants influencing post-stress fate and demonstrate that specific nutrient signaling, stress response, trafficking, and inter-organelle biomarkers are early indicators for long-term fate outcomes.

Changes in cardiometabolic functions contribute to increased morbidity and mortality after chronic spinal cord injury. Despite many advancements in discovering SCI-induced pathologies, the cardiometabolic risks and divergences in severity-related responses have yet to be elucidated. Here, we examined the effects of SCI severity on functional recovery and cardiometabolic functions following moderate (50 kdyn) and severe (75 kdyn) contusions in the thoracic-8 (T8) vertebrae in mice using imaging, morphometric, and molecular analyses. Both severities reduced hindlimbs motor functions, body weight (g), and total body fat (%) at all-time points up to 20 weeks post-injury (PI), while only severe SCI reduced the total body lean (%). Severe SCI increased liver echogenicity starting from 12 weeks PI, with an increase in liver fibrosis in both moderate and severe SCI. Severe SCI mice showed a significant reduction in left ventricular internal diameters and LV volume at 20 weeks PI, associated with increased LV ejection fraction as well as cardiac fibrosis. These cardiometabolic dysfunctions were accompanied by changes in the inflammation profile, varying with the severity of the injury, but not in the lipid profile nor cardiac or hepatic tyrosine hydroxylase innervation changes, suggesting that systemic inflammation may be involved in these SCI-induced health complications.

Introduction: Innovative motor therapies have attempted to reduce upper extremity impairment after stroke but have not made substantial improvement as over 50% of people post-stroke continue to have sensorimotor deficits affecting their self-care and participation in daily activities. Intervention studies have focused on the role of increased dosing, however recent studies have indicated that timing of rehabilitation interventions may be as important as dosing and importantly, that dosing and timing interact in mediating effectiveness. This study is designed to empirically test dosing and timing.Methods and Analysis: In this single-blinded, interventional study, subjects will be stratified on two dimensions, impairment level (Fugl-Meyer Upper Extremity Assessment (FM) and presence or absence of Motor Evoked Potentials (MEPs) as follows; (1) Severe, FM score 10–19, MEP+, (2) Severe, FM score 10–19, MEP–, (3) Moderate, FM score 20–49, MEP+, (4) Moderate, FM score 20–49, MEP–. Subjects not eligible for TMS will be assigned to either group 2 (if severe) or group 3 (if moderate). Stratified block randomization will then be used to achieve a balanced assignment. Early Robotic/VR Therapy (EVR) experimental group will receive in-patient usual care therapy plus an extra 10 h of intensive upper extremity therapy focusing on the hand using robotically facilitated rehabilitation interventions presented in virtual environments and initiated 5–30 days post-stroke. Delayed Robotic/VR Therapy (DVR) experimental group will receive the same intervention but initiated 30–60 days post-stroke. Dose-matched usual care group (DMUC) will receive an extra 10 h of usual care initiated 5–30 days post-stroke. Usual Care Group (UC) will receive the usual amount of physical/occupational therapy.Outcomes: There are clinical, neurophysiological, and kinematic/kinetic measures, plus measures of daily arm use and quality of life. Primary outcome is the Action Research Arm Test (ARAT) measured at 4 months post-stroke.Discussion: Outcome measures will be assessed to determine whether there is an early time period in which rehabilitation will be most effective, and whether there is a difference in the recapture of premorbid patterns of movement vs. the development of an efficient, but compensatory movement strategy.Ethical Considerations: The IRBs of New Jersey Institute of Technology, Rutgers University, Northeastern University, and Kessler Foundation reviewed and approved all study protocols. Study was registered in https://ClinicalTrials.gov (NCT03569059) prior to recruitment. Dissemination will include submission to peer-reviewed journals and professional presentations.

Developing a typology of heterogeneous fishing practices through the use of métier analysis is a useful step in understanding the dynamics of fishing fleets and enabling effective implementation of management outcomes. We develop a non-hierarchical clustering framework to quantitatively categorize individual fishing events to a particular métier based on corresponding catch composition, gear configuration, and spatial and temporal references. Our clustering framework has several innovations over predecessors including: (i) introducing alternative methods for encoding and transforming fisheries data; (ii) variable (feature) selection methods; (iii) complementary metrics and methods for internal métier validation; and (iv) use of a network science method to model and analyze fishing practices. To demonstrate applicability, we apply this framework to the Australian Eastern Tuna and Billfish Fishery (ETBF), a multispecies pelagic longline fishery with a diversity of fishing practices. We identified a total of seven stable métiers within the ETBF. While each métier was characterized by a predominant target species, they were differentiated more by seasonal and temporal references (e.g., time of set, month, latitude) than gear configuration (e.g., hooks per basket) or target species. By collapsing a large amount of high-dimensional operational data into a relatively uniform and limited number of components, decision-makers can more easily evaluate the likely consequences of management and design policies that target a particular métier.

The globalization of healthcare has had a significant impact on healthcare delivery and human workforce development in many countries. Consequently, many educational institutions have had to revise the content of their medical curricula to focus on training and preparing future doctors to effectively cater to the needs of the “modern patient”. The focus covers broader aspects of medicine that include environmental and professional culture and the impact of socioeconomic inequalities on the delivery of healthcare. Therefore, in most global health (GH) education programs described, students undertake a specific part of their clinical rotation in a foreign (non-western) educational setting to facilitate authentic learning experiences within a resource-constrained environment. In this paper we present a unique Global Health program for medical interns between the University Medical Center Groningen, Netherlands and the Sint Elisabeth Hospital, Curaçao, Dutch Caribbean that has, for more than 50 years, been providing a one-year internship program as part of the formal Groningen curriculum. The program we describe, illustrates the essential requirements for best practice in GH education that fully complies with recently proposed GH program objectives and competencies. The characteristics of our long running program show the basic requirements necessary for sustainable and successful health education collaboration between institutions in high- and low/middle income countries. Finally, this program, which is part of a Dutch medical curriculum, not only provided unique global health learning experiences for the interns, but importantly also contributed to improving the general healthcare delivery services on the island of Curaçao as well.

The endocannabinoid system (ECS) acts as a negative feedback mechanism to suppress synaptic transmission and plays a major role in a diverse range of brain functions including, for example, the regulation of mood, energy balance, and learning and memory. The function and dysfunction of the ECS are strongly implicated in multiple psychiatric, neurological, and neurodegenerative diseases. Cannabinoid type 1 receptor (CB1R) is the most abundant G protein-coupled receptor (GPCR) expressed in the brain and, as for any synaptic receptor, CB1R needs to be in the right place at the right time to respond appropriately to changing synaptic circumstances. While CB1R is found intracellularly throughout neurons, its surface expression is highly polarized to the axonal membrane, consistent with its functional expression at presynaptic sites. Surprisingly, despite the importance of CB1R, the interacting proteins and molecular mechanisms that regulate the highly polarized distribution and function of CB1R remain relatively poorly understood. Here we set out what is currently known about the trafficking pathways and protein interactions that underpin the surface expression and axonal polarity of CB1R, and highlight key questions that still need to be addressed.

Reliable extraction and sensitive detection of RNA from human peripheral blood mononuclear cells (PBMCs) is critical for a broad spectrum of immunology research and clinical diagnostics. RNA analysis platforms are dependent upon high-quality and high-quantity RNA; however, sensitive detection of specific responses associated with high-quality RNA extractions from human samples with limited PBMCs can be challenging. Furthermore, the comparative sensitivity between RNA quantification and best-practice protein quantification is poorly defined. Therefore, we provide herein a critical evaluation of the wide variety of current generation of RNA-based kits for PBMCs, representative of several strategies designed to maximize sensitivity. We assess these kits with a reverse transcription quantitative PCR (RT-qPCR) assay optimized for both analytically and diagnostically sensitive cell-based RNA-based applications. Specifically, three RNA extraction kits, one post-extraction RNA purification/concentration kit, four SYBR master-mix kits, and four reverse transcription kits were tested. RNA extraction and RT-qPCR reaction efficiency were evaluated with commonly used reference and cytokine genes. Significant variation in RNA expression of reference genes was apparent, and absolute quantification based on cell number was established as an effective RT-qPCR normalization strategy. We defined an optimized RNA extraction and RT-qPCR protocol with an analytical sensitivity capable of single cell RNA detection. The diagnostic sensitivity of this assay was sufficient to show a CD8+ T cell peptide epitope hierarchy with as few as 1 × 104 cells. Finally, we compared our optimized RNA extraction and RT-qPCR protocol with current best-practice immune assays and demonstrated that our assay is a sensitive alternative to protein-based assays for peptide-specific responses, especially with limited PBMCs number. This protocol with high analytical and diagnostic sensitivity has broad applicability for both primary research and clinical practice.

Social aggregation is a widespread and important phenomenon among fishes. Understanding the questions of why and how aggregations form and are subsequently maintained is a central goal for behavioral ecologists. Research in this field has shown that aggregations are typically structured, non-random associations. This indicates that fish are able to differentiate between potential group-mates and that this ability mediates their association preferences, and, ultimately, the composition of their groups. In this review, we examine the characteristics that influence the expression of social attraction among fishes, before going on to describe the recognition mechanisms that underpin social attraction. Finally, we highlight a number of outstanding questions in the field with a view to generating a more complete understanding of social aggregation in fishes.

Overcoming tolerance to tumor-associated antigens remains a hurdle for cancer vaccine-based immunotherapy. A strategy to enhance the anti-tumor immune response is the inclusion of adjuvants to cancer vaccine protocols. In this report, we generated and systematically screened over twenty gene-based molecular adjuvants composed of cytokines, chemokines, and T cell co-stimulators for the ability to increase anti-tumor antigen T cell immunity. We identified several robust adjuvants whose addition to vaccine formulations resulted in enhanced T cell responses targeting the cancer antigens STEAP1 and TERT. We further characterized direct T cell stimulation through CD80-Fc and indirect T cell targeting via the dendritic cell activator Flt3L-Fc. Mechanistically, intramuscular delivery of Flt3L-Fc into mice was associated with a significant increase in infiltration of dendritic cells at the site of administration and trafficking of activated dendritic cells to the draining lymph node. Gene expression analysis of the muscle tissue confirmed a significant up-regulation in genes associated with dendritic cell signaling. Addition of CD80-Fc to STEAP1 vaccine formulation mimicked the engagement provided by DCs and increased T cell responses to STEAP1 by 8-fold, significantly increasing the frequency of antigen-specific cells expressing IFNγ, TNFα, and CD107a for both CD8+ and CD4+ T cells. CD80-Fc enhanced T cell responses to multiple tumor-associated antigens including Survivin and HPV, indicating its potential as a universal adjuvant for cancer vaccines. Together, the results of our study highlight the adjuvanting effect of T cell engagement either directly, CD80-Fc, or indirectly, Flt3L-Fc, for cancer vaccines.

Tubulocystic renal cell carcinoma (TCC) is a rare and newly recognized variant of renal cell carcinoma, which may mimic benign cystic disease of the kidney. To our knowledge, we present the first reported case of a patient who, despite standard preoperative workup, developed TCC of his native kidney soon after receiving kidney transplantation. He was appropriately treated with native nephrectomy and has had no signs of reoccurrence 7 years postoperatively. Given the significant risk of malignancy in renal transplant patients, this case emphasizes the need for close monitoring of native cystic disease before and after transplantation, with low threshold to proceed with surgical intervention.

Even as new medical modalities, diagnostics, and technologies are rapidly changing healthcare, providing patients with safe, high-quality care remains the central focus. To provide safe patient care, healthcare providers are obligated to demonstrate and maintain the necessary competence. As more healthcare disciplines move toward a competency-based education model, it is essential to extend the competence verification from the academic educational level to the patient’s bedside. The nutrition-focused physical exam (NFPE) is a competency recently adopted by registered dietitian nutritionists (RDNs) for assessing patients’ nutritional status. Being a newly acquired skill, validated tools are required to measure NFPE competence during routine clinical practice. The Interactive Nutrition Specific Physical Exam Competency Tool (INSPECT) is a new tool developed specifically to observe and measure RDNs’ NFPE competence in clinical settings. The INSPECT was designed and validated for content using expert RDNs’ input in the first and second phases of the study. This current study aimed to assess the reliability of the INSPECT through multi-site observations by clinical supervisors evaluating RDNs’ NFPE competency during patient assessment. The INSPECT exhibited good inter-rater reliability (ICC = 0.78 for the first assessment and ICC = 0.68 for the second assessment), moderate to strong intra-rater reliability for 37 of 41 items (Spearman rho = 0.54 to 1.0), and excellent internal consistency (Cronbach’s α = 0.86 for the first assessment and α = 0.92 for the second assessment). In total, 10 out of the 11 INSPECT subsets showed good to excellent internal consistency (α ranging from 0.70 to 0.98). The results demonstrate that the INSPECT is a reliable tool, is stable over time, and has good agreement and excellent consistency between raters. The INSPECT can be a valuable tool to measure, promote and maintain RDNs’ NFPE competence in authentic acute care settings.

The Interactive Nutrition Specific Physical Exam Competency Tool (INSPECT) is a tool designed specifically to observe and measure registered dietitian nutritionists’ (RDNs) nutrition-focused physical exam (NFPE) competence in authentic acute care settings. The initial INSPECT items were generated and tested for content and face validity using expert RDNs’ input. The INSPECT was further examined for inter-rater, intra-rater, and internal consistency using clinical supervisor observations of RDNs performing NFPE on patients in real-life acute care settings. These previous studies showed the INSPECT to have excellent content validity, acceptable face validity, good inter-rater reliability, moderate to strong intra-rater reliability, and excellent internal consistency. In the current study, the Rasch measurement model was applied to examine the item-level properties of the INSPECT. Results confirm that the INSPECT measured a single construct. All items fit the established criteria for clinical observations of >0.5 and 40% variance explained by the Rasch dimension as well as a sub-dimension based on item difficulty from the principal component analysis of the first contrast Rasch residuals. Rasch rating scale analysis revealed that the rating scale and majority of the items (39/41) fit the Rasch model. Rasch item hierarchy analysis matched the a priori hypothesized hierarchy for the top-most and bottom-most items. Ceiling effects were seen for three items (hand hygiene, personal protective equipment, and patient position) and one item (handgrip using hand dynamometer) reached the floor effect. Rasch reliability assessment demonstrated high person reliability (0.86), high item reliability (0.96), and person separation of 3.56 ability levels. The principal component analysis of residuals revealed two factors based on item difficulty, one for micronutrient exam and another for macronutrient exam, initial steps, and bedside manner. The resulting two factors may likely be due to a sub-dimension of the latent NFPE trait. Overall, the INSPECT items were found to have good item-level psychometrics. Continued testing of the INSPECT with RDNs at different ability levels will help to determine cut-off scores ranging from novice to expert. Establishing cut-off scores for the INSPECT will further enhance the utility of the tool.

The purpose of this study was to improve the understanding of how perceptions of practicing specific medical specialties contribute to career decisions. Participants included medical students and residents from training programs in West Virginia. Focus groups were conducted between September 2013 and February 2015. Thematic content analysis of focus group interviews was used to identify phrases indicating perceptions of person- versus technique-oriented personality medical specialties. “Self-focused” and “other-focused” themes emerged within person-oriented comments. Factors affecting “self”, including work/life balance and salary and practice environment, made certain specialties more appealing. Technique-oriented comments included working in a hospital setting, performing technical procedures, and solving immediate problems as opposed to long-term management. Technique-oriented comments from students emphasized learning about disease, and resident comments emphasized treatment as their primary focus. The majority of participants’ comments suggested that a strong patient-doctor relationship and continuity of care are rewarding components of primary care and more compatible with person-oriented personalities. Participants selecting person-oriented specialties viewed rural practice locations more favorably, especially the opportunity to become an integral part of a community. Participants selecting technique-oriented specialties preferred limited interactions with patients and stated that problem-solving and learning about disease states were the most appealing aspects of practicing medicine.

Previous research has shown that developing second language (L2) academic writers use a limited set of reporting verbs in comparison to more advanced writers (Biber & Reppen, 1998; Hinkel, 2003; Kwon, Staples, & Partridge, 2018; Neff et al., 2003; Staples & Reppen, 2016). These writers also tend to rely on verbs that are typical for conversation (Biber et al., 1999). The present study examines the effects of corpus-informed instruction on developing L2 writers’ learning of reporting verbs in a first-year writing course by comparing drafts of literature reviews before and after a workshop. The forty-five-minute workshop was designed to improve L2 writers’ lexical and functional uses of reporting verbs using corpus-informed materials. The researchers compared the literature review drafts written by 40 students who participated in the workshop to 38 randomly chosen drafts from our corpus. The results show an increase in the experimental groups’ reporting verb lexical variety and a decrease in the use of verb types used in speech in favor of types used in academic writing. The results suggest that corpus-informed instruction may support L2 writers in the development of lexical and functional reporting verb use.

Background: Exposure to fluoride has been linked with increased prevalence of attention deficit hyperactivity disorder (ADHD) in the United States and symptoms of inattention in Mexican children. We examined the association between fluoride exposure and attention outcomes among youth living in Canada. Method: We used cross-sectional data collected from youth 6 to 17 years of age from the Canadian Health Measures Survey (Cycles 2 and 3). Urinary fluoride concentration adjusted for specific gravity (UFSG) was available for 1877 participants. Water fluoride concentration measured in tap water samples was available for 980 participants. Community water fluoridation (CWF) status was determined by viewing reports on each city’s website or contacting the water treatment plant. We used logistic regression to test the association between the three measures of fluoride exposure and ADHD diagnosis. Linear regression was used to examine the relationship between the three measures of fluoride exposure and the hyperactivity/inattention score on the Strengths and Difficulties Questionnaire (SDQ). Results: UFSG did not significantly predict ADHD diagnosis or hyperactive/inattentive symptoms. A 1 mg/L increase in tap water fluoride level was associated with a 6.1 times higher odds of an ADHD diagnosis (95% CI = 1.60, 22.8). A significant interaction between age and tap water fluoride level (p = .03) indicated a stronger association between tap water fluoride and hyperactivity/inattention symptoms among older youth. A 1 mg/L increase in water fluoride level was associated with a 1.5 SDQ score increase (95% CI: 0.23, 2.68, p = .02) for youth at the 75th percentile of age (14 years old). Similarly, there was a significant interaction between age and CWF. At the 75th percentile of age (14 years old), those living in a fluoridated region had a 0.7-point higher SDQ score (95% CI = 0.34, 1.06, p

Extensive evaluation of RNA-seq methods have demonstrated that no single algorithm consistently outperforms all others. Removal of unwanted variation (RUV) has also been proposed as a method for stabilizing differential expression (DE) results. Despite this, it remains a challenge to run multiple RNA-seq algorithms to identify significant differences common to multiple algorithms, whilst also integrating and assessing the impact of RUV into all algorithms. consensusDE was developed to automate the process of identifying significant DE by combining the results from multiple algorithms with minimal user input and with the option to automatically integrate RUV. consensusDE only requires a table describing the sample groups, a directory containing BAM files or preprocessed count tables and an optional transcript database for annotation. It supports merging of technical replicates, paired analyses and outputs a compendium of plots to guide the user in subsequent analyses. Herein, we assess the ability of RUV to improve DE stability when combined with multiple algorithms and between algorithms, through application to real and simulated data. We find that, although RUV increased fold change stability between algorithms, it demonstrated improved FDR in a setting of low replication for the intersect, the effect was algorithm specific and diminished with increased replication, reinforcing increased replication for recovery of true DE genes. We finish by offering some rules and considerations for the application of RUV in a consensus-based setting. consensusDE is freely available, implemented in R and available as a Bioconductor package, under the GPL-3 license, along with a comprehensive vignette describing functionality: http://bioconductor.org/packages/consensusDE/.

Abstract Objective The National Health Service (NHS) in England planned a national diabetes prevention programme (NHS DPP) with phased implementation. Evidence-based guidelines and service specifications support efficient and effective translation of research into practice. We aimed to evaluate the use of a structured mapping exercise to appraise how evidence, service specification and early phase practice could inform recommendations to guide subsequent implementation of the NHS DPP. Results The mapping exercise facilitated comparison and appraisal of key components from different documentary sources (evidence-based NICE guidelines, service specification, and provider documents). Key components were categorised into (A) pathways into programmes, (B) intervention content (C) inequalities and (D) quality assurance and staff training. We identified where key components were the same (accordance), where they varied (discrepancies) and where they were lacking (discontinuities), across the documentary sources. For example there was discrepancy in intervention duration and discontinuity in intervention enrolment procedures. This mapping exercise was useful to compare the fidelity in translation of evidence-based guidance into service specification and programme documents, thus identifying where future service implementation might be improved. This method may be applicable for use with other health conditions where research evidence requires translation into real world population programmes.

Owing to the propensity for uranium(III) compounds to undergo disproportionation, uranium-element multiple bonds involving uranium(III) oxidation states remain rare. Here the authors report hexauranium-methanediide rings that formally contain uranium(III)- and uranium(IV)-methanediides supported by alternating halide and arene bridges.

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease caused by mutations in the dystrophin gene. We generated induced pluripotent stem cells (iPSCs) from a 13-year-old male patient carrying a deletion mutation of exons 45–50; iPSCs were subsequently differentiated into cardiomyocytes. iPSCs exhibit expression of the pluripotent markers (SOX2, NANOG, OCT4), differentiation capacity into the three germ layers, normal karyotype, genetic identity to the skin biopsy dermal fibroblasts and the patient-specific dystrophin mutation.

Abstract Objective This paper highlights recruitment and retention problems identified during a pilot randomised controlled trial and process evaluation. The pilot trial aimed to evaluate the feasibility and acceptability of a web-delivered weight loss intervention for postnatal women and associated trial protocol. Results General practice database searches revealed low rates of eligible postnatal women per practice. 16 (10%) of the 168 identified women were recruited and randomised, seven to the intervention and nine to the control. 57% (4/7) of the intervention women completed 3 month follow-up measurements in comparison to 56% (5/9) in the control group. By 12 months, retention in the intervention group was 43% (3/7), with 2/7 women active on the website, in comparison to 44% (4/9) of the control group. Interview findings revealed the web as an acceptable method for delivery of the intervention, with the suggestion of an addition of a mobile application. Alternative recruitment strategies, using health visitor appointments, midwifery departments or mother and baby/toddler groups, should be explored. Greater involvement of potential users should enable better recruitment methods to be developed. Trial registration ISRCTN: ISRCTN48086713, Registered 26 October 2012

Abstract Background Although the Short Test of Functional Health Literacy in Adults (S-TOFHLA) is widely used, misidentification of individuals with low health literacy (HL) in specific HL dimensions, like numeracy, is a concern. We examined the degree to which individuals scored as “adequate” HL on the S-TOFHLA would be considered as having low HL by two additional numerical measures. Methods English-speaking adults aged 45–75 years were recruited from a large, urban academic medical center and a community foodbank in the United States. Participants completed the S-TOFHLA, the Subjective Numeracy Scale (SNS), and the Graphical Literacy Measure (GL), an objective measure of a person’s ability to interpret numeric information presented graphically. Established cut-points or a median split classified participants and having high and low numeracy. Results Participants (n = 187), on average were: aged 58 years; 63% female; 70% Black/African American; and 45% had a high school degree or less. Of those who scored “adequate” on the S-TOFHLA, 50% scored low on the SNS and 40% scored low on GL. Correlation between the S-TOFHLA and the SNS Total was moderate (r = 0.22, n = 186, p = 0.01), while correlation between the S-TOFHLA and the GL Total was large (r = 0.53, n = 187, p ≤ 0.01). Conclusions Findings suggest that the S-TOFHLA may not capture an individuals’ HL in the dimension of numeracy. Efforts are needed to develop more encompassing and practical strategies for identifying those with low HL for use in research and clinical practice. Trial registration NCT02151032 (retrospectively registered: May 30, 2014).

Sphingolipids constitute a dynamic metabolic network that interconnects several bioactive molecules, including ceramide (Cer), sphingosine (Sph), Sph 1-phosphate, and Cer 1-phosphate. The interconversion of these metabolites is controlled by a cohort of at least 40 enzymes, many of which respond to endogenous or exogenous stimuli. Typical probing of the sphingolipid pathway relies on sphingolipid mass levels or determination of the activity of individual enzymes. Either approach is unable to provide a complete analysis of flux through sphingolipid metabolism, which, given the interconnectivity of the sphingolipid pathway, is critical information to identify nodes of regulation. Here, we present a one-step in situ assay that comprehensively probes the flux through de novo sphingolipid synthesis, post serine palmitoyltransferase, by monitoring the incorporation and metabolism of the 17 carbon dihydrosphingosine precursor with LC/MS. Pulse labeling and analysis of precursor metabolism identified sequential well-defined phases of sphingolipid synthesis, corresponding to the activity of different enzymes in the pathway, further confirmed by the use of specific inhibitors and modulators of sphingolipid metabolism. This work establishes precursor pulse labeling as a practical tool for comprehensively studying metabolic flux through de novo sphingolipid synthesis and complex sphingolipid generation.

The reactivity of f-block complexes is primarily defined by single-electron oxidations and σ-bond metathesis. Here, Liddle and co-workers provide evidence that a uranium complex can undergo reversible oxidative addition and reductive elimination, demonstrating transition metal-like reactivity within f-block chemistry.

Substantial variability exists with regard to autism service provision around the world. Service disparities observed in many low- and middle-income countries may be driven, in part, by limited autism knowledge; however, measurement limitations have made it difficult to quantify autism knowledge across countries. The current study uses the autism stigma and knowledge questionnaire (ASK-Q) to quantify autism knowledge and stigma between different countries and demographics. The current study compiled data from 6830 participants collected using adapted versions of the ASK-Q administered in 13 different countries, representing four different continents. Structural equation modeling was used to examine how autism knowledge varied across country and individual factors. Results reveal cross country variability with a large, 17-point difference between the countries with the highest knowledge (Canada) and the lowest knowledge (Lebanon). As expected, countries with higher economies had higher levels of knowledge. We also documented differences based on country worldview, participant occupation, gender, age, and education level. These results help to identify specific regions and populations that might most need greater information about autism.

As the COVID-19 pandemic rages unabated, and with more infectious variants, vaccination may offer a way to transit out of strict restrictions on physical human interactions to curb the virus spread and prevent overwhelming the healthcare system. However, vaccine hesitancy threatens to significantly impact our progress towards achieving this. It is thus important to understand the sentiments regarding vaccination for different segments of the population to facilitate the development of effective strategies to persuade these groups. Here, we surveyed the COVID-19 vaccination sentiments among a highly educated group of graduate students from the National University of Singapore (NUS). Graduate students who are citizens of 54 different countries, mainly from Asia, pursue studies in diverse fields, with 32% expressing vaccine hesitancy. Citizenship, religion, country of undergraduate/postgraduate studies, exposure risk and field of study are significantly associated with vaccine sentiments. Students who are Chinese citizens or studied in Chinese Universities prior to joining NUS are more hesitant, while students of Indian descent or studied in India are less hesitant about vaccination. Side effects, safety issues and vaccine choice are the major concerns of the hesitant group. Hence, this study would facilitate the development of strategies that focus on these determinants to enhance vaccine acceptance.