Electrically stimulated in vitro heart cell mimic of acute exercise reveals novel immediate cellular responses to exercise: Reduced contractility and metabolism, but maintained calcium cycling and increased myofilament calcium sensitivity.

Cardiac cellular responses to acute exercise remain undescribed. We present a model for mimicking acute aerobic endurance exercise to freshly isolated cardiomyocytes by evoking exercise-like contractions over prolonged periods of time with trains of electrical twitch stimulations. We then investigated immediate contractile, Ca2+ , and metabolic responses to acute exercise in perfused freshly isolated left ventricular rat cardiomyocytes, after a matrix-design optimized protocol and induced a mimic for acute aerobic endurance exercise by trains of prolonged field twitch stimulations. Acute exercise decreased cardiomyocyte fractional shortening 50%-80% (p < .01). This was not explained by changes to intracellular Ca2+ handling (p > .05); rather, we observed a weak insignificant Ca2+ transient increase (p = .11), while myofilament Ca2+ sensitivity increased 20%-70% (p < .05). Acidic pH 6.8 decreased fractional shortening 20%-70% (p < .05) because of 20%-30% decreased Ca2+ transients (p < .05), but no difference occurred between control and acute exercise (p > .05). Addition of 1 or 10 mM La- increased fractional shortening in control (1 mM La- : no difference, p > .05; 10 mM La- : 20%-30%, p < .05) and acute exercise (1 mM La- : 40%-90%, p < .01; 10 mM La- : 50%-100%, p < .01) and rendered acute exercise indifferent from control (p > .05). Intrinsic autofluorescence showed a resting NADstate of 0.59 ± 0.04 and FADstate of 0.17 ± 0.03, while acute exercise decreased NADH/FAD ratio 8% (p < .01), indicating intracellular oxidation. In conclusion, we show a novel approach for studying immediate acute cardiomyocyte responses to aerobic endurance exercise. We find that acute exercise in cardiomyocytes decreases contraction, but Ca2+ handling and myofilament Ca2+ sensitivity compensate for this, while acidosis and reduced energy substrate and mitochondrial ATP generation explain this.

Understanding barriers to prescription retrieval and attitudes toward mental health medications at a university student health services pharmacy.

OBJECTIVES: To describe barriers affecting college students' ability to retrieve prescription medications for mental health conditions from a community pharmacy embedded within a student health center on a university campus and then describe beliefs and attitudes toward the use of those medications. METHODS: We conducted a cross-sectional study of college students who were prescribed a medication(s) for a mental health condition(s) and left the medication(s) unclaimed at the pharmacy for at least 5 days. Eligible participants were identified through routine reminder telephone calls and then provided a link to an online survey via e-mail. The survey collected information on demographics, prescription retrieval barriers, and medication beliefs and attitudes using 2 validated questionnaires. Data were summarized using descriptive statistics and scoring methods specific to the validated questionnaires. RESULTS: A total of 83 e-mails were distributed with 46 usable responses received (55.4% response rate). Participants were undergraduate students (n = 38, 82.6%) and most frequently prescribed a medication(s) for depression (n = 21, 45.7%) or anxiety (n = 16, 34.8%). Forgetting to pick up their medication was the most commonly cited reason for prescription nonretrieval (n = 32, 69.6%) followed by limited pharmacy hours (n = 21, 45.7%). Participants generally viewed the necessity of their medication(s) as outweighing their concerns about their medication(s), and they generally viewed themselves as medication adherent. CONCLUSIONS: University students taking medications for mental health conditions identified forgetfulness and limited pharmacy hours as the most common reasons for not retrieving prescriptions. Students generally viewed their medication(s) as necessary and themselves as medication adherent, suggesting that attitudes and beliefs may play a smaller role in medication use behaviors in this population. Future research is needed to develop and evaluate interventions that improve medication adherence in college students, potentially focused on reminder-based interventions.

The role of mTOR-mediated signals during haemopoiesis and lineage commitment.

The serine/threonine protein kinase mechanistic target of rapamycin (mTOR) has been implicated in the regulation of an array of cellular functions including protein and lipid synthesis, proliferation, cell size and survival. Here, we describe the role of mTOR during haemopoiesis within the context of mTORC1 and mTORC2, the distinct complexes in which it functions. The use of conditional transgenic mouse models specifically targeting individual mTOR signalling components, together with selective inhibitors, have generated a significant body of research emphasising the critical roles played by mTOR, and individual mTOR complexes, in haemopoietic lineage commitment and development. This review will describe the profound role of mTOR in embryogenesis and haemopoiesis, underscoring the importance of mTORC1 at the early stages of haemopoietic cell development, through modulation of stem cell potentiation and self-renewal, and erythroid and B cell lineage commitment. Furthermore, the relatively discrete role of mTORC2 in haemopoiesis will be explored during T cell development and B cell maturation. Collectively, this review aims to highlight the functional diversity of mTOR signalling and underline the importance of this pathway in haemopoiesis.

Improving patient safety by enhancing raising concerns at medical school.

BACKGROUND: Doctors and medical students have a professional responsibility to raise concerns. Failure to raise concerns may compromise patient safety. It is widely known that medical students frequently encounter unprofessional behaviours in the workplace, but little is known about the barriers to raising concerns amongst medical students. This paper explores these issues and discusses some innovations in the medical undergraduate curriculum, offering a good practice model for other medical and healthcare curricula. We set out to ascertain the attitudes and experiences of medical students in relation to raising concerns. This data was then used to innovate the raising concerns curriculum, and access to the raising concerns system, in order to fundamentally improve patient safety and experience, as well as the student experience. METHODS: The authors conducted a mixed methods quantitative and qualitative research study. Research was based at a UK medical school and involved data collection using an anonymous, voluntary survey emailed to all medical students (n = 363) as well as voluntary attendance focus groups (n = 24) recruited by email. Both tools investigated student attitudes towards raising concerns and explored student ideas for solutions to improving the process. The focus group data was thematically analysed by three researchers. RESULTS: The authors identified five key themes which described medical student attitudes towards raising concerns. This article discusses these themes and the resulting work to enhance medical education within the medical school curriculum. CONCLUSIONS: More research is needed to further address the barriers that medical students find in raising concerns. However, despite being a single study in one UK medical school, the authors propose some changes which they hope may inspire other educators to build upon their raising concerns curricula to foster more transparent undergraduate cultures and ultimately improve patient experience and safety.

A Creative Communication Partnership to Promote Curricula Dissemination on Social Media.

Genome Sciences Education Outreach (GSEO) has developed innovative programs that bring leading-edge science to teachers and students in K-12 schools. Disseminating educational materials equitably and accessibly to teacher stakeholders to maximize reach and impact is challenging for many programs. Traditionally, programs connect materials with teachers through local networks, in-person professional development sessions, and at regional and national conference presentations. The need for curricular changes in 2020 spurred the proliferation of online and digital educational materials and professional development opportunities. These digital materials-now available to a worldwide audience-require a shift in dissemination strategy to enhance the potential reach of these materials both locally and nationally. This manuscript reports a case study of a dissemination approach, to create a collaboration between GSEO and CommLead (the communications master's program at the University of Washington) to promote education materials developed by the publicly-funded Genes, Environment and Me Network (GEMNet) program. This manuscript describes the development and the ad hoc implementation and evaluation of a social media campaign to expand the reach of the GEMNet curricula. With a targeted social media campaign on Facebook, GSEO was able to dramatically and affordably increase the reach of the GEMNet curricula and expand the potential impact and utilization of educational materials to a nationwide teacher audience, highlighting the potential for other similar collaborations to efficiently enhance the dissemination strategy of other education outreach programs.

Feasibility, Usability, and Acceptability of Online Mind-Body Exercise Programs for Older Adults: A Scoping Review.

Objectives: Engaging in mind-body exercises (MBEs: e.g., Tai Chi and yoga) can have physical and mental health benefits particularly for older adults. Many MBEs require precise timing and coordination of complex body postures posing challenges for online instruction. Such challenges include difficulty viewing instructors as they demonstrate different movements and lack of feedback to participants. With the shift of exercise programs to online platforms during the COVID-19 pandemic, we conducted a scoping review to examine the feasibility, usability, and acceptability of online MBE classes for older adults. Materials and Methods: We followed the scoping review methodology and adhered to the PRISMA reporting checklist. We searched five databases: Medline, Embase, CINHAL, Web of Science, and ACM digital library. Screening of articles and data extraction was conducted independently by two reviewers. Settings/Location: Online/virtual. Subjects: Older adults ≥55 years of age. Outcome Measures: Feasibility measures. Results: Of 6711 studies retrieved, 18 studies were included (715 participants, mean age 66.9 years). Studies reported moderate to high retention and adherence rates (mean >75%). Older adults reported online MBE classes were easy to use and reported high satisfaction with the online format. We also identified barriers (e.g., lack of space and privacy and unstable internet connection) and facilitators (e.g., convenience and technical support) to the online format. Opinions related to social connectedness were mixed. Conclusion: Online MBE programs for older adults appear to be a feasible and acceptable alternative to in-person programs. It is important to consider the type of exercise (e.g., MBE), diverse teaching styles, and learner needs when designing online exercise classes.

mTORC1-selective activation of translation elongation promotes disease progression in chronic lymphocytic leukemia.

Targeted deletion of Raptor, a component of mechanistic target of rapamycin complex 1 (mTORC1), reveals an essential role for mTORC1 in initiation/maintenance of leukemia in a CLL model, resulting from a failure for haemopoietic stem/progenitor cells (HSPCs) to commit to the B cell lineage. Induction of Raptor-deficiency in NSG mice transplanted with Mx1-Raptor CLL progenitor cells (PKCα-KR-transduced HSPCs) after disease establishment revealed a reduction in CLL-like disease load and a significant increase in survival in the mice. Interestingly in an aggressive CLL-like disease model, rapamycin treatment reduced disease burden more effectively than AZD2014 (dual mTORC1/2 inhibitor), indicating a skew towards mTORC1 sensitivity with more aggressive disease. Rapamycin, but not ibrutinib, efficiently targeted the eEF2/eEF2K translation elongation regulatory axis, downstream of mTORC1, resulting in eEF2 inactivation through induction of eEF2T56 phosphorylation. mTOR inhibitor treatment of primary patient CLL cells halted proliferation, at least in part through modulation of eEF2K/eEF2 phosphorylation and expression, reduced protein synthesis and inhibited expression of MCL1, Cyclin A and Cyclin D2. Our studies highlight the importance of translation elongation as a driver of disease progression and identify inactivation of eEF2 activity as a novel therapeutic target for blocking CLL progression.

PKCß Facilitates Leukemogenesis in Chronic Lymphocytic Leukaemia by Promoting Constitutive BCR-Mediated Signalling.

B cell antigen receptor (BCR) signalling competence is critical for the pathogenesis of chronic lymphocytic leukaemia (CLL). Defining key proteins that facilitate these networks aid in the identification of targets for therapeutic exploitation. We previously demonstrated that reduced PKCα function in mouse hematopoietic stem/progenitor cells (HPSCs) resulted in PKCßII upregulation and generation of a poor-prognostic CLL-like disease. Here, prkcb knockdown in HSPCs leads to reduced survival of PKCα-KR-expressing CLL-like cells, concurrent with reduced expression of the leukemic markers CD5 and CD23. SP1 promotes elevated expression of prkcb in PKCα-KR expressing cells enabling leukemogenesis. Global gene analysis revealed an upregulation of genes associated with B cell activation in PKCα-KR expressing cells, coincident with upregulation of PKCßII: supported by activation of key signalling hubs proximal to the BCR and elevated proliferation. Ibrutinib (BTK inhibitor) or enzastaurin (PKCßII inhibitor) treatment of PKCα-KR expressing cells and primary CLL cells showed similar patterns of Akt/mTOR pathway inhibition, supporting the role for PKCßII in maintaining proliferative signals in our CLL mouse model. Ibrutinib or enzastaurin treatment also reduced PKCα-KR-CLL cell migration towards CXCL12. Overall, we demonstrate that PKCß expression facilitates leukemogenesis and identify that BCR-mediated signalling is a key driver of CLL development in the PKCα-KR model.

A novel transgenic mouse strain expressing PKCßII demonstrates expansion of B1 and marginal zone B cell populations.

Protein kinase Cß (PKCß) expressed in mammalian cells as two splice variants, PKCßI and PKCßII, functions in the B cell receptor (BCR) signaling pathway and contributes to B cell development. We investigated the relative role of PKCßII in B cells by generating transgenic mice where expression of the transgene is directed to these cells using the Eµ promoter (Eµ-PKCßIItg). Our findings demonstrate that homozygous Eµ-PKCßIItg mice displayed a shift from IgD+IgMdim toward IgDdimIgM+ B cell populations in spleen, peritoneum and peripheral blood. Closer examination of these tissues revealed respective expansion of marginal zone (MZ)-like B cells (IgD+IgM+CD43negCD21+CD24+), increased populations of B-1 cells (B220+IgDdimIgM+CD43+CD24+CD5+), and higher numbers of immature B cells (IgDdimIgMdimCD21neg) at the expense of mature B cells (IgD+IgM+CD21+). Therefore, the overexpression of PKCßII, which is a phenotypic feature of chronic lymphocytic leukaemia cells, can skew B cell development in mice, most likely as a result of a regulatory influence on BCR signaling.

Protoporphyrin IX is a dual inhibitor of p53/MDM2 and p53/MDM4 interactions and induces apoptosis in B-cell chronic lymphocytic leukemia cells.

p53 is a tumor suppressor, which belongs to the p53 family of proteins. The family consists of p53, p63 and p73 proteins, which share similar structure and function. Activation of wild-type p53 or TAp73 in tumors leads to tumor regression, and small molecules restoring the p53 pathway are in clinical development. Protoporphyrin IX (PpIX), a metabolite of aminolevulinic acid, is a clinically approved drug applied in photodynamic diagnosis and therapy. PpIX induces p53-dependent and TAp73-dependent apoptosis and inhibits TAp73/MDM2 and TAp73/MDM4 interactions. Here we demonstrate that PpIX is a dual inhibitor of p53/MDM2 and p53/MDM4 interactions and activates apoptosis in B-cell chronic lymphocytic leukemia cells without illumination and without affecting normal cells. PpIX stabilizes p53 and TAp73 proteins, induces p53-downstream apoptotic targets and provokes cancer cell death at doses non-toxic to normal cells. Our findings open up new opportunities for repurposing PpIX for treating lymphoblastic leukemia with wild-type TP53.

mTORC1 activity is essential for erythropoiesis and B cell lineage commitment.

Mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that mediates phosphoinositide-3-kinase (PI3K)/AKT signalling. This pathway is involved in a plethora of cellular functions including protein and lipid synthesis, cell migration, cell proliferation and apoptosis. In this study, we proposed to delineate the role of mTORC1 in haemopoietic lineage commitment using knock out (KO) mouse and cell line models. Mx1-cre and Vav-cre expression systems were used to specifically target Raptorfl/fl (mTORC1), either in all tissues upon poly(I:C) inoculation, or specifically in haemopoietic stem cells, respectively. Assessment of the role of mTORC1 during the early stages of development in Vav-cre+Raptorfl/fl mice, revealed that these mice do not survive post birth due to aberrations in erythropoiesis resulting from an arrest in development at the megakaryocyte-erythrocyte progenitor stage. Furthermore, Raptor-deficient mice exhibited a block in B cell lineage commitment. The essential role of Raptor (mTORC1) in erythrocyte and B lineage commitment was confirmed in adult Mx1-cre+Raptorfl/fl mice upon cre-recombinase induction. These studies were supported by results showing that the expression of key lineage commitment regulators, GATA1, GATA2 and PAX5 were dysregulated in the absence of mTORC1-mediated signals. The regulatory role of mTOR during erythropoiesis was confirmed in vitro by demonstrating a reduction of K562 cell differentiation towards RBCs in the presence of established mTOR inhibitors. While mTORC1 plays a fundamental role in promoting RBC development, we showed that mTORC2 has an opposing role, as Rictor-deficient progenitor cells exhibited an elevation in RBC colony formation ex vivo. Collectively, our data demonstrate a critical role played by mTORC1 in regulating the haemopoietic cell lineage commitment.

AKT/mTORC2 Inhibition Activates FOXO1 Function in CLL Cells Reducing B-Cell Receptor-Mediated Survival.

PURPOSE: To determine whether inhibition of mTOR kinase-mediated signaling represents a valid therapeutic approach for chronic lymphocytic leukemia (CLL). EXPERIMENTAL DESIGN: Stratification of mTOR activity was carried out in patients with primary CLL samples and an aggressive CLL-like mouse model. The potency of dual mTOR inhibitor AZD8055 to induce apoptosis in primary CLL cells was assessed in the presence/absence of B-cell receptor (BCR) ligation. Furthermore, we addressed the molecular and functional impact of dual mTOR inhibition in combination with BTK inhibitor ibrutinib. RESULTS: Differential regulation of basal mTORC1 activity was observed in poor prognostic CLL samples, with elevated p4EBP1T37/46 and decreased p70S6 kinase activity, suggesting that dual mTORC1/2 inhibitors may exhibit improved response in poor prognostic CLL compared with rapalogs. AZD8055 treatment of primary CLL cells significantly reduced CLL survival in vitro compared with rapamycin, preferentially targeting poor prognostic subsets and overcoming BCR-mediated survival advantages. Furthermore, AZD8055, and clinical analog AZD2014, significantly reduced CLL tumor load in mice. AKT substrate FOXO1, while overexpressed in CLL cells of poor prognostic patients in LN biopsies, peripheral CLL cells, and mouse-derived CLL-like cells, appeared to be inactive. AZD8055 treatment partially reversed FOXO1 inactivation downstream of BCR crosslinking, significantly inhibiting FOXO1T24 phosphorylation in an mTORC2-AKT-dependent manner, to promote FOXO1 nuclear localization, activity, and FOXO1-mediated gene regulation. FOXO1 activity was further significantly enhanced on combining AZD8055 with ibrutinib. CONCLUSIONS: Our studies demonstrate that dual mTOR inhibitors show promise as future CLL therapies, particularly in combination with ibrutinib.

ICP34.5-dependent and -independent activities of salubrinal in herpes simplex virus-1 infected cells.

The small molecule salubrinal has antiviral activity against herpes simplex virus-1 (HSV-1) and inhibits dephosphorylation of eIF2 alpha mediated by the HSV-1 protein ICP34.5. We investigated whether salubrinal's activities in infected cells depend on ICP34.5. An ICP34.5 deletion mutant was as sensitive as wild type HSV-1 to salubrinal inhibition of plaque formation in Vero cells. However, salubrinal induced formation of syncytia in infected Vero cells, which was enhanced by ICP34.5 mutations. Expression of HSV-1 US11 with immediate early kinetics, which is known to suppress the effects of ICP34.5 mutations, resulted in slight resistance to salubrinal in murine embryonic fibroblasts, and substantial resistance in those cells when ICP34.5 was additionally mutated. ICP34.5 mutations, but not immediate early expression of US11, prevented salubrinal's ability to increase phosphorylation of eIF2 alpha during HSV-1 infection of Vero cells. Taken together, our data indicate that salubrinal has both ICP34.5-dependent and -independent activities in HSV-1 infected cells.