Regulation of cardiomyocyte t-tubule structure by preload and afterload: Roles in cardiac compensation and decompensation.

Mechanical load is a potent regulator of cardiac structure and function. Although high workload during heart failure is associated with disruption of cardiomyocyte t-tubules and Ca2+ homeostasis, it remains unclear whether changes in preload and afterload may promote adaptive t-tubule remodelling. We examined this issue by first investigating isolated effects of stepwise increases in load in cultured rat papillary muscles. Both preload and afterload increases produced a biphasic response, with the highest t-tubule densities observed at moderate loads, whereas excessively low and high loads resulted in low t-tubule levels. To determine the baseline position of the heart on this bell-shaped curve, mice were subjected to mildly elevated preload or afterload (1 week of aortic shunt or banding). Both interventions resulted in compensated cardiac function linked to increased t-tubule density, consistent with ascension up the rising limb of the curve. Similar t-tubule proliferation was observed in human patients with moderately increased preload or afterload (mitral valve regurgitation, aortic stenosis). T-tubule growth was associated with larger Ca2+ transients, linked to upregulation of L-type Ca2+ channels, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients advanced the heart down the declining limb of the t-tubule-load relationship. This bell-shaped relationship was lost in the absence of electrical stimulation, indicating a key role of systolic stress in controlling t-tubule plasticity. In conclusion, modest augmentation of workload promotes compensatory increases in t-tubule density and Ca2+ cycling, whereas this adaptation is reversed in overloaded hearts during heart failure progression. KEY POINTS: Excised papillary muscle experiments demonstrated a bell-shaped relationship between cardiomyocyte t-tubule density and workload (preload or afterload), which was only present when muscles were electrically stimulated. The in vivo heart at baseline is positioned on the rising phase of this curve because moderate increases in preload (mice with brief aortic shunt surgery, patients with mitral valve regurgitation) resulted in t-tubule growth. Moderate increases in afterload (mice and patients with mild aortic banding/stenosis) similarly increased t-tubule density. T-tubule proliferation was associated with larger Ca2+ transients, with upregulation of the L-type Ca2+ channel, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients placed the heart on the declining phase of the t-tubule-load relationship, promoting heart failure progression. The dependence of t-tubule structure on preload and afterload thus enables both compensatory and maladaptive remodelling, in rodents and humans.

Persistent Opioid Use After Hospital Admission From Surgery in New Zealand: A Population-Based Study.

BACKGROUND: Persistent opioid use (POU) is common after surgery and is associated with an increased risk of mortality and morbidity. There have been no population-based studies exploring POU in opioid-naïve surgical patients in New Zealand (NZ). This study aimed to determine the incidence and risk factors for POU in opioid-naïve patients undergoing surgery in all NZ hospitals. METHOD: We included all opioid-naïve patients who underwent surgery without a concomitant trauma diagnosis and received opioids after discharge from any NZ hospital between January 2007 and December 2019. Patients were considered opioid naïve if no opioids had been dispensed to them or if they did not have a prior diagnosis of an opioid-use disorder up to 365 days preceding the index date. The primary outcome was the incidence of POU, defined a priori as opioid use after discharge between 91 and 365 days. We used a multivariable logistic regression to identify risk factors for POU. RESULTS: We identified 1789,407 patients undergoing surgery with no concomitant diagnosis of trauma; 377,144 (21.1%) were dispensed opioids and 260,726 patients were eligible and included in the analysis. Of those included in the final sample, 23,656 (9.1%; 95% confidence interval [CI], 9.0%-9.2%) developed POU. Risk factors related to how opioids were prescribed included: changing to different opioid(s) after discharge (adjusted odds ratio [aOR], 3.21; 95% CI, 3.04-3.38), receiving multiple opioids on discharge (aOR, 1.37; 95% CI, 1.29-1.45), and higher total oral morphine equivalents (>400 mg) (aOR, 1.23; 95% CI, 1.23-1.45). Conversely, patients who were coprescribed nonopioid analgesics on discharge had lower odds of POU (aOR, 0.91; 95% CI, 0.87-0.95). Only small differences were observed between different ethnicities. Other risk factors associated with increased risk of POU included undergoing neurosurgery (aOR, 2.02; 95% CI, 1.83-2.24), higher comorbidity burden (aOR, 1.90; 95% CI, 1.75-2.07), preoperative nonopioid analgesic use (aOR, 1.65; 95% CI, 1.60-1.71), smoking (aOR, 1.44; 95% CI, 1.35-1.54), and preoperative hypnotics use (aOR, 1.35; 95% CI, 1.28-1.42). CONCLUSIONS: Approximately 1 in 11 opioid-naïve patients who were dispensed opioids on surgical discharge, developed POU. Potentially modifiable risk factors for POU, related to how opioids were prescribed included changing opioids after discharge, receiving multiple opioids, and higher total dose of opioids given on discharge. Clinicians should discuss the possibility of developing POU with patients before and after surgery and consider potentially modifiable risk factors for POU when prescribing analgesia on discharge after surgery.

UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation.

The RING E3 ubiquitin ligase UHRF1 is an established cofactor for DNA methylation inheritance. Nucleosomal engagement through histone and DNA interactions directs UHRF1 ubiquitin ligase activity toward lysines on histone H3 tails, creating binding sites for DNMT1 through ubiquitin interacting motifs (UIM1 and UIM2). Here, we profile contributions of UHRF1 and DNMT1 to genome-wide DNA methylation inheritance and dissect specific roles for ubiquitin signaling in this process. We reveal DNA methylation maintenance at low-density CpGs is vulnerable to disruption of UHRF1 ubiquitin ligase activity and DNMT1 ubiquitin reading activity through UIM1. Hypomethylation of low-density CpGs in this manner induces formation of partially methylated domains (PMD), a methylation signature observed across human cancers. Furthermore, disrupting DNMT1 UIM2 function abolishes DNA methylation maintenance. Collectively, we show DNMT1-dependent DNA methylation inheritance is a ubiquitin-regulated process and suggest a disrupted UHRF1-DNMT1 ubiquitin signaling axis contributes to the development of PMDs in human cancers.

A phase II study of guadecitabine combined with irinotecan vs regorafenib or TAS-102 in irinotecan-refractory metastatic colorectal cancer patients.

DNA methyltransferase inhibitors (DNMTi) have demonstrated benefit in reversing resistance to systemic therapies for several cancer types. In a phase II trial of guadecitabine and irinotecan compared to regorafenib or TAS-102 in pts with advanced mCRC refractory to irinotecan. Patients with mCRC refractory to irinotecan were randomized 2:1 to guadecitabine and irinotecan (Arm A) vs standard of care regorafenib or TAS-102 (Arm B) on a 28-day cycle. Between January 15, 2016 and October 24, 2018, 104 pts were randomized at four international sites, with 96 pts undergoing treatment, 62 in Arm A and 34 in Arm B. Median overall survival was 7.15 months for Arm A and 7.66 months for Arm B (HR 0.93, 95% CI: 0.58-1.47, P = .75). The Kaplan-Meier rates of progression free survival at 4 months were 32% in Arm A and 26% in Arm B. Common ≥Grade 3 treatment related adverse events in Arm A were neutropenia (42%), anemia (18%), diarrhea (11%), compared to Arm B pts with neutropenia (12%), anemia (12%). Guadecitabine and irinotecan had similar OS compared to standard of care TAS-102 or regorafenib, with evidence of target modulation. Clinical trial information: NCT01896856.

Smoking Status and Survival in Patients With Early-Stage Primary Cutaneous Melanoma.

Importance: While smoking is associated with a decreased incidence of cutaneous melanoma, the association of smoking with melanoma progression and death is not well defined. Objective: To determine the association of smoking with survival in patients with early-stage primary cutaneous melanoma. Design, Setting, and Participants: This cohort study performed a post hoc analysis of data derived from the randomized, multinational first and second Multicenter Selective Lymphadenectomy Trials (MSLT-I and MSLT-II). Participants were accrued for MSLT-I from January 20, 1994, to March 29, 2002; MSLT-II, from December 21, 2004, to March 31, 2014. Median follow-up was 110.0 (IQR, 53.4-120.0) months for MSLT-I and 67.6 (IQR, 25.8-110.2) months for MSLT-II. Patients aged 18 to 75 years with clinical stages I or II melanoma with a Breslow thickness of 1.00 mm or greater or Clark level IV to V and available standard prognostic and smoking data were included. Analyses were performed from October 4, 2022, to March 31, 2023. Exposure: Current, former, and never smoking. Main Outcomes and Measures: Melanoma-specific survival of patients with current, former, and never smoking status was assessed for the entire cohort and for nodal observation and among subgroups with sentinel lymph node biopsy (SLNB)-negative and SLNB-positive findings. Results: Of 6279 included patients, 3635 (57.9%) were men, and mean (SD) age was 52.7 (13.4) years. The most common tumor location was an extremity (2743 [43.7%]), and mean (SD) Breslow thickness was 2.44 (2.06) mm. Smoking status included 1077 (17.2%) current, 1694 (27.0%) former, and 3508 (55.9%) never. Median follow-up was 78.4 (IQR, 30.5-119.6) months. Current smoking was associated with male sex, younger age, trunk site, thicker tumors, tumor ulceration, and SLNB positivity. Current smoking was associated with a greater risk of melanoma-associated death by multivariable analysis for the entire study (hazard ratio [HR], 1.48 [95% CI, 1.26-1.75]; P < .001). Former smoking was not. The increased risk of melanoma-specific mortality associated with current smoking was greatest for patients with SLNB-negative melanoma (HR, 1.85 [95% CI, 1.35-2.52]; P < .001), but also present for patients with SLNB-positive melanoma (HR, 1.29 [95% CI, 1.04-1.59]; P = .02) and nodal observation (HR, 1.68 [95% CI, 1.09-2.61]; P = .02). Smoking at least 20 cigarettes/d doubled the risk of death due to melanoma for patients with SLNB-negative disease (HR, 2.06 [95% CI, 1.36-3.13]; P < .001). Conclusions and Relevance: The findings of this cohort study suggest that patients with clinical stage I and II melanoma who smoked had a significantly increased risk of death due to melanoma. Smoking status should be assessed at time of melanoma diagnosis and may be considered a risk factor for disease progression.

Mouse islet-derived stellate cells are similar to, but distinct from, mesenchymal stromal cells and influence the beta cell function.

AIMS: Evidence is accumulating of the therapeutic benefits of mesenchymal stromal cells (MSCs) in diabetes-related conditions. We have identified a novel population of stromal cells within islets of Langerhans - islet stellate cells (ISCs) - which have a similar morphology to MSCs. In this study we characterize mouse ISCs and compare their morphology and function to MSCs to determine whether ISCs may also have therapeutic potential in diabetes. METHODS: ISCs isolated from mouse islets were compared to mouse bone marrow MSCs by analysis of cell morphology; expression of cell-surface markers and extracellular matrix (ECM) components; proliferation; apoptosis; paracrine activity; and differentiation into adipocytes, chondrocytes and osteocytes. We also assessed the effects of co-culture with ISCs or MSCs on the insulin secretory capacity of islet beta cells. RESULTS: Although morphological similar, ISCs were functionally distinct from MSCs. Thus, ISCs were less proliferative and more apoptotic; they had different expression levels of important paracrine factors; and they were less efficient at differentiation down multiple lineages. Co-culture of mouse islets with ISCs enhanced glucose induced insulin secretion more effectively than co-culture with MSCs. CONCLUSIONS: ISCs are a specific sub-type of islet-derived stromal cells that possess biological behaviors distinct from MSCs. The enhanced beneficial effects of ISCs on islet beta cell function suggests that they may offer a therapeutic target for enhancing beta cell functional survival in diabetes.

Kisspeptin upregulates ß-cell serotonin production during pregnancy.

During pregnancy the maternal pancreatic islets of Langerhans undergo adaptive changes to compensate for gestational insulin resistance. The lactogenic hormones are well established to play a key role in regulating the islet adaptation to pregnancy, and one of the mechanisms through which they act is through upregulating ß-cell serotonin production. During pregnancy islet serotonin levels are significantly elevated, where it is released from the ß-cells to drive the adaptive response through paracrine and autocrine effects. We have previously shown that placental kisspeptin (KP) also plays a role in promoting the elevated insulin secretion and ß-cell proliferation observed during pregnancy, although the precise mechanisms involved are unclear. In the present study we investigated the effects of KP on expression of pro-proliferative genes and serotonin biosynthesis within rodent islets. Whilst KP had limited effect on pro-proliferative gene expression at the time points tested, KP did significantly stimulate expression of the serotonin biosynthesis enzyme Tph-1. Furthermore, the islets of pregnant ß-cell-specific GPR54 knockdown mice were found to contain significantly fewer serotonin-positive ß-cells when compared to pregnant controls. Our previous studies suggested that reduced placental kisspeptin production, with consequent impaired kisspeptin-dependent ß-cell compensation, may be a factor in the development of GDM in humans. These current data suggest that, similar to the lactogenic hormones, KP may also contribute to serotonin biosynthesis and subsequent islet signalling during pregnancy. Furthermore, upregulation of serotonin biosynthesis may represent a common mechanism through which multiple signals might influence the islet adaptation to pregnancy.

Mesenchymal stromal cell secretory molecules improve the functional survival of human islets.

AIMS: Human islet transplantation as a therapy for type 1 diabetes is compromised by the loss of functional beta cells in the immediate post-transplantation period. Mesenchymal stromal cells (MSCs) and MSC-derived secretory peptides improve the outcomes of islet transplantation in rodent models of diabetes. Here, we utilized a mouse model for human islet transplantation and assessed the effects of a cocktail of MSC-secreted peptides (screened by MSC-secretome for human islet GPCRs) on the functional survival of human islets. METHODS: Human islets from nine donors (Age: 36-57; BMI: 20-35) were treated with a cocktail of human recombinant annexin A1 (ANXA1), stromal cell-derived factor-1 (SDF-1/CXCL12) and complement component C3 (C3a). Glucose-stimulated insulin secretion (GSIS) was assessed in static incubation, and cytokine-induced apoptosis was assessed by measuring caspase 3/7 activity. mRNA expression levels were determined by qPCR. Human islet function in vivo was assessed using a novel model for human islet transplantation into a T1D mouse model. Human islet function in vivo was assessed using islet transplantation under the kidney capsule of immunodeficient mice prior to STZ destruction of endogenous mouse beta cells to model T1DM. RESULTS: Pretreatment with a cocktail of MSC-secreted peptides increased GSIS in vitro and protected against cytokine-induced apoptosis in human islets isolated from nine donors. Animals transplanted with either treated or untreated human islets remained normoglycaemic for up to 28 days after STZ-administration to ablate the endogenous mouse beta cells, whereas non-transplanted animals showed significantly increased blood glucose immediately after STZ administration. Removal of the human islet graft by nephrectomy resulted in rapid increases in blood glucose to similar levels as the non-transplanted controls. Pretreating human islets with the MSC-derived cocktail significantly improved glucose tolerance in graft recipients, consistent with enhanced functional survival of the treated islets in vivo. CONCLUSION: Pretreating human islets before transplantation with a defined cocktail of MSC-derived molecules could be employed to improve the quality of human islets for transplantation therapy for type 1 diabetes.

Network analysis of inflammation and symptoms in recent onset schizophrenia and the influence of minocycline during a clinical trial.

Attempts to delineate an immune subtype of schizophrenia have not yet led to the clear identification of potential treatment targets. An unbiased informatic approach at the level of individual immune cytokines and symptoms may reveal organisational structures underlying heterogeneity in schizophrenia, and potential for future therapies. The aim was to determine the network and relative influence of pro- and anti-inflammatory cytokines on depressive, positive, and negative symptoms. We further aimed to determine the effect of exposure to minocycline or placebo for 6 months on cytokine-symptom network connectivity and structure. Network analysis was applied to baseline and 6-month data from the large multi-center BeneMin trial of minocycline (N = 207) in schizophrenia. Pro-inflammatory cytokines IL-6, TNF-α, and IFN-γ had the greatest influence in the inflammatory network and were associated with depressive symptoms and suspiciousness at baseline. At 6 months, the placebo group network connectivity was 57% stronger than the minocycline group, due to significantly greater influence of TNF-α, early wakening, and pathological guilt. IL-6 and its downstream impact on TNF-α, and IFN-γ, could offer novel targets for treatment if offered at the relevant phenotypic profile including those with depression. Future targeted experimental studies of immune-based therapies are now needed.

Derivation of CD8+ T cell infiltration potentiators in non-small-cell lung cancer through tumor microenvironment analysis.

Non-small-cell lung cancer remains a deadly form of human cancer even in the era of immunotherapy with existing immunotherapy strategies currently only benefiting a minority of patients. Therefore, the derivation of treatment options, which might extend the promise of immunotherapy to more patients, remains of paramount importance. Here, we define using TCGA lung squamous and lung adenocarcinoma RNAseq datasets a significant correlation between epigenetic therapy actionable interferon genes with both predicted tumor immune score generally, and CD8A specifically. IHC validation using primary sample tissue microarrays confirmed a pronounced positive association between CD8+ T cell tumor infiltration and the interferon-associated targets, CCL5 and MDA5. We next extended these findings to the assessment of clinical trial biopsies from patients with advanced non-small-cell lung cancer treated with epigenetic therapy with and without concurrent immunotherapy. These analyses revealed treatment-associated increases in both CD8+ T cell intratumoral infiltration and microenvironment CCL5 staining intensity.

Inflammatory subgroups of schizophrenia and their association with brain structure: A semi-supervised machine learning examination of heterogeneity.

OBJECTIVE: Immune system dysfunction is hypothesised to contribute to structural brain changes through aberrant synaptic pruning in schizophrenia. However, evidence is mixed and there is a lack of evidence of inflammation and its effect on grey matter volume (GMV) in patients. We hypothesised that inflammatory subgroups can be identified and that the subgroups will show distinct neuroanatomical and neurocognitive profiles. METHODS: The total sample consisted of 1067 participants (chronic patients with schizophrenia n = 467 and healthy controls (HCs) n = 600) from the Australia Schizophrenia Research Bank (ASRB) dataset, together with 218 recent-onset patients with schizophrenia from the external Benefit of Minocycline on Negative Symptoms of Psychosis: Extent and Mechanism (BeneMin) dataset. HYDRA (HeterogeneitY through DiscRiminant Analysis) was used to separate schizophrenia from HC and define disease-related subgroups based on inflammatory markers. Voxel-based morphometry and inferential statistics were used to explore GMV alterations and neurocognitive deficits in these subgroups. RESULTS: An optimal clustering solution revealed five main schizophrenia groups separable from HC: Low Inflammation, Elevated CRP, Elevated IL-6/IL-8, Elevated IFN-γ, and Elevated IL-10 with an adjusted Rand index of 0.573. When compared with the healthy controls, the IL-6/IL-8 cluster showed the most widespread, including the anterior cingulate, GMV reduction. The IFN-γ inflammation cluster showed the least GMV reduction and impairment of cognitive performance. The CRP and the Low Inflammation clusters dominated in the younger external dataset. CONCLUSIONS: Inflammation in schizophrenia may not be merely a case of low vs high, but rather there are pluripotent, heterogeneous mechanisms at play which could be reliably identified based on accessible, peripheral measures. This could inform the successful development of targeted interventions.

BIN1, Myotubularin, and Dynamin-2 Coordinate T-Tubule Growth in Cardiomyocytes.

BACKGROUND: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. METHODS: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4. RESULTS: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. CONCLUSIONS: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.

The association between reasons for first using cannabis, later pattern of use, and risk of first-episode psychosis: the EU-GEI case-control study.

BACKGROUND: While cannabis use is a well-established risk factor for psychosis, little is known about any association between reasons for first using cannabis (RFUC) and later patterns of use and risk of psychosis. METHODS: We used data from 11 sites of the multicentre European Gene-Environment Interaction (EU-GEI) case-control study. 558 first-episode psychosis patients (FEPp) and 567 population controls who had used cannabis and reported their RFUC.We ran logistic regressions to examine whether RFUC were associated with first-episode psychosis (FEP) case-control status. Path analysis then examined the relationship between RFUC, subsequent patterns of cannabis use, and case-control status. RESULTS: Controls (86.1%) and FEPp (75.63%) were most likely to report 'because of friends' as their most common RFUC. However, 20.1% of FEPp compared to 5.8% of controls reported: 'to feel better' as their RFUC (χ2 = 50.97; p < 0.001). RFUC 'to feel better' was associated with being a FEPp (OR 1.74; 95% CI 1.03-2.95) while RFUC 'with friends' was associated with being a control (OR 0.56; 95% CI 0.37-0.83). The path model indicated an association between RFUC 'to feel better' with heavy cannabis use and with FEPp-control status. CONCLUSIONS: Both FEPp and controls usually started using cannabis with their friends, but more patients than controls had begun to use 'to feel better'. People who reported their reason for first using cannabis to 'feel better' were more likely to progress to heavy use and develop a psychotic disorder than those reporting 'because of friends'.

Predicting treatment resistance from first-episode psychosis using routinely collected clinical information.

Around a quarter of people who experience a first episode of psychosis (FEP) will develop treatment-resistant schizophrenia (TRS), but there are currently no established clinically useful methods to predict this from baseline. We aimed to explore the predictive potential for clozapine use as a proxy for TRS of routinely collected, objective biomedical predictors at FEP onset, and to externally validate the model in a separate clinical sample of people with FEP. We developed and externally validated a forced-entry logistic regression risk prediction Model fOr cloZApine tReaTment, or MOZART, to predict up to 8-year risk of clozapine use from FEP using routinely recorded information including age, sex, ethnicity, triglycerides, alkaline phosphatase levels, and lymphocyte counts. We also produced a least-absolute shrinkage and selection operator (LASSO) based model, additionally including neutrophil count, smoking status, body mass index, and random glucose levels. The models were developed using data from two UK psychosis early intervention services (EIS) and externally validated in another UK EIS. Model performance was assessed via discrimination and calibration. We developed the models in 785 patients, and validated externally in 1,110 patients. Both models predicted clozapine use well at internal validation (MOZART: C 0.70; 95%CI 0.63,0.76; LASSO: 0.69; 95%CI 0.63,0.77). At external validation, discrimination performance reduced (MOZART: 0.63; 0.58,0.69; LASSO: 0.64; 0.58,0.69) but recovered after re-estimation of the lymphocyte predictor (C: 0.67; 0.62,0.73). Calibration plots showed good agreement between observed and predicted risk in the forced-entry model. We also present a decision-curve analysis and an online data visualisation tool. The use of routinely collected clinical information including blood-based biomarkers taken at FEP onset can help to predict the individual risk of clozapine use, and should be considered equally alongside other potentially useful information such as symptom scores in large-scale efforts to predict psychiatric outcomes.

Assessment of patient satisfaction following revision total knee arthroplasty.

BACKGROUND: Patient satisfaction is becoming an increasingly important measure of healthcare quality. Satisfaction following RTKA is poorly described within the literature, resulting in difficulty for clinicians to manage patient expectations and obtain informed consent. METHODS: We investigated postoperative satisfaction of RTKA patients by a single surgeon, using a single prosthesis, at a single institution. Patient satisfaction was assessed using structured telephone assessment questionnaires and review of orthopaedic/hospital records. The effect of patient and surgical characteristics on satisfaction was assessed using correlation coefficients and binary logistic regression in SPSS. RESULTS: Two hundred and two RTKAs were performed in 178 patients between 2004 and 2015 inclusive. One hundred and twenty-four patients (143 RTKAs) were contactable to complete satisfaction assessment. Eighty-five percent of patients were satisfied and would have the RTKA again, 8% were unsure, 7% would not. Mean reported satisfaction on a numerical scale (1-10) was 8.17 (range 1-10), with 74% of patients scoring 8 or above, and 35% of patients scoring 10. The Mahomed Satisfaction Scale outcomes demonstrated a mean score of 87.7. High positive correlation was found between assessment tools. Logistic regression analysis identified factors contributing to satisfaction included ROM, OKS, BMI, and surgical time. CONCLUSION: This cohort demonstrated high patient satisfaction rate following RTKA, utilizing simple and reliable outcome measurement tools. We found a high positive correlation between methods of assessment, and moderate positive correlation between satisfaction and functional outcomes. These results contribute to the understanding of satisfaction in RTKA patients, which may assist in informing patients of expected post-operative outcomes.

A Phase II Trial of Guadecitabine plus Atezolizumab in Metastatic Urothelial Carcinoma Progressing after Initial Immune Checkpoint Inhibitor Therapy.

PURPOSE: On the basis of preclinical evidence of epigenetic contribution to sensitivity and resistance to immune checkpoint inhibitors (ICI), we hypothesized that guadecitabine (hypomethylating agent) and atezolizumab [anti-programmed cell death ligand 1 (PD-L1)] together would potentiate a clinical response in patients with metastatic urothelial carcinoma (UC) unresponsive to initial immune checkpoint blockade therapy. PATIENTS AND METHODS: We designed a single arm phase II study (NCT03179943) with a safety run-in to identify the recommended phase II dose of the combination therapy of guadecitabine and atezolizumab. Patients with recurrent/advanced UC who had previously progressed on ICI therapy with programmed cell death protein 1 or PD-L1 targeting agents were eligible. Preplanned correlative analysis was performed to characterize peripheral immune dynamics and global DNA methylation, transcriptome, and immune infiltration dynamics of patient tumors. RESULTS: Safety run-in enrolled 6 patients and phase II enrolled 15 patients before the trial was closed for futility. No dose-limiting toxicity was observed. Four patients, with best response of stable disease (SD), exhibited extended tumor control (8-11 months) and survival (>14 months). Correlative analysis revealed lack of DNA demethylation in tumors after 2 cycles of treatment. Increased peripheral immune activation and immune infiltration in tumors after treatment correlated with progression-free survival and SD. Furthermore, high IL6 and IL8 levels in the patients' plasma was associated with short survival. CONCLUSIONS: No RECIST responses were observed after combination therapy in this trial. Although we could not detect the anticipated tumor-intrinsic effects of guadecitabine, the addition of hypomethylating agent to ICI therapy induced immune activation in a few patients, which associated with longer patient survival.

The Switch and Reciprocating Models for the Function of ABC Multidrug Exporters: Perspectives on Recent Research.

ATP-binding cassette (ABC) transporters comprise a large superfamily of primary active transporters, which are integral membrane proteins that couple energy to the uphill vectorial transport of substrates across cellular membranes, with concomitant hydrolysis of ATP. ABC transporters are found in all living organisms, coordinating mostly import in prokaryotes and export in eukaryotes. Unlike the highly conserved nucleotide binding domains (NBDs), sequence conservation in the transmembrane domains (TMDs) is low, with their divergent nature likely reflecting a need to accommodate a wide range of substrate types in terms of mass and polarity. An explosion in high resolution structural analysis over the past decade and a half has produced a wealth of structural information for ABCs. Based on the structures, a general mechanism for ABC transporters has been proposed, known as the Switch or Alternating Access Model, which holds that the NBDs are widely separated, with the TMDs and NBDs together forming an intracellular-facing inverted "V" shape. Binding of two ATPs and the substrate to the inward-facing conformation induces a transition to an outward conformation. Despite this apparent progress, certainty around the transport mechanism for any given ABC remains elusive. How substrate binding and transport is coupled to ATP binding and hydrolysis is not known, and there is a large body of biochemical and biophysical data that is at odds with the widely separated NBDs being a functional physiological state. An alternative Constant Contact model has been proposed in which the two NBSs operate 180 degrees out of phase with respect to ATP hydrolysis, with the NBDs remaining in close proximity throughout the transport cycle and operating in an asymmetric allosteric manner. The two models are discussed in the light of recent nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses of three ABC exporters.

The COVID-19 e-lective: using innovation to manage disrupted medical student clinical placements.

BACKGROUND: The COVID-19 pandemic changed the way we work, spend, live, and learn. The impact was felt in the health sector where hospitals cancelled elective surgery, put on hold outpatient services, and implemented new social distancing procedures and telehealth systems, to enable hospitals to increase bed capacity. For medical students, these factors meant significant disruption to their clinical placements, remote delivery of their education, cessation of international and interstate placements, complicated by significant travel restrictions and border closures. There were concerns that final year students might be unable to graduate that year due to this lack of clinical exposure. INNOVATION: As a result of this disruption in late March 2020 we developed an innovative 6 week 'COVID-19 e-lective' rotation, consisting of online modules, virtual clinical tutorials and a COVID project totalling the equivalent of 200 h of work. RESULTS: An evaluation was undertaken that found it to be remarkably successful in meeting the students' learning needs and alleviating concerns about disrupted placements. It was also conducted during 2021 for all Year 4 students to help expand clinical placement opportunities. OUTCOMES: This paper describes the e-lective, its innovations, its challenges, and its evaluation findings, for others to learn from.

RNA mis-splicing drives viral mimicry response after DNMTi therapy in SETD2-mutant kidney cancer.

Tumors with mutations in chromatin regulators present attractive targets for DNA hypomethylating agent 5-aza-2'-deoxycytidine (DAC) therapy, which further disrupts cancer cells' epigenomic fidelity and reactivates transposable element (TE) expression to drive viral mimicry responses. SETD2 encodes a histone methyltransferase (H3K36me3) and is prevalently mutated in advanced kidney cancers. Here, we show that SETD2-mutant kidney cancer cells are especially sensitive in vitro and in vivo to DAC treatment. We find that the viral mimicry response are direct consequences of mis-splicing events, such as exon inclusions or extensions, triggered by DAC treatment in an SETD2-loss context. Comprehensive epigenomic analysis reveals H3K9me3 deposition, rather than DNA methylation dynamics, across intronic TEs might contribute to elevated mis-splicing rates. Through epigenomic and transcriptomic analyses, we show that SETD2-deficient kidney cancers are prone to mis-splicing, which can be therapeutically exacerbated with DAC treatment to increase viral mimicry activation and provide synergy with combinatorial immunotherapy approaches.

Commiphora myrrha stimulates insulin secretion from ß-cells through activation of atypical protein kinase C and mitogen-activated protein kinase.

ETHNOPHARMACOLOGICAL RELEVANCE: Ayurvedic medicine has been used in the treatment of diabetes mellitus for centuries. In Arabia and some areas of Africa, Commiphora myrrha (CM) has been extensively used as a plant-based remedy. We have previously shown that an aqueous CM resin solution directly stimulates insulin secretion from MIN6 cells, a mouse ß-cell line, and isolated mouse and human islets. However, the signaling pathways involved in CM-induced insulin secretion are completely unknown. Insulin secretion is normally triggered by elevations in intracellular Ca2+ ([Ca2+]i) through voltage gated Ca2+ channels (VGCC) and activation of protein kinases. Protein and lipid kinases such as protein kinase A (PKA), Ca2+-calmodulin dependent protein kinase II (CaMKII), phosphoinositide 3-kinases (PI3Ks), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), specifically extracellular signal-regulated kinases (ERK1/2), may be involved in receptor-operated insulin secretion. Therefore, we hypothesized that CM may induce insulin secretion by modulating the activity of VGCC and/or one or more of the above kinases. AIM OF THE STUDY: To investigate the possible molecular mechanism of action of CM-induced insulin secretion. The effects of aqueous CM resin extract on [Ca2+]i and protein kinase activation from ß-cells were examined. METHODS: The effect of aqueous CM resin solution on [Ca2+]i was assessed using Ca2+ microfluorimetry. The involvement of VGCC in CM-induced insulin secretion was investigated using static and perifusion insulin secretion experiments in the presence of either EGTA, a Ca2+ chelator, or nifedipine, a blocker of VGCC. The involvement of kinase activation in the stimulatory effect of CM on insulin secretion was examined by using static and perifusion insulin secretion experiments in the presence of known pharmacological inhibitors and/or downregulation of specific kinases. The effects of CM on phosphorylation of PKCζ and ERK1/2 were also assessed using the Wes™ capillary-based protein electrophoresis. RESULTS: Ca2+ microfluorimetry measurements showed that exposing MIN6 cells to CM (0.5-2 mg/mL) was not associated with changes in [Ca2+]i. Similarly, incubating MIN6 cells and mouse islets with EGTA and nifedipine, respectively, did not attenuate the insulin secretion induced by CM. However, incubating mouse and human islets with CM in the presence of staurosporine, a non-selective protein kinase inhibitor, completely blocked the effect of CM on insulin secretion. Exposing mouse islets to CM in the presence of H89, KN62 and LY294002, inhibitors of PKA, CaMKII and PI3K, respectively, did not reduce CM-induced insulin secretion. However, incubating mouse and human islets with CM in the presence of Ro 31-8220, a pan-PKC inhibitor, diminished insulin secretion stimulated by CM, whereas inhibiting the action of typical PKC (with Go6976) and PLCß (with U73122) did not affect CM-stimulated insulin secretion. Similarly, downregulating typical and novel PKC by chronic exposure of mouse islets to phorbol 12-myristate 13-acetate (PMA) was also not associated with a decrease in the stimulatory effect of CM on insulin secretion. Interestingly, CM-induced insulin secretion from mouse islets was inhibited in the presence of the PKCζ inhibitor ZIP and a MAPK inhibitor PD 98059. In addition, Wes™ capillary-based protein electrophoresis indicated that expression of the phosphorylated forms of PKCζ and ERK1/2, a MAPK, was significantly increased following exposure of INS-1832/13 cells, a rat insulinoma cell line, to CM. CONCLUSIONS: Our data indicate that CM directly stimulates insulin secretion through activating known downstream effectors of insulin-stimulus secretion coupling. Indeed, the increase in insulin secretion seen with CM is independent of changes in [Ca2+]i and does not involve activation of VGCC. Instead, the CM stimulatory effect on insulin secretion is completely dependent on protein kinase activation. Our findings indicate that CM could induce insulin exocytosis by stimulating the phosphorylation and activation of PKCζ, which in turn phosphorylates and activates ERK1/2.