Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.

Picalm, a novel regulator of GLUT4-trafficking in adipose tissue.

OBJECTIVE: Picalm (phosphatidylinositol-binding clathrin assembly protein), a ubiquitously expressed clathrin-adapter protein, is a well-known susceptibility gene for Alzheimer's disease, but its role in white adipose tissue (WAT) function has not yet been studied. Transcriptome analysis revealed differential expression of Picalm in WAT of diabetes-prone and diabetes-resistant mice, hence we aimed to investigate the potential link between Picalm expression and glucose homeostasis, obesity-related metabolic phenotypes, and its specific role in insulin-regulated GLUT4 trafficking in adipocytes. METHODS: Picalm expression and epigenetic regulation by microRNAs (miRNAs) and DNA methylation were analyzed in WAT of diabetes-resistant (DR) and diabetes-prone (DP) female New Zealand Obese (NZO) mice and in male NZO after time-restricted feeding (TRF) and alternate-day fasting (ADF). PICALM expression in human WAT was evaluated in a cross-sectional cohort and assessed before and after weight loss induced by bariatric surgery. siRNA-mediated knockdown of Picalm in 3T3-L1-cells was performed to elucidate functional outcomes on GLUT4-translocation as well as insulin signaling and adipogenesis. RESULTS: Picalm expression in WAT was significantly lower in DR compared to DP female mice, as well as in insulin-sensitive vs. resistant NZO males, and was also reduced in NZO males following TRF and ADF. Four miRNAs (let-7c, miR-30c, miR-335, miR-344) were identified as potential mediators of diabetes susceptibility-related differences in Picalm expression, while 11 miRNAs (including miR-23a, miR-29b, and miR-101a) were implicated in TRF and ADF effects. Human PICALM expression in adipose tissue was lower in individuals without obesity vs. with obesity and associated with weight-loss outcomes post-bariatric surgery. siRNA-mediated knockdown of Picalm in mature 3T3-L1-adipocytes resulted in amplified insulin-stimulated translocation of the endogenous glucose transporter GLUT4 to the plasma membrane and increased phosphorylation of Akt and Tbc1d4. Moreover, depleting Picalm before and during 3T3-L1 differentiation significantly suppressed adipogenesis, suggesting that Picalm may have distinct roles in the biology of pre- and mature adipocytes. CONCLUSIONS: Picalm is a novel regulator of GLUT4-translocation in WAT, with its expression modulated by both genetic predisposition to diabetes and dietary interventions. These findings suggest a potential role for Picalm in improving glucose homeostasis and highlight its relevance as a therapeutic target for metabolic disorders.

Constitutive activation of the PI3K-Akt-mTORC1 pathway sustains the m.3243 A > G mtDNA mutation.

Mutations of the mitochondrial genome (mtDNA) cause a range of profoundly debilitating clinical conditions for which treatment options are very limited. Most mtDNA diseases show heteroplasmy - tissues express both wild-type and mutant mtDNA. While the level of heteroplasmy broadly correlates with disease severity, the relationships between specific mtDNA mutations, heteroplasmy, disease phenotype and severity are poorly understood. We have carried out extensive bioenergetic, metabolomic and RNAseq studies on heteroplasmic patient-derived cells carrying the most prevalent disease related mtDNA mutation, the m.3243 A > G. These studies reveal that the mutation promotes changes in metabolites which are associated with the upregulation of the PI3K-Akt-mTORC1 axis in patient-derived cells and tissues. Remarkably, pharmacological inhibition of PI3K, Akt, or mTORC1 reduced mtDNA mutant load and partially rescued cellular bioenergetic function. The PI3K-Akt-mTORC1 axis thus represents a potential therapeutic target that may benefit people suffering from the consequences of the m.3243 A > G mutation.

Staphylococcal driveline infections are the predominant type of left ventricular assist device associated infections in Singapore.

BACKGROUND: Left ventricular assist device (LVAD) associated infections (LVADIs) have substantial morbidity and mortality. We aim to describe the incidence and epidemiology of LVADIs in an Asian cohort. This is currently not well studied. METHODS: We conducted a retrospective review of 52 patients who underwent LVAD implantation from 1 May 2009-31 December 2014 in National Heart Centre Singapore. LVADIs were defined based on definitions proposed by the International Society for Heart and Lung Transplantation. RESULTS: There were 39 males and 13 females. Seventy-three percent had Heartmate II LVAD implant while 27% received Heartware HVAD. Eighty-one percent were implanted as bridge to heart transplantation, 19% as destination therapy. Forty-five episodes of LVADIs occurred in 25 patients. Overall LVADI incidence was 47.5 cases per 100 patient-years. Driveline infections (58%) were the commonest type of LVADI. The commonest causative organisms were coagulase-negative staphylococci (33%), Staphylococcus aureus (31%) and Corynebacterium species (19%). Twelve percent of patients with LVADI required surgical debridement and one patient required pump exchange due to pump pocket infection. All-cause mortality was 13%. CONCLUSIONS: The findings of our study add to the understanding and epidemiology of LVADIs, particularly in the Asian setting. This can contribute to the development of evidence based strategies to prevent and manage LVADIs.

Effectiveness of interventions for the assessment and prevention of falls in adult psychiatric patients: A systematic review.

BACKGROUND: Patient fall is among the top five sentinel events in hospitals due to the resultant functional loss and injury sustained. Precise fall risk assessment and prevention strategies are important components of a fall prevention program. Due to psychiatric conditions and medications, these patients may require a different fall management program compared to other patient populations. OBJECTIVE: The objective of this review was to identify the best available evidence for the effectiveness of nursing fall risk assessment tools, interventions to reduce incidence of falls, and common risk factors of adult psychiatric patients who fall. INCLUSION CRITERIA: Adults (19 to 64 years) diagnosed with mental illnessEvaluation of nursing fall risk assessment tools in adult psychiatric settings, and interventions, which minimised fall risk or fall rates.Number of patient falls during hospitalisation was the main outcome.Primary quantitative studies published in English language. SEARCH STRATEGY: The literature search sought published studies, and was limited to English language reports. There were no date restrictions applied to the search. Electronic databases searched included:CINAHLPubMedCochrane Central Register of Controlled TrialsPsycINFOScienceDirectScopusWeb of ScienceWiley-InterScienceProQuestMedNar METHODOLOGICAL QUALITY: Studies retrieved were assessed by two independent reviewers for methodological validity prior to inclusion in the review using standardised critical appraisal instruments from the Joanna Briggs Institute System for the Unified Management, Assessment and Review of Information (JBI-SUMARI). DATA EXTRACTION: Data including specific details about the methods, settings, purposes, populations, interventions, and outcomes significant to the review's objectives were extracted by two independent reviewers using standardised data extraction tools from JBI-SUMARI. RESULTS: Eleven studies were included in this review: three before-and-after studies, four descriptive studies, two case control studies and two cohort studies. Evidence with regards to the effectiveness of fall risk assessment tools and prevention strategies was inconclusive. Certain risk factors which were more frequently associated with falls included diagnoses of depression, bipolar disorder, and dementia / Alzheimer's disease, altered mental status, physiological symptoms, past history of falls, mobility and gait problems, concurrent medical conditions, polypharmacy, and taking of certain medications such as sedatives, antidepressants and mood stabilisers, particularly lithium. CONCLUSION: Evidence on the effectiveness of fall risk assessment tools and prevention strategies in psychiatric setting was inconclusive due to a paucity of studies. However, certain risk factors were found to be more commonly associated with falls in adult psychiatric patients (Level III Evidence).Constant observation of the side effects of medications, particularly orthostatic hypotension, and review of patients' medical profile by doctors or pharmacists may be helpful in preventing falls in psychiatric patients. Findings on common risk factors can better aid healthcare professionals in identifying psychiatric patients who are at risk for falls.More research is needed on the evaluation of fall risk assessment tools and fall prevention strategies, specifically for the adult psychiatric patients. More prospective and better quality studies examining fall risk factors in psychiatric patients are needed.