Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

The role of FAK and related signalling in adipocytes and the pathogenesis of obesity and diabetes

Obesity and diabetes, key elements of the metabolic syndrome, are fast expanding global epidemics. While obesity is the strongest risk factor for type 2 diabetes, most obese people do not develop diabetes, and many people with diabetes are not obese. This suggests a complex relationship between excess body weight, primarily adipose tissue, and diabetes, characterized by insulin resistance. Adipocyte cell death has been hypothesized to be an initiating factor in chronic, obesity-associated inflammation thought to lead to insulin resistance and diabetes, but the mechanisms regulating this process remain poorly understood. Thus, in this work, I sought to better understand the cell survival pathways regulating adipose tissue and its role in glucose and energy homeostasis. Focal adhesion kinase (FAK) plays a central role in integrin signaling which regulates growth and survival of tumors, but its role in growth in other contexts is less well known. In Chapter IV, I find that FAK is increased in adipose tissue of mice and humans with obesity and insulin resistance. Using adipose tissue-specific FAK knockout mice I report that FAK in adipose tissue is required to maintain insulin sensitivity, particularly in the setting of metabolic stress induced by high fat diet feeding or genetic models of obesity. In Chapter V, I demonstrate that disruption of FAK impairs adipocyte survival both in vitro in 3T3-L1 adipocytes and in vivo, contributing to inflammation and insulin resistance, which can be attenuated by pharmacologic or genetic inhibition of apoptosis. In Chapter VI, I further identify the specific role of apoptotic protein caspase 8 in adipose tissue regulation of glucose and energy homeostasis. I show that disruption of caspase 8 improves glucose tolerance and protects from weight gain in mouse models of obesity and insulin resistance. Our results identify novel roles for FAK in promoting adipocyte survival and insulin sensitivity with metabolic stress, whereas caspase 8 promotes cell death and inflammation contributing to insulin resistance with metabolic stress. Characterizing the interplay of specific signaling pathways helps us to better understand the complex interaction between obesity and diabetes. These findings improve our knowledge of the signaling pathways regulating adipose tissue and their role in obesity, diabetes and metabolic syndrome.Ph.D.2020-02-09 00:00:0