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

Clinical Characteristics, Racial Inequities, and Outcomes in Patients with Breast Cancer and COVID-19: A COVID-19 and Cancer Consortium (CCC19) Cohort Study

BACKGROUND: Limited information is available for patients with breast cancer (BC) and coronavirus disease 2019 (COVID-19), especially among underrepresented racial/ethnic populations. METHODS: This is a COVID-19 and Cancer Consortium (CCC19) registry-based retrospective cohort study of females with active or history of BC and laboratory-confirmed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection diagnosed between March 2020 and June 2021 in the US. Primary outcome was COVID-19 severity measured on a five-level ordinal scale, including none of the following complications, hospitalization, intensive care unit admission, mechanical ventilation, and all-cause mortality. Multivariable ordinal logistic regression model identified characteristics associated with COVID-19 severity. RESULTS: 1383 female patient records with BC and COVID-19 were included in the analysis, the median age was 61 years, and median follow-up was 90 days. Multivariable analysis revealed higher odds of COVID-19 severity for older age (aOR per decade, 1.48 [95% CI, 1.32-1.67]); Black patients (aOR 1.74; 95 CI 1.24-2.45), Asian Americans and Pacific Islander patients (aOR 3.40; 95 CI 1.70-6.79) and Other (aOR 2.97; 95 CI 1.71-5.17) racial/ethnic groups; worse ECOG performance status (ECOG PS ≥2: aOR, 7.78 [95% CI, 4.83-12.5]); pre-existing cardiovascular (aOR, 2.26 [95% CI, 1.63-3.15])/pulmonary comorbidities (aOR, 1.65 [95% CI, 1.20-2.29]); diabetes mellitus (aOR, 2.25 [95% CI, 1.66-3.04]); and active and progressing cancer (aOR, 12.5 [95% CI, 6.89-22.6]). Hispanic ethnicity, timing, and type of anti-cancer therapy modalities were not significantly associated with worse COVID-19 outcomes. The total all-cause mortality and hospitalization rate for the entire cohort was 9% and 37%, respectively however, it varied according to the BC disease status. CONCLUSIONS: Using one of the largest registries on cancer and COVID-19, we identified patient and BC-related factors associated with worse COVID-19 outcomes. After adjusting for baseline characteristics, underrepresented racial/ethnic patients experienced worse outcomes compared to non-Hispanic White patients. FUNDING: This study was partly supported by National Cancer Institute grant number P30 CA068485 to Tianyi Sun, Sanjay Mishra, Benjamin French, Jeremy L Warner; P30-CA046592 to Christopher R Friese; P30 CA023100 for Rana R McKay; P30-CA054174 for Pankil K Shah and Dimpy P Shah; KL2 TR002646 for Pankil Shah and the American Cancer Society and Hope Foundation for Cancer Research (MRSG-16-152-01-CCE) and P30-CA054174 for Dimpy P Shah. REDCap is developed and supported by Vanderbilt Institute for Clinical and Translational Research grant support (UL1 TR000445 from NCATS/NIH). The funding sources had no role in the writing of the manuscript or the decision to submit it for publication. CLINICAL TRIAL NUMBER: CCC19 registry is registered on ClinicalTrials.gov, NCT04354701

Compatibility of Norepinephrine Bitartrate with Levofloxacin and Moxifloxacin During Simulated Y-site Administration

The purpose of this study was to determine the physical compatibility and chemical stability of norepinephrine bitartrate with selected fluoroquinolones during simulated intravenous Y-site administration. Simulation of Y-compatibility of intravenous fluids can be experimentally demonstrated by mixing equal volumes of two drugs of interest. Hence, we prepared 1:1 mixture of norepinephrine bitartrate and levofloxacin or moxifloxacin by mixing 64 .g/mL of norepinephrine bitartrate individually with clinically relevant concentrations of levofloxacin (5 mg/mL) and moxifloxacin (1.6 mg/mL). The physical stability of these mixtures was assessed via measurement of change in turbidity, and visual inspection for color change, haziness, and precipitate formation. The chemical stability of these mixtures was assessed via high-performance liquid chromatography by evaluating the change in concentration for norepinephrine bitartrate and individual fluoroquinolones, from 0 to 4 hours after mixing. The visual evaluation and turbidity measurements revealed that norepinephrine bitartrate is compatible with individual fluoroquinolones. No color change, haziness, or precipitate formation was observed at 0 and 4 hours when the drugs were mixed at equal volume. The change in turbidity for the mixture as compared to the individual drugs was very minimal (0.001 to 0.004 nephelometric turbidity unit). No significant change in drug concentrations was observed during the 4-hour period for norepinephrine bitartrate and the individual fluoroquinolones following high-performance liquid chromatography analysis of the norepinephrine bitartrate/levofloxacin and norepinephrine bitartrate/moxifloxacin mixtures, which further confirms the stability of this mixture. Our data indicates that norepinephrine bitartrate is compatible with levofloxacin and moxifloxacin, and can be used for intravenous Y-site administration with either of these fluoroquinolone antibiotics

Influence of Manufacturing Process on the Microstructure, Stability, and Sensorial Properties of a Topical Ointment Formulation

The manufacturing process for ointments typically involves a series of heating, cooling, and mixing steps. Precise control of the level of mixing through homogenization and the cooling rate, as well as temperature at different stages, is important in delivering ointments with the desired quality attributes, stability, and performance. In this work, we investigated the influence of typical plant processing conditions on the microstructure, stability, and sensorial properties of a model ointment system through a Design of Experiments (DoE) approach. Homogenization speed at the cooling stage after the addition of the solvent (propylene glycol, PG) was found to be the critical processing parameter that affects stability and the rheological and sensorial properties of the ointment. A lower PG addition temperature was also found to be beneficial. The stabilization of the ointment at a lower PG addition temperature was hypothesized to be due to more effective encapsulation by crystallizing mono- and diglycerides at the lower temperature. The in vitro release profiles were found to be not influenced by the processing parameters, suggesting that for the ointment platform studied, processing affects the microstructure, but the effects do not translate into the release profile, a key performance indicator. Our systematic study represents a Quality-by-Design (QbD) approach to the design of a robust manufacturing process for delivering stable ointments with the desired performance attributes and properties

Safety, Pharmacokinetics, & Efficacy Signals of Larsucosterol (DUR-928) in Alcohol-associated Hepatitis

This study is to evaluate safety and pharmacokinetics (PK) of larsucosterol (DUR-928 or 25HC3S) in subjects with alcohol-associated hepatitis (AH), a devastating acute illness without FDA-approved therapies. This Phase 2a, multicenter, open-label, dose escalation study evaluated safety, PK, and efficacy signals of larsucosterol in 19 clinically diagnosed AH subjects. Based on MELD (Model for End-stage Liver Disease) score, 7 subjects were considered to have moderate AH and 12 to have severe AH. All subjects received 1 or 2 intravenous (IV) infusions (72 hours apart) of larsucosterol at a dose of 30, 90, or 150mg, and were followed for 28 days. Efficacy signals from a subgroup of severe AH subjects were compared with two matched arms of severe AH subjects treated with standard of care (SOC), including corticosteroids (CS), from a contemporaneous study. All 19 larsucosterol-treated subjects survived the 28-day study. Fourteen (74%) of all subjects including 8 (67%) of the severe AH subjects were discharged ≤72 hours after receiving a single infusion. There were no drug-related serious adverse events (SAEs) nor early terminations due to the treatment. PK profiles were not affected by disease severity. Biochemical parameters improved in most subjects. Serum bilirubin levels declined notably from baseline to Day7 and Day28, and MELD scores were reduced at Day28. The efficacy signals compared favorably with two matched groups treated with SOC. Lille scores at Day7 were <0.45 in 16 of the 18 (89%) subjects with Day7 samples. Lille scores from 8 severe AH subjects who received 30 or 90mg larsucosterol (doses used in Phase 2b trial) were statistically significantly lower (p<0.01) than those severe AH subjects treated with SOC from the contemporaneous study. Larsucosterol was well tolerated at all 3 doses in AH subjects without safety concerns. Data from this pilot study showed promising efficacy signals in AH subjects. Larsucosterol is being evaluated in a Phase 2b multicenter, randomized, double-blinded, placebo-controlled (AHFIRM) trial

The CoVID- TE risk assessment model for venous thromboembolism in hospitalized patients with cancer and COVID- 19

BackgroundHospitalized patients with COVID- 19 have increased risks of venous (VTE) and arterial thromboembolism (ATE). Active cancer diagnosis and treatment are well- known risk factors; however, a risk assessment model (RAM) for VTE in patients with both cancer and COVID- 19 is lacking.ObjectivesTo assess the incidence of and risk factors for thrombosis in hospitalized patients with cancer and COVID- 19.MethodsAmong patients with cancer in the COVID- 19 and Cancer Consortium registry (CCC19) cohort study, we assessed the incidence of VTE and ATE within 90 days of COVID- 19- associated hospitalization. A multivariable logistic regression model specifically for VTE was built using a priori determined clinical risk factors. A simplified RAM was derived and internally validated using bootstrap.ResultsFrom March 17, 2020 to November 30, 2020, 2804 hospitalized patients were analyzed. The incidence of VTE and ATE was 7.6% and 3.9%, respectively. The incidence of VTE, but not ATE, was higher in patients receiving recent anti- cancer therapy. A simplified RAM for VTE was derived and named CoVID- TE (Cancer subtype high to very- high risk by original Khorana score +1, VTE history +2, ICU admission +2, D- dimer elevation +1, recent systemic anti- cancer Therapy +1, and non- Hispanic Ethnicity +1). The RAM stratified patients into two cohorts (low- risk, 0- 2 points, n = 1423 vs. high- risk, 3+ points, n = 1034) where VTE occurred in 4.1% low- risk and 11.3% high- risk patients (c statistic 0.67, 95% confidence interval 0.63- 0.71). The RAM performed similarly well in subgroups of patients not on anticoagulant prior to admission and moderately ill patients not requiring direct ICU admission.ConclusionsHospitalized patients with cancer and COVID- 19 have elevated thrombotic risks. The CoVID- TE RAM for VTE prediction may help real- time data- driven decisions in this vulnerable population.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/170302/1/jth15463_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/170302/2/jth15463.pd

The CoVID-TE risk assessment model for venous thromboembolism in hospitalized patients with cancer and COVID-19

BACKGROUND: Hospitalized patients with COVID-19 have increased risks of venous (VTE) and arterial thromboembolism (ATE). Active cancer diagnosis and treatment are well-known risk factors; however, a risk assessment model (RAM) for VTE in patients with both cancer and COVID-19 is lacking. OBJECTIVES: To assess the incidence of and risk factors for thrombosis in hospitalized patients with cancer and COVID-19. METHODS: Among patients with cancer in the COVID-19 and Cancer Consortium registry (CCC19) cohort study, we assessed the incidence of VTE and ATE within 90 days of COVID-19-associated hospitalization. A multivariable logistic regression model specifically for VTE was built using a priori determined clinical risk factors. A simplified RAM was derived and internally validated using bootstrap. RESULTS: From March 17, 2020 to November 30, 2020, 2804 hospitalized patients were analyzed. The incidence of VTE and ATE was 7.6% and 3.9%, respectively. The incidence of VTE, but not ATE, was higher in patients receiving recent anti-cancer therapy. A simplified RAM for VTE was derived and named CoVID-TE (Cancer subtype high to very-high risk by original Khorana score +1, VTE history +2, ICU admission +2, D-dimer elevation +1, recent systemic anti-cancer Therapy +1, and non-Hispanic Ethnicity +1). The RAM stratified patients into two cohorts (low-risk, 0-2 points, n = 1423 vs. high-risk, 3+ points, n = 1034) where VTE occurred in 4.1% low-risk and 11.3% high-risk patients (c statistic 0.67, 95% confidence interval 0.63-0.71). The RAM performed similarly well in subgroups of patients not on anticoagulant prior to admission and moderately ill patients not requiring direct ICU admission. CONCLUSIONS: Hospitalized patients with cancer and COVID-19 have elevated thrombotic risks. The CoVID-TE RAM for VTE prediction may help real-time data-driven decisions in this vulnerable population

Human distal airways contain a multipotent secretory cell that can regenerate alveoli

The human lung differs substantially from its mouse counterpart, resulting in a distinct distal airway architecture affected by disease pathology in chronic obstructive pulmonary disease. In humans, the distal branches of the airway interweave with the alveolar gas-exchange niche, forming an anatomical structure known as the respiratory bronchioles. Owing to the lack of a counterpart&nbsp;in mouse, the cellular and molecular mechanisms that govern&nbsp;respiratory bronchioles in the human lung remain uncharacterized. Here we show that human respiratory bronchioles contain a unique secretory cell population that is distinct from cells in larger proximal airways. Organoid modelling reveals that these respiratory airway secretory (RAS) cells act as unidirectional progenitors for alveolar type 2 cells, which are essential for maintaining and regenerating the alveolar niche. RAS cell lineage differentiation into alveolar type 2 cells is regulated by Notch and Wnt signalling. In chronic obstructive pulmonary disease, RAS cells are altered transcriptionally, corresponding to abnormal alveolar type 2 cell states, which are associated with smoking exposure in both humans and ferrets. These data identify a distinct progenitor in a region of the human lung that is not found in mouse that has a critical role in maintaining the gas-exchange compartment and is altered in chronic lung disease