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

Editorial: Clinical Application of Artificial Intelligence in Emergency and Critical Care Medicine, Volume I

10.3389/fmed.2021.809478Frontiers in Medicine880947

L-arginine attenuates Interleukin-1β (IL-1β) induced Nuclear Factor Kappa-Beta (NF-κB) activation in Caco-2 cells.

BACKGROUND:Specific nutrients like L-arginine (L-Arg) ameliorate intestinal inflammation, however the exact mechanisms of this effect are unclear. We hypothesized the anti-inflammatory effects of L-Arg require active transport and metabolism by inducible nitric oxide synthase (iNOS) to generate nitric oxide (NO). To test this hypothesis we examined the effects of L-Arg, L-Arg transport activity, NO production and iNOS inhibitor on IL-1β-mediated NF-κB-activation in Caco-2 cells. METHODS:Caco-2 cells were cultured, transfected with a NF-κB promoter luciferase vector, incubated ± L-Arg, ± IL-1β and luciferase activity was measured. Using siRNA we inhibited the L-Arg cationic amino acid transporter system y+ (CAT1) expression and examined its effects on L-Arg transport activity and IL-1β-mediated NF-κB-activation. Finally, the effects of sodium nitroprusside (SNP, a NO donor) and Nω-nitro-L-arginine (NNA, an iNOS inhibitor) on IL-1β-mediated NF-κB-activation were examined. RESULTS:IL-1β increased NF-κB luciferase activity (8-fold) and NF-κB expression (mRNA and protein), both of these were significantly decreased by L-Arg. System y+ CAT1 siRNA decreased CAT1 expression, L-Arg transport activity and attenuated the inhibitory effects of L-Arg on NF- κB activity. SNP attenuated the IL-1β-induced increase in NF-κB luciferase activity and expression, whereas NNA diminished the inhibitory effects of L-Arg on IL-1β-inducible NF- κB luciferase activity. CONCLUSION:The inhibitory effects of L-Arg on IL-1β-mediated NF-κB-activation in Caco-2 cells involve L-Arg transport activity by CAT1, regulation of IL-1β-mediated increases in NF-κB expression, changes in iNOS expression and NO production. Our data suggest the inhibitory effects of L-Arg on NF-κB activation are mediated in part by iNOS since SNP preserves and NNA attenuates the effects of L-Arg on IL-1β-mediated NF-κB-activation and expression

A Small Surge in Incidence of SARS-CoV-2 Omicron Variant in the “Dynamic Zero” Period

To describe the epidemiological characteristics and transmission dynamics of SARS-CoV-2 Omicron variant during “Dynamic Zero” period, we analyzed data on the 108 laboratory-confirmed SARS-CoV-2 cases during 14 to 30 May 2022 in Beichen district, Tianjin, China. We collected information on demographic characteristics, exposure history, and illness timelines of the 108 cases. We described characteristics of the patients and estimated the key epidemiological parameters, including serial interval and the time-dependent reproduction number of the Omicron variant, Rt. Among the 108 laboratory-confirmed patients, the median age was 38 years old, and 50.9% were females. Obvious symptoms were observed among 67.6% (73/108) of all cases, and major clinical manifestations included fever, sore throat, and cough, which occurred in 31.5%, 26.9%, and 19.4% of the 108 cases, respectively. The mean and standard deviation of the SI were estimated as 2.89 and 0.95 days, the Rt varied from 1.24 to 0.27 for a 7-day timelapse. The low reproduction number and the Omicron outbreak being suppressed within a short time marked the effectiveness of the implemented public health measures, such as nucleic acid screening, social distancing, masking, vaccination, medical treatment of patients, and isolation of close contacts. These measures play an important role in fulfilling the goal of controlling the spread of the disease

Roux-en-Y gastric bypass alters small intestine glutamine transport in the obese Zucker rat

The metabolic effects of Roux-en-Y gastric bypass (RYGB) are caused by postsurgical changes in gastrointestinal anatomy affecting gut function. Glutamine is a critical gut nutrient implicated in regulating glucose metabolism as a substrate for intestinal gluconeogenesis. The present study examines the effects of obesity and RYGB on intestinal glutamine transport and metabolism. First, lean and obese Zucker rats (ZRs) were compared. Then the effects of RYGB and sham surgery with pair feeding (PF) in obese ZRs were studied. Segments of small intestine (biliopancreatic limb, Roux limb, and common channel) mucosa were harvested and brush border membrane vesicles (BBMVs) were isolated on postoperative day 28. Glutamine transporter activity and abundance, B0AT1 protein, and mRNA levels were measured. Levels of glutaminase, cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), and glucose-6-phosphatase (G6Pase) were measured to assess glutamine metabolism and intestinal gluconeogenesis. Obesity increased glutamine transport and B0AT1 expression throughout the intestine. RYGB increased glutamine transport activity in the biliopancreatic (3.8-fold) and Roux limbs (1.4-fold) but had no effect on the common channel. The relative abundance of B0AT1 mRNA and protein were increased in the biliopancreatic (6-fold) and Roux limbs (10-fold) after RYGB (P < 0.05 vs. PF), but not the common channel. Glutaminase levels were increased, whereas the relative abundance of PEPCK-C and G6Pase were decreased in all segments of intestine after RYGB. RYGB selectively increased glutamine absorption in biliopancreatic and Roux limbs by a mechanism involving increased B0AT1 expression. Post-RYGB glutaminase levels were increased, but the reductions in PEPCK-C and G6Pase suggest that RYGB downregulates intestinal gluconeogenesis