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

A disconnect between precursor frequency, expansion potential, and site-specific CD4+ T cell responses in aged mice.

T cell recognition of peptides presented within self-major histocompatibility complex (pMHC) molecules is essential for long-lived protective immunity. As mice age the number of naïve CD4+ and CD8+ T cells declines. However, unlike for CD8+ T cells, there are more naïve and memory phenotype CD4+ T cells that bind foreign pMHCII in old mice (18-22 months) than adults (12-15 weeks), suggesting increased promiscuity of pMHCII recognition with aging. Here we asked if CD4+ T cell responses to immunization or infection increase with aging since the magnitude of a CD4+ T cell response to a foreign pMHCII is proportional to the size of the precursor population in adult mice. We observed no difference in the number of pMHCII-specific CD4+ T cells in adult versus old mice for pooled secondary lymphoid organs after immunization, bacterial infection, or viral infection, but we did observe diminished numbers of pMHCII-specific CD4+ T cells in both the draining lymph node and brain of old mice after West Nile virus infection. These data indicate that an increased precursor frequency does not translate into more robust responses upon immunization or infection in old mice

Larger precursor population does not equate to a larger response upon <i>Lm</i> infection.

<p>Adult and old C57Bl/6 mice were infected i.v. with 10⁷ CFU <i>Lm</i>E641-OVA in 100μl PBS. On day 6 post-infection, pooled spleen plus lymph node cells were incubated with E641:I-A^b, OVA:I-A^b and MCC:I-E^k tetramers, enriched, and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198354#pone.0198354.g001" target="_blank">Fig 1</a>. (A) Absolute number of responding E641:I-Ab⁺, OVA:I-A^b−, MCC:I-E^k− CD4⁺ T-cells in adult and old mice (ns p>0.05; Mann-Whitney). The average number of cells (±SEM) is shown below the respective label. (B) Representative concatenated contour plot showing BrdU incorporation by E641:I-A^b only cells in adult and old mice. Numbers indicate percent (±SEM) E641:I-A^b+ CD4⁺ T cells in the respective gates. Shown are the aggregate results of two experiments with 3–4 mice/group.</p

Fewer WNV-specific CD4⁺ T cells are present in the draining lymph node of old WNV-infected mice compared to adult.

<p>C57Bl/6 mice were infected with 1000 PFU 385–99 WNV, sc (foot pad). ndLN, dLN and the spleen were harvested on day 8 post-infection for lymphocyte analysis by flow cytometry (FCM). Representative gating strategy for CD4⁺ tetramer⁺ T cells via live, forward and side scatter, CD3⁺ T cells, CD8α^- CD4⁺ T cells and E641:I-A^b+ CD4⁺ T cells harvested from (A) ndLN, (B) dLN, and (C) spleen are shown on day 8 post-WNV infection. Absolute numbers of E641:I-A^b+ CD4⁺ T cells in adult and old C57BL/6 and DEREG mice enumerated for (D) ndLN, (E) dLN, and (F) spleen are shown as labeled. (G) Percent CD4⁺ INF-γ⁺ T cells in dLN of adult and old C57BL/6 and DEREG mice. Shown are the aggregate results of two experiments with (A-F) 6–9 mice/group and (G) 5–8 mice/group. Each dot represents a single mouse (*p<0.05; **p<0.01; Mann-Whitney).</p

Disconnect between precursor number and response magnitude after immunization.

<p>Adult and old C57Bl/6 mice were immunized subcutaneously with 20μg peptide and 10μg LPS on both sides of the base of the tail and mice were kept on BrdU drinking water during the course of the challenge. Day 6 post immunization, pooled spleen and lymph node cells were incubated with E641:I-A^b, OVA:I-A^b and MCC:I-E^k tetramers, enriched using anti-His magnetic beads, and enumerated by flow cytometry after antibody staining and gating on: lymphocytes via forward and side scatter; then CD3⁺ CD19⁻, CD11c⁻, F4/80⁻, CD8⁻ T-cells; then CD3⁺ CD4⁺ T-cells; and finally E641:I-Ab⁺, OVA:I-A^b−, MCC:I-E^k− T-cells in the tetramer enrichment bound fraction from adult and old mice [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198354#pone.0198354.ref015" target="_blank">15</a>]. (A) Absolute number of E641:I-A^b+, OVA:I-A^b−, MCC:I-E^k− cells from adult and old mice after immunization with E641+LPS. Bars represent median values (ns p>0.05; Mann-Whitney). The average number per group (±SEM) is also shown below the respective label. (B) Concatenated contour plots showing BrdU incorporation by E641:I-Ab⁺, OVA:I-A^b−, MCC:I-E^k− cells in adult and old mice (gated as above). E641:I-A^b+ cells are divided into BrdU negative (neg), intermediate (int) and high (hi) subsets. Numbers indicate percent (±SEM) E641:I-A^b+ CD4⁺ T cells in the respective gates. Shown are the aggregate results of two experiments with 4 mice/group.</p

Delivering health interventions to women, children, and adolescents in conflict settings: What have we learned from ten country case studies?

Armed conflict disproportionately affects the morbidity, mortality, and wellbeing of women, newborns, children, and adolescents. Our study presents insights from a collection of ten country case studies aiming to assess the provision of sexual, reproductive, maternal, newborn, child, and adolescent health and nutrition interventions in ten conflict-affected settings in Afghanistan, Colombia, Democratic Republic of the Congo, Mali, Nigeria, Pakistan, Somalia, South Sudan, Syria, and Yemen. We found that despite large variations in contexts and decision making processes, antenatal care, basic emergency obstetric and newborn care, comprehensive emergency obstetric and newborn care, immunisation, treatment of common childhood illnesses, infant and young child feeding, and malnutrition treatment and screening were prioritised in these ten conflict settings. Many lifesaving women\u27s and children\u27s health (WCH) services, including the majority of reproductive, newborn, and adolescent health services, are not reported as being delivered in the ten conflict settings, and interventions to address stillbirths are absent. International donors remain the primary drivers of influencing the what, where, and how of implementing WCH interventions. Interpretation of WCH outcomes in conflict settings are particularly context-dependent given the myriad of complex factors that constitute conflict and their interactions. Moreover, the comprehensiveness and quality of data remain limited in conflict settings. The dynamic nature of modern conflict and the expanding role of non-state armed groups in large geographic areas pose new challenges to delivering WCH services. However, the humanitarian system is creative and pluralistic and has developed some novel solutions to bring lifesaving WCH services closer to populations using new modes of delivery. These solutions, when rigorously evaluated, can represent concrete response to current implementation challenges to modern armed conflicts

Integrative Profiling of T790M-Negative EGFR-Mutated NSCLC Reveals Pervasive Lineage Transition and Therapeutic Opportunities

Purpose: Despite the established role of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutated NSCLC, drug resistance inevitably ensues, with a paucity of treatment options especially in EGFRT790M-negative resistance. Experimental Design: We performed whole-exome and transcriptome analysis of 59 patients with first- and second-generation EGFR TKI-resistant metastatic EGFR-mutated NSCLC to characterize and compare molecular alterations mediating resistance in T790M-positive (T790M(+)) and -negative (T790M(-)) disease. Results: Transcriptomic analysis revealed ubiquitous loss of adenocarcinoma lineage gene expression in T790M(-) tumors, orthogonally validated using multiplex IHC. There was enrichment of genomic features such as TP53 alterations, 3q chromosomal amplifications, whole-genome doubling and nonaging mutational signatures in T790M(-) tumors. Almost half of resistant tumors were further classified as immune(hot), with clinical outcomes conditional on immune cell-infiltration state and T790M status. Finally, using a Bayesian statistical approach, we explored how T790M(-) and T790M(+) disease might be predicted using comprehensive genomic and transcriptomic profiles of treatment-naive patients. Conclusions: Our results illustrate the interplay between genetic alterations, cell lineage plasticity, and immune microenvironment in shaping divergent TKI resistance and outcome trajectories in EGFR-mutated NSCLC. Genomic and transcriptomic profiling may facilitate the design of bespoke therapeutic approaches tailored to a tumor's adaptive potential

Effects of once-weekly exenatide on cardiovascular outcomes in type 2 diabetes

BACKGROUND: The cardiovascular effects of adding once-weekly treatment with exenatide to usual care in patients with type 2 diabetes are unknown. METHODS: We randomly assigned patients with type 2 diabetes, with or without previous cardiovascular disease, to receive subcutaneous injections of extended-release exenatide at a dose of 2 mg or matching placebo once weekly. The primary composite outcome was the first occurrence of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke. The coprimary hypotheses were that exenatide, administered once weekly, would be noninferior to placebo with respect to safety and superior to placebo with respect to efficacy. RESULTS: In all, 14,752 patients (of whom 10,782 [73.1%] had previous cardiovascular disease) were followed for a median of 3.2 years (interquartile range, 2.2 to 4.4). A primary composite outcome event occurred in 839 of 7356 patients (11.4%; 3.7 events per 100 person-years) in the exenatide group and in 905 of 7396 patients (12.2%; 4.0 events per 100 person-years) in the placebo group (hazard ratio, 0.91; 95% confidence interval [CI], 0.83 to 1.00), with the intention-to-treat analysis indicating that exenatide, administered once weekly, was noninferior to placebo with respect to safety (P<0.001 for noninferiority) but was not superior to placebo with respect to efficacy (P=0.06 for superiority). The rates of death from cardiovascular causes, fatal or nonfatal myocardial infarction, fatal or nonfatal stroke, hospitalization for heart failure, and hospitalization for acute coronary syndrome, and the incidence of acute pancreatitis, pancreatic cancer, medullary thyroid carcinoma, and serious adverse events did not differ significantly between the two groups. CONCLUSIONS: Among patients with type 2 diabetes with or without previous cardiovascular disease, the incidence of major adverse cardiovascular events did not differ significantly between patients who received exenatide and those who received placebo