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

Stroke genetics informs drug discovery and risk prediction across ancestries

Previous genome-wide association studies (GWASs) of stroke — the second leading cause of death worldwide — were conducted predominantly in populations of European ancestry1,2. Here, in cross-ancestry GWAS meta-analyses of 110,182 patients who have had a stroke (five ancestries, 33% non-European) and 1,503,898 control individuals, we identify association signals for stroke and its subtypes at 89 (61 new) independent loci: 60 in primary inverse-variance-weighted analyses and 29 in secondary meta-regression and multitrait analyses. On the basis of internal cross-ancestry validation and an independent follow-up in 89,084 additional cases of stroke (30% non-European) and 1,013,843 control individuals, 87% of the primary stroke risk loci and 60% of the secondary stroke risk loci were replicated (P < 0.05). Effect sizes were highly correlated across ancestries. Cross-ancestry fine-mapping, in silico mutagenesis analysis3, and transcriptome-wide and proteome-wide association analyses revealed putative causal genes (such as SH3PXD2A and FURIN) and variants (such as at GRK5 and NOS3). Using a three-pronged approach4, we provide genetic evidence for putative drug effects, highlighting F11, KLKB1, PROC, GP1BA, LAMC2 and VCAM1 as possible targets, with drugs already under investigation for stroke for F11 and PROC. A polygenic score integrating cross-ancestry and ancestry-specific stroke GWASs with vascular-risk factor GWASs (integrative polygenic scores) strongly predicted ischaemic stroke in populations of European, East Asian and African ancestry5. Stroke genetic risk scores were predictive of ischaemic stroke independent of clinical risk factors in 52,600 clinical-trial participants with cardiometabolic disease. Our results provide insights to inform biology, reveal potential drug targets and derive genetic risk prediction tools across ancestries

Synthesis and characterization of some organoruthenium complexes with macrocyclic amine ligands

published_or_final_versionChemistryMasterMaster of Philosoph

A Role for Mitochondrial Translation in Promotion of Viability in K-Ras Mutant Cells

Activating mutations in the KRAS oncogene are highly prevalent in tumors, especially those of the colon, lung, and pancreas. To better understand the genetic dependencies that K-Ras mutant cells rely upon for their growth, we employed whole-genome CRISPR loss-of-function screens in two isogenic pairs of cell lines. Since loss of essential genes is uniformly toxic in CRISPR-based screens, we also developed a small hairpin RNA (shRNA) library targeting essential genes. These approaches uncovered a large set of proteins whose loss results in the selective reduction of K-Ras mutant cell growth. Pathway analysis revealed that many of these genes function in the mitochondria. For validation, we generated isogenic pairs of cell lines using CRISPR-based genome engineering, which confirmed the dependency of K-Ras mutant cells on these mitochondrial pathways. Finally, we found that mitochondrial inhibitors reduce the growth of K-Ras mutant tumors in vivo, aiding in the advancement of strategies to target K-Ras-driven malignancy

The Oklahoma State Capital

Daily newspaper from Guthrie, Oklahoma that includes local, territorial, and national news along with advertising

Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation.

Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific "activator" for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity

Overexpression of transferrin receptor CD71 and its tumorigenic properties in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer in endemic Asian regions. In the present study, we investigated the clinical implication and role of transferrin receptor CD71 in ESCC. CD71 has a physiological role in cellular iron intake and is implicated in the carcinogenesis of various types of tumors. In our cohort, more than a 2-fold upregulation of the CD71 transcript was detected in 61.5% of patients using quantitative polymerase chain reaction. Immunohistochemical analysis also showed strong membranous and cytoplasmic localization of CD71 in paraffin-embedded tumors. Staining parallel tumor sections with the proliferative marker Ki-67 revealed that the pattern of Ki-67 staining was associated with CD71 expression. Analysis of clinicopathological data indicated that CD71 overexpression can be used as an indicator for advanced T4 stage (P=0.0307). These data suggested a strong link between CD71 and ESCC. Subsequent in vitro assays using short interfering RNA (siRNA) to suppress CD71 expression confirmed the tumorigenic properties of CD71 in ESCC; cell growth inhibition and cell cycle arrest at S phase were observed in CD71-suppressed cells. The underlying mechanism involved activation of the MEK/ERK pathway. In summary, the present study provides evidence showing the tumorigenic properties of CD71 in ESCC with clinical correlations and suggests targeting CD71 as a strategy for the treatment of ESCC.Link_to_subscribed_fulltex