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

Epidemiology of basal-like breast cancer

Risk factors for the newly identified “intrinsic” breast cancer subtypes (luminal A, luminal B, basal-like and human epidermal growth factor receptor 2-positive/estrogen receptor-negative) were determined in the Carolina Breast Cancer Study, a population-based, case–control study of African-American and white women. Immunohistochemical markers were used to subtype 1,424 cases of invasive and in situ breast cancer, and case subtypes were compared to 2,022 controls. Luminal A, the most common subtype, exhibited risk factors typically reported for breast cancer in previous studies, including inverse associations for increased parity and younger age at first full-term pregnancy. Basal-like cases exhibited several associations that were opposite to those observed for luminal A, including increased risk for parity and younger age at first term full-term pregnancy. Longer duration breastfeeding, increasing number of children breastfed, and increasing number of months breastfeeding per child were each associated with reduced risk of basal-like breast cancer, but not luminal A. Women with multiple live births who did not breastfeed and women who used medications to suppress lactation were at increased risk of basal-like, but not luminal A, breast cancer. Elevated waist-hip ratio was associated with increased risk of luminal A in postmenopausal women, and increased risk of basal-like breast cancer in pre- and postmenopausal women. The prevalence of basal-like breast cancer was highest among premenopausal African-American women, who also showed the highest prevalence of basal-like risk factors. Among younger African-American women, we estimate that up to 68% of basal-like breast cancer could be prevented by promoting breastfeeding and reducing abdominal adiposity

Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry techniques are well-suited to high-throughput characterization of natural products, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social molecular networking (GNPS, http://gnps.ucsd.edu), an open-access knowledge base for community wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of ‘living data’ through continuous reanalysis of deposited data

Insights into Alpha-Hemolysin (Hla) Evolution and Expression among <i>Staphylococcus aureus</i> Clones with Hospital and Community Origin

<div><p>Background</p><p>Alpha-hemolysin (Hla) is a major virulence factor in the pathogenesis of <i>Staphylococcus aureus</i> infection, being active against a wide range of host cells. Although <i>hla</i> is ubiquitous in <i>S. aureus</i>, its genetic diversity and variation in expression in different genetic backgrounds is not known. We evaluated nucleotide sequence variation and gene expression profiles of <i>hla</i> among representatives of hospital (HA) and community-associated (CA) <i>S. aureus</i> clones.</p><p>Methods</p><p>51 methicillin-resistant <i>S. aureus</i> and 22 methicillin-susceptible <i>S. aureus</i> were characterized by PFGE, <i>spa</i> typing, MLST and SCC<i>mec</i> typing. The internal regions of <i>hla</i> and the <i>hla</i> promoter were sequenced and gene expression was assessed by RT-PCR.</p><p>Results</p><p>Alpha-hemolysin encoding- and promoter sequences were diverse, with 12 and 23 different alleles, respectively. Based on phylogenetic analysis, we suggest that <i>hla</i> may have evolved together with the <i>S. aureus</i> genetic background, except for ST22, ST121, ST59 and ST93. Conversely, the promoter region showed lack of co-evolution with the genetic backgrounds. Four non-synonymous amino acid changes were identified close to important regions of <i>hla</i> activity. Amino acid changes in the RNAIII binding site were not associated to <i>hla</i> expression. Although expression rates of <i>hla</i> were in general strain-specific, we observed CA clones showed significantly higher <i>hla</i> expression (p = 0.003) when compared with HA clones.</p><p>Conclusion</p><p>We propose that the <i>hla</i> gene has evolved together with the genetic background. Overall, CA genetic backgrounds showed higher levels of <i>hla</i> expression than HA, and a high strain-to-strain variation of gene expression was detected in closely related strains.</p></div

Summary of molecular characterization, sequence variation and relative expression rates of <i>S. aureus</i> strains collection.

1<p>H: High polymorphism; L: Low polymorphism;</p>2<p>Mean Delta C_t1–3 =  Average (Delta C_t1; Delta C_t2; Delta C_t3), Delta C_t = C_t hla−C_t 16S; Not valid: only one C_t reading;</p>3<p>*low reproducibility between three C_T values (Stddv≤2). nt: non typable; Stddv: standard deviation.</p

HA and CA strains relative expression distribution.

<p>Mean of expression rates from three biological replicates. Dashed line corresponding to the mean Ct value 5.73 results from the regression tree analysis which split strains in two distinct groups, at <i>spa</i> type level: a) high expression group - corresponding to strains with Mean Delta Ct≤5.73 and b) low expression group- corresponding to strains with Mean Delta Ct>5.73). Highlighted in red are the high expressing strains.</p

Strains data distribution based on promoter allotypes.

<p>(*)(**) relative expression values not valid (SDV≤2 or only one C_T reading).</p