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

Female chromosome X mosaicism is age-related and preferentially affects the inactivated X chromosome

To investigate large structural clonal mosaicism of chromosome X, we analysed the SNP microarray intensity data of 38,303 women from cancer genome-wide association studies (20,878 cases and 17,425 controls) and detected 124 mosaic X events >2 Mb in 97 (0.25%) women. Here we show rates for X-chromosome mosaicism are four times higher than mean autosomal rates; X mosaic events more often include the entire chromosome and participants with X events more likely harbour autosomal mosaic events. X mosaicism frequency increases with age (0.11% in 50-year olds; 0.45% in 75-year olds), as reported for Y and autosomes. Methylation array analyses of 33 women with X mosaicism indicate events preferentially involve the inactive X chromosome. Our results provide further evidence that the sex chromosomes undergo mosaic events more frequently than autosomes, which could have implications for understanding the underlying mechanisms of mosaic events and their possible contribution to risk for chronic diseases

Female chromosome X mosaicism is age-related and preferentially affects the inactivated X chromosome

To investigate large structural clonal mosaicism of chromosome X, we analysed the SNP microarray intensity data of 38,303 women from cancer genome-wide association studies (20,878 cases and 17,425 controls) and detected 124 mosaic X events42Mb in 97 (0.25%) women. Here we show rates for X-chromosome mosaicism are four times higher than mean autosomal rates; X mosaic events more often include the entire chromosome and participants with X events more likely harbour autosomal mosaic events. X mosaicism frequency increases with age (0.11% in 50-year olds; 0.45% in 75-year olds), as reported for Y and autosomes. Methylation array analyses of 33 women with X mosaicism indicate events preferentially involve the inactive X chromosome. Our results provide further evidence that the sex chromosomes undergo mosaic events more frequently than autosomes, which could have implications for understanding the underlying mechanisms of mosaic events and their possible contribution to risk for chronic diseases

Structure of the Varicella Zoster Virus Thymidylate Synthase Establishes Functional and Structural Similarities as the Human Enzyme and Potentiates Itself as a Target of Brivudine.

Varicella zoster virus (VZV) is a highly infectious human herpesvirus that is the causative agent for chicken pox and shingles. VZV encodes a functional thymidylate synthase (TS), which is the sole enzyme that produces dTMP from dUMP de novo. To study substrate binding, the complex structure of TSVZV with dUMP was determined to a resolution of 2.9 Å. In the absence of a folate co-substrate, dUMP binds in the conserved TS active site and is coordinated similarly as in the human encoded TS (TSHS) in an open conformation. The interactions between TSVZV with dUMP and a cofactor analog, raltitrexed, were also studied using differential scanning fluorimetry (DSF), suggesting that TSVZV binds dUMP and raltitrexed in a sequential binding mode like other TS. The DSF also revealed interactions between TSVZV and in vitro phosphorylated brivudine (BVDUP), a highly potent anti-herpesvirus drug against VZV infections. The binding of BVDUP to TSVZV was further confirmed by the complex structure of TSVZV and BVDUP solved at a resolution of 2.9 Å. BVDUP binds similarly as dUMP in the TSHS but it induces a closed conformation of the active site. The structure supports that the 5-bromovinyl substituent on BVDUP is likely to inhibit TSVZV by preventing the transfer of a methylene group from its cofactor and the subsequent formation of dTMP. The interactions between TSVZV and BVDUP are consistent with that TSVZV is indeed a target of brivudine in vivo. The work also provided the structural basis for rational design of more specific TSVZV inhibitors

DSF melting curves of TS_VZV with BVDU_P.

<p>(a) BVDU could not stabilize TS_VZV before phosphorylation but after an <i>in vitro</i> phosphorylation with TK_HS, BVDU_P increased the T_m of TS_VZV by a larger extent than dUMP. (b) Varying concentrations of BVDU_P increased the T_m of TS_VZV in a dose responsive manner. (c) Further stabilization of TS_VZV was observed in the presence of 100 μM raltitrexed with 100 μM of BVDU_P.</p

Stereo views of the TS_VZV and TS_HS active sites.

<p>Corresponding stereo views of the ligands and amino acids lining the TS active sites in the structures of (a) apo-TS_VZV with a phosphate ion, (b) TS_VZV with dUMP, (c) TS_HS with dUMP (PDB ID: 1HVY) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143947#pone.0143947.ref035" target="_blank">35</a>] and (d) TS_VZV with BVDU_P. The polar interactions between the amino acids and the different ligands are illustrated by orange dotted lines. 2F₀-F_C electron densities of the binding (e) BVDU_P and (f) dUMP with the surrounding hydrophobic amino acids are also shown (contoured at 1σ with carved = 2.0).</p

Data collection and refinement statistics on the apo and complex structures of TS_VZV.

<p>^a The values in the parentheses are for the highest resolution shell (3.1–3.2 Å).</p><p>^b The values in the parentheses are for the highest resolution shell (2.9–3.0 Å).</p><p>^c<math><mrow><msub><mi>R</mi><mrow><mi>m</mi><mi>e</mi><mi>r</mi><mi>g</mi><mi>e</mi></mrow></msub><mo>=</mo><mrow><mrow><mo>[</mo><mrow><msub><mo>∑</mo><mrow><mi>h</mi><mi>k</mi><mi>l</mi></mrow></msub><msub><mo>∑</mo><mi>i</mi></msub><mrow><mo>|</mo><mrow><msub><mi>I</mi><mi>i</mi></msub><mrow><mo>(</mo><mrow><mi>h</mi><mi>k</mi><mi>l</mi></mrow><mo>)</mo></mrow><mo>−</mo><mi>I</mi><mo>¯</mo><mrow><mo>(</mo><mrow><mi>h</mi><mi>k</mi><mi>l</mi></mrow><mo>)</mo></mrow></mrow><mo>|</mo></mrow></mrow><mo>]</mo></mrow></mrow><mrow><mrow><mo>[</mo><mrow><msub><mo>∑</mo><mrow><mi>h</mi><mi>k</mi><mi>l</mi></mrow></msub><msub><mo>∑</mo><mi>i</mi></msub><mi>I</mi><mo>¯</mo><mrow><mo>(</mo><mrow><mi>h</mi><mi>k</mi><mi>l</mi></mrow><mo>)</mo></mrow></mrow><mo>]</mo></mrow></mrow><mo>×</mo><mn>100</mn></mrow></math>, where <i>I</i>_<i>i</i> is the <i>i</i>th intensity measurement of reflection <i>hkl</i>, <i>Ī(hkl)</i> is the mean intensity measurement of the symmetry related or replicated reflections of the unique reflection <i>hkl</i>.</p><p>^d<math><mrow><msub><mi>R</mi><mrow><mi>f</mi><mi>a</mi><mi>c</mi><mi>t</mi><mi>o</mi><mi>r</mi></mrow></msub><mo>=</mo><mrow><mrow><mo>[</mo><mrow><msub><mo>∑</mo><mrow><mi>h</mi><mi>k</mi><mi>l</mi></mrow></msub><mrow><mo>|</mo><mrow><msub><mi>F</mi><mrow><mi>o</mi><mi>b</mi><mi>s</mi></mrow></msub><mrow><mo>(</mo><mrow><mi>h</mi><mi>k</mi><mi>l</mi></mrow><mo>)</mo></mrow><mo>−</mo><msub><mi>F</mi><mrow><mi>c</mi><mi>a</mi><mi>l</mi><mi>c</mi></mrow></msub><mrow><mo>(</mo><mrow><mi>h</mi><mi>k</mi><mi>l</mi></mrow><mo>)</mo></mrow></mrow><mo>|</mo></mrow></mrow><mo>]</mo></mrow></mrow><mrow><mrow><mo>[</mo><mrow><msub><mo>∑</mo><mrow><mi>h</mi><mi>k</mi><mi>l</mi></mrow></msub><msub><mi>F</mi><mrow><mi>o</mi><mi>b</mi><mi>s</mi></mrow></msub><mrow><mo>(</mo><mrow><mi>h</mi><mi>k</mi><mi>l</mi></mrow><mo>)</mo></mrow></mrow><mo>]</mo></mrow></mrow><mo>×</mo><mn>100</mn></mrow></math>, where <i>F</i>_<i>obs</i> and <i>F</i>_<i>calc</i> are the observed and calculated structure factors respectively.</p><p>^e R_free is equivalent to R_factor but 5% of the measured reflections have been excluded from refinement and set aside for cross validation.</p><p>Data collection and refinement statistics on the apo and complex structures of TS_VZV.</p