Normal growth in <i>Gja1</i>^<i>–/+</i>;<i>Sost</i>^<i>–/+</i> compound heterozygous mice relative to control littermates.

<p>(A) Body weight in 1 (open bars) vs. 3 months (solid) old male mice (n = 4–13 per group per time-point) and (B) 1 vs. 3 months old female mice (n = 3–13 per group per time-point); two-way ANOVA p<0.001 for age in both genders, genotype p = 0.016 in females, ns in males (C) tibial length measured by in-vivo CT at 1 vs. 3 months of age in males (n = 4–9 per group per time-point) and (D) in female mice (n = 3–7 per group per time-point); # Dunnett’s post-hoc test p<0.01 relative to WT at one month of age; two-way ANOVA p<0.001 for age in both genders, genotype p<0.01 in females, p = 0.055 in males.</p

Gene expression in cortical bones of <i>Gja1</i>^<i>–/+</i> and <i>Sost</i>^<i>–/+</i> mutant mice.

<p>Abundance of mRNA for sclerostin (<i>Sost</i>) (A), osteocalcin (<i>Bglap</i>) (B), TRAP5b (<i>Acp5</i>) (C), and RANKL (<i>Tnfsf11</i>) (D) extracted from the tibial diaphysis and assessed by RT-qPCR. Average and standard deviation of biological triplicates normalized to WT. * p<0.05 and **p<0.01 relative to WT in Dunnett’s test for multiple comparisons after ANOVA.</p

Biomechanical properties in <i>Gja1</i>^<i>–/+</i> and <i>Sost</i>^<i>–/+</i> mutant mice.

<p>Polar moment of inertia (pMOI) at 1 (A) and 3 (B) months of age measured by μCT analysis of the tibia as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187980#pone.0187980.g002" target="_blank">Fig 2</a> (n = 7–14 per group). Three-point bending biomechanical testing in femurs of 3 month old mice, reporting ultimate force (C), fracture force (D), and stiffness (E) (n = 6–12 per group). * p<0.05 and **p<0.01 relative to WT in Dunnett’s test for multiple comparisons in ANOVA.</p

In vivo μCT analysis of tibial diaphysis at the mid-point.

<p>In vivo μCT analysis of tibial diaphysis at the mid-point.</p

In vivo μCT analysis of cortical bone in <i>Gja1</i>^<i>–/+</i> and <i>Sost</i>^<i>–/+</i> mutant mice.

<p>In vivo analysis of cortical bone at the mid-point of tibial diaphysis in 1 (left: A, C, E, G) and 3 month old mice (right: B, D, F, H). (A-B) marrow area; (C-D) Total area; (E-F) Cortical thickness; (G-H) representative images. **p<0.01 relative to WT in Dunnett’s test for multiple comparisons in ANOVA; data from males and females (n = 6–14 per group). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187980#pone.0187980.t001" target="_blank">Table 1</a> for gender-specific analyses (two-way ANOVA for gender ns).</p

Static cellular histomorphometric parameters in <i>Gja1</i>^<i>–/+</i> and <i>Sost</i>^<i>–/+</i> mutant mice.

<p>Static cellular histomorphometric parameters in <i>Gja1</i>^<i>–/+</i> and <i>Sost</i>^<i>–/+</i> mutant mice.</p

Helical Sense-Responsive and Substituent-Sensitive Features in Vibrational and Electronic Circular Dichroism, in Circularly Polarized Luminescence, and in Raman Spectra of Some Simple Optically Active Hexahelicenes

Four different hexahelicenes, 5-aza-hexahelicene (<b>1</b>), hexahelicene (<b>2</b>), 2-methyl-hexahelicene (<b>3</b>), and 2-bromo-hexahelicene (<b>4</b>), were prepared and their enantiomers, which are stable at r.t., were separated. Vibrational circular dichroism (VCD) spectra were measured for compound <b>1</b>; for all the compounds, electronic circular dichroism (ECD) and circularly polarized luminescence (CPL) spectra were recorded. Each type of experimental spectrum was compared with the corresponding theoretical spectrum, determined via Density Functional Theory (DFT). Following the recent papers by Nakai et al., this comparison allowed to identify some features related to the helicity and some other features typical of the substituent groups on the helical backbone. The Raman spectrum of compound <b>1</b> is also examined from this point of view

A streamlined workflow for single-cells genome-wide copy-number profiling by low-pass sequencing of LM-PCR whole-genome amplification products

<div><p>Chromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Single-cell genome analysis has gained interest in latest years as a source of biomarkers for targeted-therapy selection and drug resistance, and several methods have been developed to amplify the genomic DNA and to produce libraries suitable for Whole Genome Sequencing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key factors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplification (WGA) method, for low-pass genome sequencing with the Ion Torrent^™ platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on single cells isolated from 6 aberrant cell lines of the NCI-H series. In addition, to demonstrate the feasibility of the workflow on clinical samples, we analyzed single circulating tumor cells (CTCs) and white blood cells (WBCs) isolated from the blood of patients affected by prostate cancer or lung adenocarcinoma. The results obtained show that the developed workflow generates data accurately representing whole genome absolute copy number profiles of single cell and allows alterations calling at resolutions down to 100 Kbp with as few as 200,000 reads. The presented data demonstrate the feasibility of the <i>Ampli</i>1^™ WGA-based low-pass workflow for detection of CNAs in single tumor cells which would be of particular interest for genome-driven targeted therapy selection and for monitoring of disease progression.</p></div

CNA detection by low-pass experiments at different read depths and resolution.

<p>Two cells (a-d & e-h) from cell line NCI1650 were analyzed at different window size/resolutions (a,e = 100Kb; b,f = 200Kb; c,g = 500Kb; d,h = 2,000Kb). A dataset at 3,500,000 reads served as reference for ROC analysis.</p

Comparison of LowPass copy number profiles and CNA calling with aCGH.

<p>Example profiles from one single cell of aberrant cell line NCI-H23 generated by <i>Ampli</i>1^™ LowPass (a) and aCGH of <i>Ampli</i>1^™ amplified DNA (b). In c-p): ROC curves comparing <i>Ampli</i>1^™ LowPass CNA calls with aCGH calls from single cell of 6 cell lines of the NCI-H series.</p