PhyloWGS: Reconstructing subclonal composition and evolution from whole-genome sequencing of tumors

Tumors often contain multiple subpopulations of cancerous cells defined by distinct somatic mutations. We describe a new method, PhyloWGS, which can be applied to whole-genome sequencing data from one or more tumor samples to reconstruct complete genotypes of these subpopulations based on variant allele frequencies (VAFs) of point mutations and population frequencies of structural variations. We introduce a principled phylogenic correction for VAFs in loci affected by copy number alterations and we show that this correction greatly improves subclonal reconstruction compared to existing methods. PhyloWGS is free, open-source software, available at https://github.com/morrislab/phylowgs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0602-8) contains supplementary material, which is available to authorized users.     

Association of CXCL10 and CXCL13 levels with disease activity and cutaneous manifestation in active adult-onset Still’s disease

IntroductionC-X-C motif chemokine 10 (CXCL10) is produced in response to interferon-γ, and tumor necrosis factor-α (TNF-α) triggers the accumulation of activated lymphocytes. CXCL13 is constitutively expressed in secondary lymphoid tissues, and the expression is upregulated by TNF-α, via T cell stimulation. It appears that CXCL10 and CXCL13 could play a potential role in the pathogenesis of adult-onset Still’s disease (AOSD), therefore, we investigated the associations between CXCL10 and CXCL13 levels and clinical manifestations in patients with active AOSD.MethodsBlood samples were collected from 39 active AOSD patients, 32 rheumatoid arthritis (RA) patients and 40 healthy controls (HC). Of the AOSD patients, follow-up samples were collected from 15 9.6 ± 9.2 months later. Serum levels of CXCL10 and CXCL13 were determined using enzyme-linked immunosorbent assay. CXCL10, CXCL13, and C-X-C chemokine receptor type 3 (CXCR3) expression levels in biopsy specimens obtained from 26 AOSD patients with skin rashes were investigated via immunohistochemistry.ResultsThe CXCL10 levels in AOSD patients (1,031.3 ± 2,019.6 pg/mL) were higher than in RA (146.3 ± 91.4 pg/mL, p = 0.008) and HC (104.4 ± 47.9 pg/mL, p = 0.006). Also, the CXCL13 levels of AOSD patients (158.8 ± 151.2 pg/mL) were higher than those of RA (54.4 ± 61.1 pg/mL, p < 0.001) and HC (23.5 ± 18.1 pg/mL, p < 0.001). Serum CXCL10 levels correlated with ferritin and systemic scores. Serum CXCL13 levels correlated with those of hemoglobin, C-reactive protein, ferritin, and albumin, and systemic scores. In follow-up AOSD patients, the levels of CXCL10 and CXCL13 fell significantly (153.7 ± 130.1 pg/mL, p = 0.002, and 89.1 ± 117.4 pg/mL, p = 0.001, respectively). On immunohistochemistry, the percentages of inflammatory cells expressing CXCL10 ranged from 1 to 85 %, CXCL13 from 1 to 72 %, and CXCR3 from 2 to 65 %. The percentage of CXCL10-positive inflammatory cells was higher in skin biopsy samples exhibiting mucin deposition than in those that did not (p = 0.01). CXCL13 levels were correlated with those of CD4 and CD68.ConclusionsSerum CXCL10 and CXCL13 levels may serve as clinical markers for assessment of disease activity in AOSD. CXCL10/CXCR3 and CXCL13 may contribute to the inflammatory response, especially skin manifestations thereof, in AOSD.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0773-4) contains supplementary material, which is available to authorized users.     

In Salmonella enterica, the sirtuin-dependent protein acylation/deacylation system (SDPADS) maintains energy homeostasis during growth on low concentrations of acetate

Acetyl-coenzyme A synthetase (Acs) activates acetate into acetyl-coenzyme A (Ac-CoA) in most cells. In Salmonella enterica, acs expression and Acs activity are controlled. It is unclear why the sirtuin-dependent protein acylation/deacylation system (SDPADS) controls the activity of Acs. Here we show that, during growth on 10 mM acetate, acs(+) induction in a S. enterica strain that cannot acetylate (i.e. inactivate) Acs leads to growth arrest, a condition that correlates with a drop in energy charge (0.17) in the acetylation-deficient strain, relative to the energy charge in the acetylation-proficient strain (0.71). Growth arrest was caused by elevated Acs activity, a conclusion supported by the isolation of a single-amino-acid variant (Acs(G266S)), whose overproduction did not arrest growth. Acs-dependent depletion of ATP, coupled with the rise in AMP levels, prevented the synthesis of ADP needed to replenish the pool of ATP. Consistent with this idea, overproduction of ADP-forming Ac-CoA-synthesizing systems did not affect the growth behaviour of acetylation-deficient or acetylation-proficient strains. The Acs(G266S) variant was >2 orders of magnitude less efficient than the Acs(WT) enzyme, but still supported growth on 10 mM acetate. This work provides the first evidence that SDPADS function helps cells maintain energy homeostasis during growth on acetate.     

Multiwalled carbon nanotubes dispersed in carminic acid for the development of catalase based biosensor for selective amperometric determination of H(2)O(2) and iodate

We report the preparation of stable dispersion of multiwalled carbon nanotubes (MWCNTs) using carminic acid (CA) as a dispersing agent. The transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) results confirmed that MWCNT is well dispersed in CA aqueous solution and CA has been well adsorbed at MWCNT walls. Fourier transform infrared (FTIR) and UV-vis absorption spectra results also confirmed the adsorption of CA at MWCNT. To develop a highly selective amperometric biosensor for H(2)O(2) and iodate, the model enzyme catalase (CAT) was immobilized at CACNT modified glassy carbon electrode surface. The immobilized CAT exhibits well defined quasi reversible redox peaks at a formal potential (E°') of -0.559V in 0.05M pH 7 phosphate buffer solution (PBS). The proposed CAT/CACNT biosensor exhibits excellent amperometric response towards H(2)O(2) and iodate in the linear concentration range between 10μM to 3.2mM and 0.01-2.16mM. The sensitivity values are 287.98μAmM(-1)cm(-2) and 0.253mAmM(-1)cm(-2), respectively. Moreover, the developed CAT biosensor exhibits high affinity for H(2)O(2) and iodate with good selectivity.     

Enhancing immunoassay detection of antigens with multimeric protein Gs

This paper describes a method for the effective and self-oriented immobilization of antibodies on magnetic silica-nanoparticles using a multimeric protein G. Cysteine-tagged recombinant dimers and trimers of protein G were produced in Escherichia coli BL21 by repeated linking of protein G monomers with a flexible (GGGGS)(3) linker. Amino-functionalized silica-coated magnetic nanoparticles (SiO(2)-MNPs, Fe(3)O(4)@SiO(2)) were prepared and coupled to the protein G multimers, giving the final magnetic immunosensor. The optimal conditions for the reaction between the protein Gs and the SiO(2)-MNPs was a time of 60 min and a concentration of 100 μg/mL, resulting in coupling efficiencies of 77%, 67% and 55% for the monomeric, dimeric and trimeric protein Gs, respectively. Subsequently, anti-hepatitis B surface antigen (HBsAg) was immobilized onto protein G-coupled SiO(2)-MNPs. The quantitative efficiency of antibody immobilization found the trimeric protein G to be the best, followed by the dimeric and monomeric proteins, which differs from the coupling efficiencies. Using all three protein constructs in an HBsAg fluoroimmunoassay, the lowest detectable concentrations were 500, 250 and 50 ng/mL for the monomeric, dimeric and trimeric protein G-coupled SiO(2)-MNPs, respectively. Therefore, multimeric protein Gs, particularly the trimeric form, can be employed to improve antibody immobilization and, ultimately, enhance the sensitivity of immunoassays. In addition, the multimeric protein Gs devised in this study can be utilized in other immunosensors to bind the antibodies at a high efficiency and in the proper orientation.     

Combining near-infrared-excited autofluorescence and Raman spectroscopy improves in vivo diagnosis of gastric cancer

This study aims to evaluate the diagnostic utility of the combined near-infrared (NIR) autofluorescence (AF) and Raman spectroscopy for improving in vivo detection of gastric cancer at clinical gastroscopy. A rapid Raman endoscopic technique was employed for in vivo spectroscopic measurements of normal (n=1098) and cancer (n=140) gastric tissues from 81 gastric patients. The composite NIR AF and Raman spectra in the range of 800-1800 cm(-1) were analyzed using principal component analysis (PCA) and linear discriminant (LDA) to extract diagnostic information associated with distinctive spectroscopic processes of gastric malignancies. High quality in vivo composite NIR AF and Raman spectra can routinely be acquired from the gastric within 0.5s. The integrated intensity over the range of 800-1800 cm(-1) established the diagnostic implications (p=1.6E-14) of the change of NIR AF intensity associated with neoplastic transformation. PCA-LDA diagnostic modeling on the in vivo tissue NIR AF and Raman spectra acquired yielded a diagnostic accuracy of 92.2% (sensitivity of 97.9% and specificity of 91.5%) for identifying gastric cancer from normal tissue. The integration area under the receiver operating characteristic (ROC) curve using the combined NIR AF and Raman spectroscopy was 0.985, which is superior to either the Raman spectroscopy or NIR AF spectroscopy alone. This work demonstrates that the complementary Raman and NIR AF spectroscopy techniques can be integrated together for improving the in vivo diagnosis and detection of gastric cancer at endoscopy.     

Liquid chromatographic resolution of 3-amino-1,4-benzodiazepin-2-one derivatives on various Pirkle-type chiral stationary phases

The two enantiomers of N-acyl amide and N-ureide derivatives of 3-amino-5-phenyl-1,4-benzodiazepin-2-ones, which have been known to show anti-respiratory syncytial virus (RSV) activity, were resolved on seven different Pirkle-type chiral stationary phases (CSPs) with the use of 10% isopropyl alcohol in hexane as a mobile phase. Among the seven Pirkle-type CSPs, the one based on (S)-leucine derivative named as N-Phe-L-Leu was found to be most successful, the separation factors (α) and the resolutions (R(S) ) for seven analytes being in the range of 1.78-4.21 and 5.94-15.08, respectively. By resolving N-benzyloxycarbonyl derivatives of 3-amino-5-phenyl(or 5-methyl)-1,4-benzodiazepin-2-ones on Pirkle-type CSPs, the phenyl ring at the 5-position and the N?H hydrogen at the 1-position of analytes were found to play an important role in the chiral recognition.     

A colorimetric chiral sensor based on chiral crown ether for the recognition of the two enantiomers of primary amino alcohols and amines

A new colorimetric chiral sensor material consisting of three different functional sites such as chromophore (2,4-dinitrophenylazophenol dye), binding site (crown ether), and chiral barrier (3,3'-diphenyl-1,1'-binaphthyl group) was prepared and applied to the recognition of the two enantiomers of primary amino alcohols and amines. Among five primary amino alcohols and two primary amines tested, the two enantiomers of phenylalaninol show the highest difference in the absorption maximum wavelength (Δλ(max)=43.5 nm) and in the association constants (K(S)/K(R)=2.51) upon complexation with the colorimetric chiral sensor material and, consequently, the two enantiomers of phenylalaninol were clearly distinguished from each other by the color difference.     

Mechanism of hydrogen peroxide-induced keratinocyte migration in a scratch-wound model

Recent studies have shown that low concentrations of H₂O₂ are produced endogenously by nonphagocytes after wounding. We observed that H₂O₂ at such concentrations can stimulate proliferation as well as migration of keratinocytes in a scratch-wound assay. Both wounding and H₂O₂ can induce phosphorylation of ERK1/2 via EGFR, but the activation of ERK1/2 by H₂O₂ is more sustained and can last more than 8 h. Sustained ERK1/2 activation is required for the increased proliferation and migration induced by H₂O₂. The p38 MAPK was also found to be phosphorylated upon treatment with H₂O₂ but it was not required for H₂O₂-induced migration or proliferation. Furthermore, it was observed that there is a cross talk between the ERK1/2 and the p38 pathways whereby inhibition of either pathway can lead to activation of the other. As a result, the motogenic effects of H₂O₂ were further enhanced when p38 was inhibited. Our data are consistent with the view that H₂O₂ may play an important signaling role in wound healing.