A Reliable Method for the Selection of Exploitable Melanoma Archival Paraffin Embedded Tissues for Transcript Biomarker Profiling

The source tissue for biomarkers mRNA expression profiling of tumors has traditionally been fresh-frozen tissue. The adaptation of formalin-fixed, paraffin-embedded (FFPE) tissues for routine mRNA profiling would however be invaluable in view of their abundance and the clinical information related to them. However, their use in the clinic remains a challenge due to the poor quality of RNA extracted from such tissues. Here, we developed a method for the selection of melanoma archival paraffin-embedded tissues that can be reliably used for transcript biomarker profiling. For that, we used qRT-PCR to conduct a comparative study in matched pairs of frozen and FFPE melanoma tissues of the expression of 25 genes involved in angiogenesis/tumor invasion and 15 housekeeping genes. A classification method was developed that can select the samples with a good frozen/FFPE correlation and identify those that should be discarded on the basis of paraffin data for four reference genes only. We propose therefore a simple and inexpensive assay which improves reliability of mRNA profiling in FFPE samples by allowing the identification and analysis of “good” samples only. This assay which can be extended to other genes would however need validation at the clinical level and on independent tumor series

Accurate Prediction of DnaK-Peptide Binding via Homology Modelling and Experimental Data

Molecular chaperones are essential elements of the protein quality control machinery that governs translocation and folding of nascent polypeptides, refolding and degradation of misfolded proteins, and activation of a wide range of client proteins. The prokaryotic heat-shock protein DnaK is the E. coli representative of the ubiquitous Hsp70 family, which specializes in the binding of exposed hydrophobic regions in unfolded polypeptides. Accurate prediction of DnaK binding sites in E. coli proteins is an essential prerequisite to understand the precise function of this chaperone and the properties of its substrate proteins. In order to map DnaK binding sites in protein sequences, we have developed an algorithm that combines sequence information from peptide binding experiments and structural parameters from homology modelling. We show that this combination significantly outperforms either single approach. The final predictor had a Matthews correlation coefficient (MCC) of 0.819 when assessed over the 144 tested peptide sequences to detect true positives and true negatives. To test the robustness of the learning set, we have conducted a simulated cross-validation, where we omit sequences from the learning sets and calculate the rate of repredicting them. This resulted in a surprisingly good MCC of 0.703. The algorithm was also able to perform equally well on a blind test set of binders and non-binders, of which there was no prior knowledge in the learning sets. The algorithm is freely available at http://limbo.vib.be

Use of complementary/alternative therapies by women with advanced-stage breast cancer

BACKGROUND: This study sought to describe the pattern of complementary/alternative medicine (CAM) use among a group of patients with advanced breast cancer, to examine the main reasons for their CAM use, to identify patient's information sources and their communication pattern with their physicians. METHODS: Face-to-face structured interviews of patients with advanced-stage breast cancer at a comprehensive oncology center. RESULTS: Seventy three percent of patients used CAM; relaxation/meditative techniques and herbal medicine were the most common. The most commonly cited primary reason for CAM use was to boost the immune system, the second, to treat cancer; however these reasons varied depending on specific CAM therapy. Friends or family members and mass media were common primary information source's about CAM. CONCLUSIONS: A high proportion of advanced-stage breast cancer patients used CAM. Discussion with doctors was high for ingested products. Mass media was a prominent source of patient information. Credible sources of CAM information for patients and physicians are needed

1H, 15N, and 13C chemical shift assignments of neuronal calcium sensor-1 homolog from fission yeast

The neuronal calcium sensor (NCS) proteins regulate signal transduction processes and are highly conserved from yeast to humans. We report complete NMR chemical shift assignments of the NCS homolog from fission yeast (Schizosaccharomyces pombe), referred to in this study as Ncs1p. (BMRB no. 16446)

Wnt5a Increases Cardiac Gene Expressions of Cultured Human Circulating Progenitor Cells via a PKC Delta Activation

Background: Wnt signaling controls the balance between stem cell proliferation and differentiation and body patterning throughout development. Previous data demonstrated that non-canonical Wnts (Wnt5a, Wnt11) increased cardiac gene expression of circulating endothelial progenitor cells (EPC) and bone marrow-derived stem cells cultured in vitro. Since previous studies suggested a contribution of the protein kinase C (PKC) family to the Wnt5a-induced signalling, we investigated which PKC isoforms are activated by non-canonical Wnt5a in human EPC. Methodology/Principal Findings: Immunoblot experiments demonstrated that Wnt5a selectively activated the novel PKC isoform, PKC delta, as evidenced by phosphorylation and translocation. In contrast, the classical Ca2+-dependent PKC isoforms, PKC alpha and beta2, and one of the other novel PKC isoforms, PKC epsilon, were not activated by Wnt5a. The PKC delta inhibitor rottlerin significantly blocked co-culture-induced cardiac differentiation in vitro, whereas inhibitors directed against the classical Ca2+-dependent PKC isoforms or a PKC epsilon-inhibitory peptide did not block cardiac differentiation. In accordance, EPC derived from PKC delta heterozygous mice exhibited a significant reduction of Wnt5a-induced cardiac gene expression compared to wild type mice derived EPC. Conclusions/Significance: These data indicate that Wnt5a enhances cardiac gene expressions of EPC via an activation of PKC delta

Non-specific interstitial pneumonia in cigarette smokers: a CT study

The goal of this study was to seek indirect evidence that smoking is an aetiological factor in some patients with non-specific interstitial pneumonia (NSIP). Ten current and eight ex-smokers with NSIP were compared to controls including 137 current smokers with no known interstitial lung disease and 11 non-smokers with NSIP. Prevalence and extent of emphysema in 18 smokers with NSIP were compared with subjects meeting GOLD criteria for chronic obstructive pulmonary disease (COPD; group A; n = 34) and healthy smokers (normal FEV1; group B; n = 103), respectively. Emphysema was present in 14/18 (77.8%) smokers with NSIP. Emphysema did not differ in prevalence between NSIP patients and group A controls (25/34, 73.5%), but was strikingly more prevalent in NSIP patients than in group B controls (18/103, 17.5%, P < 0.0005). On multiple logistic regression, the likelihood of emphysema increased when NSIP was present (OR = 18.8; 95% CI = 5.3–66.3; P < 0.0005) and with increasing age (OR = 1.04; 95% CI = 0.99–1.11; P = 0.08). Emphysema is as prevalent in smokers with NSIP as in smokers with COPD, and is strikingly more prevalent in these two groups than in healthy smoking controls. The association between NSIP and emphysema provides indirect support for a smoking pathogenesis hypothesis in some NSIP patients

Association of FcγRIIa R131H polymorphism with idiopathic pulmonary fibrosis severity and progression

<p>Abstract</p> <p>Background</p> <p>A significant genetic component has been described for idiopathic pulmonary fibrosis (IPF). The R131H (rs1801274) polymorphism of the IgG receptor FcγRIIa determines receptor affinity for IgG subclasses and is associated with several chronic inflammatory diseases. We investigated whether this polymorphism is associated with IPF susceptibility or progression.</p> <p>Methods</p> <p>In a case-control study, we compared the distribution of FcγRIIa R131H genotypes in 142 patients with IPF and in 218 controls using allele-specific PCR amplification.</p> <p>Results</p> <p>No differences in the frequency of FcγRIIa genotypes were evident between IPF patients and control subjects. However, significantly impaired pulmonary function at diagnosis was observed in HH compared to RR homozygotes, with evidence of more severe restriction (reduced forced vital capacity (FVC)) and lower diffusing capacity for carbon monoxide (D<smcaps>L</smcaps>_CO). Similarly, increased frequency of the H131 allele was observed in patients with severe disease (D<smcaps>L</smcaps>_CO< 40% predicted) (0.53 vs. 0.38; p = 0.03). Furthermore, the H131 allele was associated with progressive pulmonary fibrosis as determined by > 10% drop in FVC and/or > 15% fall in D<smcaps>L</smcaps>_COat 12 months after baseline (0.48 vs. 0.33; p = 0.023).</p> <p>Conclusions</p> <p>These findings support an association between the FcγRIIa R131H polymorphism and IPF severity and progression, supporting the involvement of immunological mechanisms in IPF pathogenesis.</p

Cloning and characterization of the ecto-nucleotidase NTPDase3 from rat brain: Predicted secondary structure and relation to other members of the E-NTPDase family and actin

The protein family of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase family) contains multiple members that hydrolyze nucleoside 5’-triphosphates and nucleoside 5’-diphosphates with varying preference for the individual type of nucleotide. We report the cloning and functional expression of rat NTPDase3. The rat brain-derived cDNA has an open reading frame of 1590 bp encoding 529 amino acid residues, a calculated molecular mass of 59.1 kDa and predicted N- and C-terminal hydrophobic sequences. It shares 94.3% and 81.7% amino acid identity with the mouse and human NTPDase3, respectively, and is more closely related to cell surface-located than to the intracellularly located members of the enzyme family. The NTPDase3 gene is allocated to chromosome 8q32 and organized into 11 exons. Rat NTPDase3 expressed in CHO cells hydrolyzed both nucleoside triphosphates and nucleoside diphosphates with hydrolysis ratios of ATP:ADP of 5:1 and UTP:UDP of 8:1. After addition of ATP, ADP is formed as an intermediate product that is further hydrolyzed to AMP. The enzyme is preferentially activated by Ca2+ over Mg2+ and reveals an alkaline pH optimum. Immunocytochemistry confirmed expression of heterologously expressed NTPDase3 to the surface of CHO cells. PC12 cells express endogenous surface-located NTPDase3. An immunoblot analysis detects NTPDase3 in all rat brain regions investigated. An alignment of the secondary structure domains of actin conserved within the actin/HSP70/sugar kinase superfamily to those of all members of the NTPDase family reveals apparent similarity. It infers that NTPDases share the two-domain structure with members of this enzyme superfamily

Crystal Structures of the ATPase Domains of Four Human Hsp70 Isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78

The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1

Building an immune-mediated coagulopathy consensus: early recognition and evaluation to enhance post-surgical patient safety

Topical hemostats, fibrin sealants, and surgical adhesives are regularly used in a variety of surgical procedures involving multiple disciplines. Generally, these adjuncts to surgical hemostasis are valuable means for improving wound visualization, reducing blood loss or adding tissue adherence; however, some of these agents are responsible for under-recognized adverse reactions and outcomes. Bovine thrombin, for example, is a topical hemostat with a long history of clinical application that is widely used alone or in combination with other hemostatic agents. Hematologists and coagulation experts are aware that these agents can lead to development of an immune-mediated coagulopathy (IMC). A paucity of data on the incidence of IMC contributes to under-recognition and leaves many surgeons unaware that this clinical entity, originating from normal immune responses to foreign antigen exposure, requires enhanced post-operative vigilance and judicious clinical judgment to achieve best outcomes