Molecular recognition of HER‐1 in whole‐blood samples

Multimode sensing was proposed for molecular screening and recognition of HER‐1 in whole blood. The tools used for molecular recognition were platforms based on nanostructured materials such as the complex of Mn(III) with meso‐tetra (4‐carboxyphenyl) porphyrin, and maltodextrin (dextrose equivalence between 4 and 7), immobilized in diamond paste, graphite paste or C₆₀ fullerene paste. The identification of HER‐1 in whole‐blood samples, at molecular level, is performed using stochastic mode and is followed by the quantification of it using stochastic and differential pulse voltammetry modes. HER‐1 can be identified in the concentration range between 280 fg/ml and 4.86 ng/ml using stochastic mode, this making possible the early detection of cancers such as gastrointestinal, pancreatic and lung cancers. The recovery tests performed using whole‐blood samples proved that the platforms can be used for identification and quantification of HER‐1 with high sensitivity and reliability in such samples, these making them good molecular screening tools. Copyright © 2014 John Wiley & Sons, Ltd.     

Finding disease variants in Mendelian disorders by using sequence data: methods and applications

Many sequencing studies are now underway to identify the genetic causes for both Mendelian and complex traits. Via exome-sequencing, genes harboring variants implicated in several Mendelian traits have already been identified. The underlying methodology in these studies is a multistep algorithm based on filtering variants identified in a small number of affected individuals and depends on whether they are novel (not yet seen in public resources such as dbSNP), shared among affected individuals, and other external functional information on the variants. Although intuitive, these filter-based methods are nonoptimal and do not provide any measure of statistical uncertainty. We describe here a formal statistical approach that has several distinct advantages: (1) it provides fast computation of approximate p values for individual genes, (2) it adjusts for the background variation in each gene, (3) it allows for incorporation of functional or linkage-based information, and (4) it accommodates designs based on both affected relative pairs and unrelated affected individuals. We show via simulations that the proposed approach can be used in conjunction with the existing filter-based methods to achieve a substantially better ranking of a gene relevant for disease when compared to currently used filter-based approaches, this is especially so in the presence of disease locus heterogeneity. We revisit recent studies on three Mendelian diseases and show that the proposed approach results in the implicated gene being ranked first in all studies, and approximate p values of 10⁻⁶ for the Miller Syndrome gene, 1.0 × 10⁻⁴ for the Freeman-Sheldon Syndrome gene, and 3.5 × 10⁻⁵ for the Kabuki Syndrome gene.     

An unusual brachiopod assemblage in a Late Jurassic (Kimmeridgian) stromatactis mud-mound of the Eastern Carpathians (Hăghimaş Mountains), Romania

An unusual fossil assemblage dominated by superabundant rhynchonellid brachiopods in a stromatactis mud-mound is recorded from the Hăghimaş Mountains (Eastern Carpathians), Romania. The mound mainly consists of bioclastic wackestones to packstones with a very rich macrofauna including crinoids, sponges, juvenile ammonites, and echinoids. The brachiopods represent a low-diversity but high-abundance association, dominated by the rhynchonellids Lacunosella and Septaliphoria. The taphonomical features of the fossil assemblage indicate an autochthonous fauna, with successive generations of brachiopods in life position and complete well-preserved individuals in different growth stage alongside an accessory population of crinoids and sponges. Brachiopod-brachiopod endosymbiotic life strategy is documented for the first time from a post-Paleozoic brachiopod assemblage. The mound reveals abundant stromatactis, filled by radiaxial fibrous or drusy calcite cement and internal polymud sediments. This is the first Late Jurassic (Kimmeridgian) stromatactis mud-mound identified in the Eastern Carpathians.     

Production of multiple brain-like ganglioside species is dispensable for fas-induced apoptosis of lymphoid cells

Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells. The present study was undertaken to clarify the role of this enzyme in the generation of gangliosides during apoptosis triggered by Fas ligation. The issue was addressed by using aSMase-deficient and aSMase-corrected cell lines derived from Niemann-Pick disease (NPD) patients. Fas cross-linking elicited a rapid production of large amounts of complex a- and b-series species of gangliosides with a pattern and a chromatographic behavior as single bands reminiscent of brain gangliosides. The gangliosides were synthesized within the first ten minutes and completely disappeared within thirty minutes after stimulation. Noteworthy is the observation that GD3 was not the only ganglioside produced. The production of gangliosides and the onset of apoptotic hallmarks occurred similarly in both aSMase-deficient and aSMase-corrected NPD lymphoid cells, indicating that aSMase activation is not accountable for ganglioside generation. Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process. In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.     

Carbon source‐induced reprogramming of the cell wall proteome and secretome modulates the adherence and drug resistance of the fungal pathogen Candida albicans

The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose‐grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C. albicans cell wall proteome and secretome. The regulation of cell wall structural proteins (e.g. Cht1, Phr1, Phr2, Pir1) correlated with extensive cell wall remodeling in lactate‐grown cells and with their increased resistance to stresses and antifungal drugs, compared with glucose‐grown cells. Moreover, changes in other proteins (e.g. Als2, Gca1, Phr1, Sap9) correlated with the increased adherence and biofilm formation of lactate‐grown cells. We identified mating and pheromone‐regulated proteins that were exclusive to lactate‐grown cells (e.g. Op4, Pga31, Pry1, Scw4, Yps7) as well as mucosa‐specific and other niche‐specific factors such as Lip4, Pga4, Plb5, and Sap7. The analysis of the corresponding null mutants confirmed that many of these proteins contribute to C. albicans adherence, stress, and antifungal drug resistance. Therefore, the cell wall proteome and secretome display considerable plasticity in response to carbon source. This plasticity influences important fitness and virulence attributes known to modulate the behavior of C. albicans in different host microenvironments during infection.     

Immunoproteomic identification and serological responses to novel Chlamydia pneumoniae antigens that are associated with persistent C. pneumoniae infections

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.     

Multitrait GWAS to connect disease variants and biological mechanisms

Genome-wide association studies (GWASs) have uncovered a wealth of associations between common variants and human phenotypes. Here, we present an integrative analysis of GWAS summary statistics from 36 phenotypes to decipher multitrait genetic architecture and its link with biological mechanisms. Our framework incorporates multitrait association mapping along with an investigation of the breakdown of genetic associations into clusters of variants harboring similar multitrait association profiles. Focusing on two subsets of immunity and metabolism phenotypes, we then demonstrate how genetic variants within clusters can be mapped to biological pathways and disease mechanisms. Finally, for the metabolism set, we investigate the link between gene cluster assignment and the success of drug targets in randomized controlled trials.     

Lettuce‐produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination

The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases (e.g. cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV‐related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca sativa) using Agrobacterium‐mediated transient expression technology. The wild‐type dimer (E1E2) and a variant without an N‐glycosylation site in the E2 polypeptide (E1E2?N6) were expressed, and appropriate N‐glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell‐expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti‐HCV IgM for both antigens; however, the E1E2?N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N‐glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti‐HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low‐cost oral vaccine development.     

On the frequency of copy number variants

MOTIVATION: Estimating the frequency distribution of copy number variants (CNVs) is an important aspect of the effort to characterize this new type of genetic variation. Currently, most studies report a strong skew toward low-frequency CNVs. In this article, our goal is to investigate the frequencies of CNVs. We employ a two-step procedure for the CNV frequency estimation process. We use family information a posteriori to select only the most reliable CNV regions, i.e. those showing high rates of Mendelian transmission. RESULTS: Our results suggest that the current skew toward low-frequency CNVs may not be representative of the true frequency distribution, but may be due, among other reasons, to the non-negligible false negative rates that characterize CNV detection methods. Moreover, false positives are also likely, as low-frequency CNVs are hard to detect with small sample sizes and technologies that are not ideally suited for their detection. Without appropriate validation methods, such as incorporation of biologically relevant information (for example, in our case, the transmission of heritable CNVs from parents to offspring), it is difficult to assess the validity of specific CNVs, and even harder to obtain reliable frequency estimates.