Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: myosin-V assay

The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 oC, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.     

Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

Recent studies have demonstrated that tocotrienol (T3) is superior to tocopherol (Toc) for cancer chemoprevention. However, there is little information on whether Toc influences the anticancer property of T3. In this study, we investigated the influence of Toc on the cytotoxic effects of δ-T3 in DLD-1 human colorectal adenocarcinoma cells. Toc, especially α-Toc, attenuated δ-T3-induced cytotoxicity and apoptosis in DLD-1 cells, whereas Toc alone did not exhibit any cytotoxic effect. δ-T3-induced cell cycle arrest and proapoptotic gene/protein expression (e.g., p21, p27, and caspases) were abrogated by α-Toc. Furthermore, coadministration of α-Toc decreased δ-T3 uptake into DLD-1 cells in a dose-dependent manner. These results indicate that α-Toc is not only less cytotoxic to cancer cells, but it also reduces the cytotoxicity of δ-T3 by inhibiting its cellular uptake.     

Younger siblings play a major role in Helicobacter pylori transmission among children from a low-income community in the Northeast of Brazil

Background and Aims: To further evaluate intrafamilial transmission of H. pylori infection during childhood, we investigated the prevalence of H. pylori in family members from a poor H. pylori high‐prevalence urban community in the Northeast of Brazil. Methods: H. pylori infection was investigated in 570 members of 128 households, by ¹³C‐urea breath test in children and by ELISA in mothers and other adult relatives. Results: The overall prevalence of H. pylori infection (376/570) increased with age (p < .001) and ranged from 28.9%, in children aged 6 months to 5 years, to 82% in adults over 40 years. An H. pylori positive mother and the number of infected siblings are independent risk factors for childhood H. pylori infection (OR = 2.2, 95% CI = 1.0–4.6 and OR = 4.3, 95% CI = 2.3–8.1, respectively) The number of siblings, number of younger siblings, and number of infected younger siblings were also associated with the infection in the univariate analysis. The number of infected younger siblings remained independently associated with the infection (p = .000), even after controlling for all the above cited variables, in addition to the H. pylori status of siblings and mothers, age, number of people per room, and number of children in the household. Conclusion: The transmission of H. pylori occurs from infected mothers to their offspring and among siblings, notably from younger siblings to the older ones.     

Screening anion-exchange chromatographic matrices for isolation of onco-retroviral vectors

The adsorption kinetics of retroviral vectors to several chromatographic media, DEAE FF, Streamlinetrade mark Q XL and CHTtrade mark Ceramic Hydroxyapatite, in batch mode was investigated. The effects of buffer type, pH and operational temperature were studied. A mathematical model describing viral adsorption kinetics that considers viral degradation in solution was developed. The best results, either in terms of speed and extent of adsorbed infectious particles, were obtained with DEAE FF and Streamlinetrade mark Q XL. Fixed-bed chromatography was further investigated using DEAE FF, Q XL and Q FF, for validation of the batch adsorption process. Fixed-bed DEAE FF and Q XL proved to be good candidates for purification of MoMLV derived vectors due to resulting high yields, 53+/-13% and 51+/-7%, respectively, while removing more than 99% of protein and 90% of the DNA contaminants.     

Isolation of Helicobacter pylori from the intestinal mucosa of patients with Crohn's disease

BACKGROUND: Helicobacter species are associated with inflammatory bowel disease in rodents and in nonhuman primates. Therefore, we prospectively investigated the presence of Helicobacter species in the intestinal mucosa of patients with and without Crohn's disease by culture and polymerase chain reaction (PCR) assays. MATERIALS AND METHODS: Mucosal fragments were obtained from the ileum, different colon regions, and rectum of 43 patients with Crohn's disease and of 74 patients without inflammatory bowel disease. RESULTS: Helicobacter pylori strains, identified by 16S rRNA gene sequencing, were more frequently isolated and PCR-detected in the intestinal mucosa of patients with ulcerative colitis-like Crohn's disease than in intestinal mucosa of the control group. Otherwise, anti-H. pylori immunoglobulin G levels were significantly lower in fibrostenosing and fistulating Crohn's disease subgroups. No other Helicobacter species were found in the intestinal mucosa of the patients. CONCLUSIONS: Although our results suggest an association between the presence of H. pylori in the intestine and ulcerative colitis-like phenotype of Crohn's disease, H. pylori infection in the actual causality of Crohn's disease is still to be determined.     

Karyotype characterization of three species of the genus Trichomycterus (Teleostei,Siluriformes) from Iguaçu river basin

Three species belonging to the genus Trichomycterus were collected from Igua?u river basin and analyzed from the cytogenetic point of view. Although the species studied had the same diploid number 2n = 54 chromosomes and fundamental number 108, they differed in their karyotypic formulae. C-band patterns showed presence of specific-species markers in T. davisi and T. stawiarski. Results of analysis from the nucleolus organizer region, obtained by silver nitrate staining and CMA3, showed that size heteromorphism observed in this region may be due to gene duplication. Evolution aspects of the Trichomycteridae family and the presence of B chromosome in T. davisi are discussed.     

Membrane topology of the ZntB efflux system of Salmonella enterica serovar Typhimurium

The membrane topology of the ZntB Zn(2+) transport protein of Salmonella enterica serovar Typhimurium was determined by constructing deletion derivatives of the protein and genetically fusing them to blaM or lacZ cassettes. The enzymatic activities of the hybrid proteins indicate that ZntB is a bitopic integral membrane protein consisting largely of two independent domains. The first 266 amino acids form a large, highly charged domain within the cytoplasm, while the remaining 61 residues form a small membrane domain containing two membrane-spanning segments. The overall orientation towards the cytoplasm is consistent with the ability of ZntB to facilitate zinc efflux.     

Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis

The evolution of the ancestral eukaryotic flagellum is an example of a cellular organelle that became dispensable in some modern eukaryotes while remaining an essential motile and sensory apparatus in others. To help define the repertoire of specialized proteins needed for the formation and function of cilia, we used comparative genomics to analyze the genomes of organisms with prototypical cilia, modified cilia, or no cilia and identified approximately 200 genes that are absent in the genomes of nonciliated eukaryotes but are conserved in ciliated organisms. Importantly, over 80% of the known ancestral proteins involved in cilia function are included in this small collection. Using Drosophila as a model system, we then characterized a novel family of proteins (OSEGs: outer segment) essential for ciliogenesis. We show that osegs encode components of a specialized transport pathway unique to the cilia compartment and are related to prototypical intracellular transport proteins.     

Detection of Toxoplasma gondii DNA by polymerase chain reaction in experimentally desiccated tissues

Despite toxoplasmosis being a common infection among human and other warm-blooded animals worldwide, there are no findings about Toxoplasma gondii evolutionary forms in ancient populations. The molecular techniques used for amplification of genetic material have allowed recovery of ancient DNA (aDNA) from parasites contained in mummified tissues. The application of polymerase chain reaction (PCR) to paleoparasitological toxoplasmosis research becomes a promising option, since it might allow diagnosis, acquisition of paleoepidemiological data, access to toxoplasmosis information related origin, evolution, and distribution among the ancient populations. Furthermore, it makes possible the analysis of parasite aDNA aiming at phylogenetic studies. To standardize and evaluate PCR applicability to toxoplasmosis paleodiagnostic, an experimental mummification protocol was tested using desiccated tissues from mice infected with the ME49 strain cysts, the chronic infection group (CIG), or infected with tachyzoites (RH strain), the acute infection group (AIG). Tissues were subjected to DNA extraction followed by PCR amplification of T. gondii B1 gene. PCR recovered T. gondii DNA in thigh muscle, encephalon, heart, and lung samples. AIG presented PCR positivity in encephalon, lungs, hearts, and livers. Based on this results, we propose this molecular approach for toxoplasmosis research in past populations.     

Glycoproteogenomics: Setting the Course for Next-generation Cancer Neoantigen Discovery for Cancer Vaccines

Molecular-assisted precision oncology gained tremendous ground with high-throughput next-generation sequencing(NGS), supported by robust bioinformatics. The quest for genomicsbased cancer medicine set the foundations for improved patient stratification, while unveiling a wide array of neoantigens for immunotherapy. Upfront pre-clinical and clinical studies have successfully used tumor-specific peptides in vaccines with minimal off-target effects. However, the low mutational burden presented by many lesions challenges the generalization of these solutions, requiring the diversification of neoantigen sources. Oncoproteogenomics utilizing customized databases for protein annotation by mass spectrometry(MS) is a powerful tool toward this end. Expanding the concept toward exploring proteoforms originated from post-translational modifications(PTMs)will be decisive to improve molecular subtyping and provide potentially targetable functional nodes with increased cancer specificity. Walking through the path of systems biology, we highlight that alterations in protein glycosylation at the cell surface not only have functional impact on cancer progression and dissemination but also originate unique molecular fingerprints for targeted therapeutics. Moreover, we discuss the outstanding challenges required to accommodate glycoproteomics in oncoproteogenomics platforms. We envisage that such rationale may flag a rather neglected research field, generating novel paradigms for precision oncology and immunotherapy.