Cultivable Microbiome Approach Applied to Cervical Cancer Exploration.

Traditional microbiological methodology is valuable and essential for microbiota composition description and microbe role assignations at different anatomical sites, including cervical and vaginal tissues; that, combined with molecular biology strategies and modern identification approaches, could give a better perspective of the microbiome under different circumstances. This pilot work aimed to describe the differences in microbiota composition in non-cancer women and women with cervical cancer through a culturomics approach combining culture techniques with Vitek mass spectrometry and 16S rDNA sequencing. To determine the possible differences, diverse statistical, diversity, and multivariate analyses were applied; the results indicated a different microbiota composition between non-cancer women and cervical cancer patients. The Firmicutes phylum dominated the non-cancer (NC) group, whereas the cervical cancer (CC) group was characterized by the predominance of Firmicutes and Proteobacteria phyla; there was a depletion of lactic acid bacteria, an increase in the diversity of anaerobes, and opportunistic and non-typical human microbiota isolates were present. In this context, we hypothesize and propose a model in which microbial composition and dynamics may be essential for maintaining the balance in the cervical microenvironment or can be pro-oncogenesis microenvironmental mediators in a process called Ying-Yang or have a protagonist/antagonist microbiota role.

Design of a Multi-Epitope Vaccine against Tuberculosis from Mycobacterium tuberculosis PE_PGRS49 and PE_PGRS56 Proteins by Reverse Vaccinology.

Tuberculosis is a disease caused by Mycobacterium tuberculosis, representing the second leading cause of death by an infectious agent worldwide. The available vaccine against this disease has insufficient coverage and variable efficacy, accounting for a high number of cases worldwide. In fact, an estimated third of the world's population has a latent infection. Therefore, developing new vaccines is crucial to preventing it. In this study, the highly antigenic PE_PGRS49 and PE_PGRS56 proteins were analyzed. These proteins were used for predicting T- and B-cell epitopes and for human leukocyte antigen (HLA) protein binding efficiency. Epitopes GGAGGNGSLSS, FAGAGGQGGLGG, GIGGGTQSATGLG (PE_PGRS49), and GTGWNGGKGDTG (PE_PGRS56) were selected based on their best physicochemical, antigenic, non-allergenic, and non-toxic properties and coupled to HLA I and HLA II structures for in silico assays. A construct with an adjuvant (RS09) plus each epitope joined by GPGPG linkers was designed, and the stability of the HLA-coupled construct was further evaluated by molecular dynamics simulations. Although experimental and in vivo studies are still necessary to ensure its protective effect against the disease, this study shows that the vaccine construct is dynamically stable and potentially effective against tuberculosis.

HP0953 - hypothetical virulence factor overexpresion and localization during Helicobacter pylori infection of gastric epithelium.

BACKGROUND: The high prevalence and persistence of Helicobacter pylori (H. pylori) infection, as well as the diversity of pathologies related to it, suggest that the virulence factors used by this microorganism are varied. Moreover, as its proteome contains 340 hypothetical proteins, it is important to investigate them to completely understand the mechanisms of its virulence and survival. We have previously reported that the hypothetical protein HP0953 is overexpressed during the first hours of adhesion to inert surfaces, under stress conditions, suggesting its role in the environmental survival of this bacterium and perhaps as a virulence factor. AIM: To investigate the expression and localization of HP0953 during adhesion to an inert surface and against gastric (AGS) cells. METHODS: Expression analysis was performed for HP0953 during H. pylori adhesion. HP0953 expression at 0, 3, 12, 24, and 48 h was evaluated and compared using the Kruskal-Wallis equality-of-populations rank test. Recombinant protein was produced and used to obtain polyclonal antibodies for immunolocalization. Immunogold technique was performed on bacterial sections during adherence to inert surfaces and AGS cells, which was analyzed by transmission electron microscopy. HP0953 protein sequence was analyzed to predict the presence of a signal peptide and transmembrane helices, both provided by the ExPASy platform, and using the GLYCOPP platform for glycosylation sites. Different programs, via, I-TASSER, RaptorX, and HHalign-Kbest, were used to perform three-dimensional modeling. RESULTS: HP0953 exhibited its maximum expression at 12 h of infection in gastric epithelium cells. Immunogold technique revealed HP0953 localization in the cytoplasm and accumulation in some peripheral areas of the bacterial body, with greater expression when it is close to AGS cells. Bioinformatics analysis revealed the presence of a signal peptide that interacts with the transmembrane region and then allows the release of the protein to the external environment. The programs also showed a similarity with the Tip-alpha protein of H. pylori. Tip-alpha is an exotoxin that penetrates cells and induces tumor necrosis factor alpha production, and HP0953 could have a similar function as posttranslational modification sites were found; modifications in turn require enzymes located in eukaryotic cells. Thus, to be functional, HP0953 may necessarily need to be translocated inside the cell where it can trigger different mechanisms producing cellular damage. CONCLUSION: The location of HP0953 around infected cells, the probable posttranslational modifications, and its similarity to an exotoxin suggest that this protein is a virulence factor.

Characterization and use in neutralization assays of avian influenza codon-optimized H5 and H7 retroviral pseudotypes.

Influenza is a relevant problem for public and animal health, with a significant economic impact. In recent years, outbreaks of avian influenza virus have resulted in devastating losses in the poultry industry worldwide, and although its transmission to humans is very rare, there is always a potential risk for an even more severe outbreak. Currently, vaccination is considered the most effective tool for the control and prevention of influenza infections in both humans and animals. The maintenance of animal welfare and the successful implementation of animal health programs depend on the timely administration of vaccines, which must comply with quality specifications indicated by health authorities; for example, the capability to ensure a minimum antibody titer. The production of viral antigens used in these tests can pose a biosafety risk, and some viral strains can be difficult to grow. Therefore, new biotechnological alternatives are required to overcome these disadvantages. In this study, we produced pseudotypes carrying H5 and H7 hemagglutinins from lowly and highly pathogenic avian influenza viruses. These pseudotypes were used in neutralization assays to detect neutralizing antibodies in avian sera, which were confirmed positive by inhibition of the hemagglutination test. Our results showed that the pseudotype neutralization assay is a viable alternative for the detection of neutralizing antibodies, by demonstrating subtype specificity and requiring reduced biosafety requirements. Therefore, it represents a versatile platform that can facilitate technology transfer protocols between laboratories, and an immediate application in serological tools for quality control of veterinary vaccines against avian influenza.

Chikungunya nsP4 homology modeling reveals a common motif with Zika and Dengue RNA polymerases as a potential therapeutic target.

Among the diseases transmitted by vectors, there are those caused by viruses named arboviruses (arthropod-borne viruses). In past years, viruses transmitted by mosquitoes have been of relevance in global health, such as Chikungunya (CHIKV), Dengue (DENV), and Zika (ZIKV), which have Aedes aegypti as a common vector, thus raising the possibility of multi-infection. Previous reports have described the general structure of RNA-dependent RNA polymerases termed right-hand fold, which is conserved in positive single-stranded RNA viruses. Here, we report a comparison between sequences and the computational structure of RNA-dependent RNA polymerases from CHIKV, DENV, and ZIKV and the conserved sites to be considered for the design of an antiviral drug against the three viruses. We show that the sequential identity between consensus sequences from CHIKV and DENV is 8.1% and the similarity is 15.1%; the identity between CHIKV and ZIKV is 9.3%, and the similarity is 16.6%; and the identity between DENV and ZIKV is 68.6%, and the similarity is 79.2%. Nevertheless, the structural alignment shows that the root-mean-square deviation (RMSD) measurement value in general structure comparison between CHIKV RdRp and ZIKV RdRp was 1.248 Å, RMSD between CHIKV RdRp and DENV RdRp was 1.070 Å, and RMSD between ZIKV RdRp and DENV RdRp was 1.106 Å. Despite the low identity and similarity of CHIKV sequence with DENV and ZIKV, we show that A, B, C, and E motifs are structurally well conserved. These structural similarities offer a window into drug design against these arboviruses giving clues about critical target sites.

In silico strategies for modeling RNA aptamers and predicting binding sites of their molecular targets.

RNA aptamers are single-stranded nucleic acids of 20-100 nucleotides, with high sensitivity and specificity against particular molecular targets. In vitro production and selection of aptamers can be performed using the SELEX method. However, this procedure requires considerable time and cost. In this sense, bioinformatics tools play an important role in reducing the time and cost associated with development and production of aptamers. In this article, we propose bioinformatics strategies for modeling and analysis of the interaction with molecular targets for two RNA aptamers: ATP binding RNA aptamer and iSpinach aptamer. For this purpose, molecular modeling of the tertiary structure of the aptamers was performed with two servers (SimRNA and RNAComposer); and AutoDock Vina and rDock programs were used to dock their respective ligands. The predictions developed with these methods could be used for in silico design of RNA aptamers, through a simple and accessible methodology.Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.1951754 .

The phosphorelay signal transduction system in Candida glabrata: an in silico analysis.

Signaling systems allow microorganisms to sense and respond to different stimuli through the modification of gene expression. The phosphorelay signal transduction system in eukaryotes involves three proteins: a sensor protein, an intermediate protein and a response regulator, and requires the transfer of a phosphate group between two histidine-aspartic residues. The SLN1-YPD1-SSK1 system enables yeast to adapt to hyperosmotic stress through the activation of the HOG1-MAPK pathway. The genetic sequences available from Saccharomyces cerevisiae were used to identify orthologous sequences in Candida glabrata, and putative genes were identified and characterized by in silico assays. An interactome analysis was carried out with the complete genome of C. glabrata and the putative proteins of the phosphorelay signal transduction system. Next, we modeled the complex formed between the sensor protein CgSln1p and the intermediate CgYpd1p. Finally, phosphate transfer was examined by a molecular dynamic assay. Our in silico analysis showed that the putative proteins of the C. glabrata phosphorelay signal transduction system present the functional domains of histidine kinase, a downstream response regulator protein, and an intermediate histidine phosphotransfer protein. All the sequences are phylogenetically more related to S. cerevisiae than to C. albicans. The interactome suggests that the C. glabrata phosphorelay signal transduction system interacts with different proteins that regulate cell wall biosynthesis and responds to oxidative and osmotic stress the same way as similar systems in S. cerevisiae and C. albicans. Molecular dynamics simulations showed complex formation between the response regulator domain of histidine kinase CgSln1 and intermediate protein CgYpd1 in the presence of a phosphate group and interactions between the aspartic residue and the histidine residue. Overall, our research showed that C. glabrata harbors a functional SLN1-YPD1-SSK1 phosphorelay system.

Seroprevalence of Pandemic A(H1N1) pmd09 Virus Antibodies in Mexican Health Care Workers Before and After Vaccination.

BACKGROUND AND AIMS: In April 2009, a new strain of influenza A(H1N1) was identified in Mexico and in the U.S. In June 2009, WHO declared this a pandemic. Health care workers constituted a risk group for their close contact with infected individuals. The aim was to estimate seropositivity for A(H1N1)pdm09 in health staff at the Instituto Mexicano del Seguro Social. METHODS: A two-stage cross-sectional study, before and after vaccination in the same workers, was performed on a random sample of health-care workers. A socio-occupational questionnaire was applied and serum antibodies against influenza A(H1N1)pdm09 were determined through neutralization of retroviral pseudotypes; two logistic regression models for both were constructed. RESULTS: The average (median/mean) age of 1378 participants from 13 work centers was 41.7 years and 68.7% (947) were women. Seroprevalence for the first stage was 26.5% (365) (7.4-43%) vs. 20.8% (11) in a control group from the blood bank; for the second stage, the vaccinated group was 33% (215) (18.2-47%) and 27% (196) (11.6-50%) for the unvaccinated group. In regression models, seropositivity was associated with occupational exposure to suspected influenza infected patients, being physicians, and being vaccinated. CONCLUSIONS: Seropositivity against pandemic virus is similar to what was reported, both for vaccinated (2.8-40.9%) and unvaccinated (18.8-64.7%). Low seroprevalence in the vaccinated group indicates that between 67% and 73% were susceptible to infection. Given the relatively low vaccine-induced seropositivity, it is imperative to increase, hygiene and safety for health staff and at-risk populations, and strengthen epidemiological surveillance.