Bacillus cereus in the Artisanal Cheese Production Chain in Southwestern Mexico.

BACKGROUND: Bacillus cereus is associated with milk, dairy product, and dairy farm contamination. The aim of this study was to characterize strains of B. cereus in the small-scale artisanal cheese production chain in southwestern Mexico. METHODS: 130 samples were collected. B. cereus isolation was performed on Mannitol Egg Yolk Polymyxin (MYP) agar. Genotyping, enterotoxigenic profile, and determination of genes involved in the formation of B. cereus biofilm were performed by PCR. An antimicrobial susceptibility test was made by broth microdilution assay. The phylogenetic analysis was performed by amplification and sequencing of 16s rRNA. RESULTS: B. cereus sensu lato was isolated and molecularly identified in 16 samples and B. cereus sensu stricto (B. cereus) was the most frequently isolated and identified species (81.25%). Of all the isolated B. cereus sensu lato strains, 93.75% presented at least one gene for some diarrheagenic toxins, 87.5% formed biofilms, and 18.75% were amylolytic. All B. cereus sensu lato strains were resistant to beta-lactams and folate inhibitors. A close phylogenetic relationship between isolates was found between the cheese isolates and the air isolates. CONCLUSIONS: Strains of B. cereus sensu lato were found in small-scale artisanal cheeses on a farm in southwestern Mexico.

DNMT3B overexpression downregulates genes with CpG islands, common motifs, and transcription factor binding sites that interact with DNMT3B.

DNA methylation is a key epigenetic modification to regulate gene expression in mammalian cells. Abnormal DNA methylation in gene promoters is common across human cancer types. DNMT3B is the main de novo methyltransferase enhanced in several primary tumors. How de novo methylation is established in genes related to cancer is poorly understood. CpG islands (CGIs), common sequences, and transcription factors (TFs) that interact with DNMT3B have been associated with abnormal de novo methylation. We initially identified cis elements associated with DNA methylation to investigate the contribution of DNMT3B overexpression to the deregulation of its possible target genes in an epithelial cell model. In a set of downregulated genes (n = 146) from HaCaT cells with DNMT3B overexpression, we found CGI, common sequences, and TFs Binding Sites that interact with DNMT3B (we called them P-down-3B). PPL1, VAV3, IRF1, and BRAF are P-down-3B genes that are downregulated and increased their methylation in DNMT3B presence. Together these findings suggest that methylated promoters aberrantly have some cis elements that could conduce de novo methylation by DNMT3B.

Utility of in silico-identified-peptides in spike-S1 domain and nucleocapsid of SARS-CoV-2 for antibody detection in COVID-19 patients and antibody production.

SARS-CoV-2 contains four structural proteins, two of which, the spike and nucleocapsid, are commonly used for the standardization of novel methods for antibody detection; however, some limitations in their use have been observed due to the homology of this virus with other phylogenetically-related viruses. We performed in silico analysis to search for novel immunogenic and antigenic peptides. A total of twenty-five peptides were preliminarily selected, located in the 3D structure of both proteins. Finally, eight peptides were selected: one located in the N protein and seven in the S1 domain of the spike protein. Additionally, the localization of selected peptides in 2D structures and possible changes in the sequences of these peptides in SARS-CoV-2 variants of concern were analyzed. All peptides were synthetized in MAP8 format, and recombinant S (trimer and RBD) and N proteins were used as antigens to search for antibodies in serum samples derived from COVID-19 patients, and for antibody response in New Zealand rabbits. Results showed high recognition of the serum derived from COVID-19 patients to all selected peptides; however, only the RBD3 peptide induced antibody production. In conclusion, this work provides evidence for a new strategy in peptide selection and its use for antibody detection or antibody production in animals.

In Peripheral Blood Mononuclear Cells Helicobacter pylori Induces the Secretion of Soluble and Exosomal Cytokines Related to Carcinogenesis.

Helicobacter pylori promotes the secretion of cytokines that regulate inflammation and carcinogenesis. Immune cells secrete cytokines into the extracellular medium or packaged in exosomes. The objective of this study was to analyze the profile of soluble and exosomal cytokines that were secreted by human peripheral blood mononuclear cells (PBMCs) that were infected with H. pylori and to build a network of interaction between cytokines and cellular proteins. PBMCs were obtained by density gradient centrifugation and infected with H. pylori for 24 h. The infection was verified by immunofluorescence and Western blot for CagA. The exosomes were obtained from culture supernatant by ultracentrifugation and characterized by transmission electron microscopy, particle size analysis, and Western blot for CD9 and CD81. Cytokines were quantified using a multiplex immunoassay in the culture supernatant, intact exosomes, and lysed exosomes. H. pylori adheres to lymphocytes and translocates CagA. In PBMCs, H. pylori induces an increase in the soluble and exosomal IL-1ß, IL-6, TNF-α, IL-10, IL-17A, IL-21, and IL-22. The protein-protein interaction (PPI) network shows that soluble and exosomal cytokines interact with proteins that participate in signaling pathways such as NF-κB, MAPK, PI3K-Akt, Jak-STAT, FoxO, and mTOR, that are related to carcinogenesis; moreover, TNF-α had the highest number of interactions. Cytokine-loaded exosomes represent another means of intercellular communication that is activated by H. pylori to stimulate inflammation, carcinogenesis, or cancer progression. Cytokine-loaded exosomes are likely to be associated with extragastrointestinal diseases of inflammatory origin.

Changes in the Expression of Insulin Pathway, Neutrophil Elastase and Alpha 1 Antitrypsin Genes from Leukocytes of Young Individuals with Insulin Resistance.

Background: Chronic hyperinsulinemia is a hallmark of insulin resistance that affects a diversity of cells, including leukocytes modifying the expression of some genes involved in insulin signaling. Purpose: The aim of this study was to evaluate how hyperinsulinemia affects the expression of genes involved in the proximal insulin signaling pathway in leukocytes from 45 young individuals grouped: normal weight with not insulin resistance (NIR), with insulin resistance (IR) and with obesity (OB-IR). Methods: qPCR was performed to analyze the expression of insulin receptor (INSR), insulin receptor substrate 1 and 2 (IRS-1 and IRS-2), neutrophil elastase (NE), alpha 1 antitrypsin (A1AT), glucose transporters 1, 3 and 4 (GLUT-1, GLUT-3 and GLUT-4) by the 2-ΔCt method, and the correlation between the genes was determined by Spearman's test. Results: The mRNA expression analysis of all genes between NIR and IR individuals revealed no differences. However, when comparing NIR and IR individuals with OB-IR, an increase in NE and A1AT expression and a clear trend towards a decrease in IRS-2 expression was observed, whereas the comparison of IR and OB-IR showed a decrease in GLUT-3 expression. Overall, the correlation analysis showed that in the IR group there was a positive correlation only between NE with IRS-1 (r = 0.72, p = 0.003), while in the OB-IR group, there was a positive correlation between the NE and A1AT with INSR (r = 0.62, p = 0.01 and r = 0.74, p = 0.002, respectively) and with IRS-2 (r = 0.74, p = 0.002 and r = 0.76, p = 0.001, respectively). Conclusion: These results suggest that hyperinsulinemia and obesity are associated with changes in the expression of genes in leukocytes involved in the insulin pathway that are related to NE and A1AT.

The regulatory role of insulin in energy metabolism and leukocyte functions.

Insulin is the hormone responsible for maintaining glucose homeostasis in the body, in addition to participating in lipid metabolism, protein synthesis, and the inhibition of gluconeogenesis. These functions are well characterized in the classic organ target cells that are responsible for general energy regulation: the liver, skeletal muscle, and adipose tissue. However, these actions are not restricted to these tissues because insulin has been shown to affect most cells in the body. This review describes the role of insulin in leukocyte signaling pathways, metabolism and functions, and how insulin resistance could affect this signaling and deteriorate leukocyte metabolism and function, in addition to showing evidence that suggests leukocytes may substantially contribute to the development of systemic insulin resistance.

Mobility and distribution of whey solutions in soil columns / Mobilidade e distribição de solutos de soro de leite em colunas de solo

In Brazil's food industry, dairy production is one of the most important sectors, whose most relevant byproduct is whey. Due to the difficulties of reuse and environmental impacts caused when discarded as effluent in water bodies, an alternative for its final destination would be the application of this residue in the soil. The purpose of this study was to determine chemical changes and mobility and distribution of solutes in the soil after applications of whey rates, as well as to analyze the leachate collected after each application. The test was carried out in a laboratory, in PVC columns filled with soil. The treatments consisted of 2 x 2 samples of a typical dystrophic Red-Yellow Oxisol (Oxisol) and a typical dystrophic fluvic Inceptisol (Inceptisol), sampled in the layers 0- 20 and 20-40 cm. Each experimental unit consisted of 11 PVC rings (diameter of 6.6 cm, height of 7 cm). The columns were arranged in a randomized complete block design with five replications. Four whey rates were applied, corresponding to a soil pore volume of 0.2, at intervals of six days. The leachate was collected 24 and 120 hours after each application to measure pH, electrical conductivity (EC), chemical oxygen demand (COD), contents of total N, N-NH4+, N-NO3-, Na, K, Ca, and Mg. Six days after the last whey application, the columns were opened and the soil of each ring was analyzed for pH, EC, total N, N-NH4+, N-NO3-, Na, K, Ca, and Mg. The high electrolyte concentrations of whey resulted in a general increase in soil EC. The increase of N-NH4+ and N-NO3- in the soil was high due to mineralization. High concentrations of K, Na and Ca caused displacement of Mg from the exchange complex. It was concluded that from an environmental standpoint, whey soil application is a viable alternative, given that problems of salinization and leaching of undesirable elements are avoided by an adequate management.

Entre os setores da indústria alimentícia, o segmento de laticínios é um dos mais importantes do Brasil, sendo o soro de leite o seu maior subproduto. Devido às dificuldades de reaproveitamento e aos impactos ambientais causados, quando descartado como efluente em corpos de água, uma alternativa para a sua destinação final seria a aplicação desse resíduo no solo. Objetivou-se com este trabalho determinar alterações químicas e a mobilidade e distribuição de solutos no solo após aplicação fracionada de soro de leite, assim como a caracterização dos percolados recolhidos após cada aplicação. O ensaio, em laboratório, foi conduzido em colunas de PVC preenchidas com solo. Os tratamentos corresponderam a um fatorial 2 x 2, sendo amostras de um Latossolo Vermelho-Amarelo distrófico típico (LVAd) e de um Cambissolo Flúvico Tb distrófico (CYbd), coletados em duas profundidades: 0­20 e 20­40 cm. Cada unidade experimental foi constituída de 11 anéis de PVC, com 6,6 cm de diâmetro interno e 7 cm de altura. As colunas foram dispostas em um delineamento experimental em blocos casualizados, com cinco repetições. Foram realizadas quatro aplicações de soro de leite, correspondentes a 0,2 volume de poros de solo, a cada intervalo de seis dias. O percolado foi recolhido após 24 e 120 horas de cada aplicação sendo realizadas as seguintes determinações: pH, condutividade elétrica (CE), demanda química de oxigênio (DQO), teores de N total, N-NH4+, N-NO3-, Na, K, Ca e Mg. Após seis dias da última aplicação de soro de leite, as colunas foram desmontadas e o solo de cada anel analisado. As análises compreenderam: pH em água, CE (1:5), N total, N-NH4+, N-NO3-, Na, K, Ca e Mg. As elevadas concentrações eletrolíticas do soro de leite provocaram aumento generalizado da CE no solo. Houve incremento maior do nitrogênio assimilável (N-NH4+ e N-NO3-) no solo em decorrência de processos de mineralização. Elevadas concentrações de K, Na e Ca causaram deslocamento de Mg do complexo de troca. Conclui-se que a aplicação de soro de leite no solo é uma alternativa viável do ponto de vista ambiental, desde que seja feito um correto manejo, a fim de evitar problemas de salinização e perdas por lixiviação de elementos indesejáveis.

In silico prediction of structural changes in human papillomavirus type 16 (HPV16) E6 oncoprotein and its variants.

BACKGROUND: HPV16 infection is one of the main risk factors involved in the development of cervical cancer, mainly due to the high oncogenic potential of the viral proteins E6 and E7, which are involved in the different processes of malignant transformation. There is a broad spectrum of intratypical variation of E6, which is reflected in its high diversity, biological behavior, global distribution and risk of causing cervical cancer. Experimental studies have shown that the intratypical variants of the protein E6 from the European variants (E-G350, E-A176/G350, E-C188/G350) and Asian-American variants (AAa and AAc), are capable of inducing the differential expression of genes involved in the development of cervical cancer. RESULTS: An in silico analysis was performed to characterize the molecular effects of these variations using the structure of the HPV16 E6 oncoprotein (PDB: 4XR8; chain H) as a template. In particular, we evaluated the 3D structures of the intratypical variants by structural alignment, ERRAT, Ramachandran plots and prediction of protein disorder, which was further validated by molecular dynamics simulations. Our results, in general, showed no significant changes in the protein 3D structure. However, we observed subtle changes in protein physicochemical features and structural disorder in the N- and C-termini. CONCLUSIONS: Our results showed that mutations in the viral oncogene E6 of six high-risk HPV16 variants are effectively neutral and do not cause significant structural changes except slight variations of structural disorder. As structural disorder is involved in rewiring protein-protein interactions, these results suggest a differential pattern of interaction of E6 with the target protein P53 and possibly different patterns of tumor aggressiveness associated with certain types of variants of the E6 oncoprotein.

Extracellular-Signal Regulated Kinase: A Central Molecule Driving Epithelial-Mesenchymal Transition in Cancer.

Epithelial-mesenchymal transition (EMT) is a reversible cellular process, characterized by changes in gene expression and activation of proteins, favoring the trans-differentiation of the epithelial phenotype to a mesenchymal phenotype. This process increases cell migration and invasion of tumor cells, progression of the cell cycle, and resistance to apoptosis and chemotherapy, all of which support tumor progression. One of the signaling pathways involved in tumor progression is the MAPK pathway. Within this family, the ERK subfamily of proteins is known for its contributions to EMT. The ERK subfamily is divided into typical (ERK 1/2/5), and atypical (ERK 3/4/7/8) members. These kinases are overexpressed and hyperactive in various types of cancer. They regulate diverse cellular processes such as proliferation, migration, metastasis, resistance to chemotherapy, and EMT. In this context, in vitro and in vivo assays, as well as studies in human patients, have shown that ERK favors the expression, function, and subcellular relocalization of various proteins that regulate EMT, thus promoting tumor progression. In this review, we discuss the mechanistic roles of the ERK subfamily members in EMT and tumor progression in diverse biological systems.

Production and characterization of monoclonal antibodies against the DNA binding domain of the RE1-silencing transcription factor.

The use of monoclonal antibodies for the detection of cellular biomarkers during carcinogenesis provides new strategies for cancer diagnosis or prognosis in patients. Loss of the Restrictive Element 1-Silencing Transcription (REST) factor has been observed in previous molecular and immunological approaches in aggressive breast cancer, small cell lung cancer, liver carcinoma, and colo-rectal cancer; however, for clinic diagnosis, monoclonal antibodies for REST recognition are unavailable. The goal of this work was to design, produce and characterize monoclonal antibodies against the REST DNA binding damain (DBD) that would be suitable for immunoassays. We searched for conserved domains, and immunogenic and antigenic sites in the REST structure via in silico analysis. For mice immunization, we used a recombinant REST DBD purified by affinity chromatography, and then Hybridomas were generated by mouse spleen fusion with myeloma cells. Finally, for monoclonal antibody characterization, we performed enzyme-linked immunosorbent (ELISA), western blot, dot blot, immunocytochemistry (ICC) and immunoprecipitation assays. Results showed that the DBD is conserved in REST isoforms and contains immunogenic and antigenic sites. We generated three clones producing monoclonal antibodies against REST DBD, one of them specifically recognized native REST and was suitable for ICC in samples from patients.

Methylation of the L1 gene and integration of human papillomavirus 16 and 18 in cervical carcinoma and premalignant lesions.

High-risk human papillomavirus (HPV) is the primary cause of cervical carcinoma (CC). Viral integration into the host chromosomes is associated with neoplastic progression, and epigenetic changes may occur as a result. The objective of the present study was to analyze HPV L1 gene methylation and to compare the use of quantitative polymerase chain reaction (qPCR), in situ hybridization (ISH) and L1 methylation analysis as methods for detecting HPV integration. Cervical scrapes or biopsy samples positive for HPV 16 or 18, from 187 female patients with CC, squamous intraepithelial lesions (SILs) or no intraepithelial lesion (non-IL) were analyzed. Methylation of the L1 gene was determined using bisulfite modification followed by PCR, and HPV integration was subsequently analyzed. HPV 16 L1 gene methylation was revealed to increase with histological grade, with statistically significant differences observed as follows: Low-grade SIL vs. CC, P<0.0001 and non-IL vs. CC, P<0.0001. HPV 18 L1 gene methylation also increased according to histological grade, however, no statistically significant differences were observed. Methylation at CpG site 5608 of the HPV 16 L1 gene was associated with all grades of cervical lesions, whereas methylation at CpG site 5617 demonstrated the strongest association with CC (odds ratio, 42.5; 95% confidence interval, 4.7-1861; P<0.0001). The concordance rates between the various methods for the detection of the physical status of HPV 16 and HPV 18 were 96.1% for qPCR and ISH, 76.7% for qPCR and L1 gene methylation, and 84.8% for ISH and L1 gene methylation. In conclusion, methylation of the HPV 16 L1 gene increases significantly according to the grade of the cervical lesion, and methylation at CpG sites 5608 and 5617 of this gene may be used as prognostic biomarkers. ISH and L1 gene methylation have good concordance with qPCR with regards to the detection of HPV integration. Therefore, these are useful methods in determining the physical state of HPV.