Transcriptional Profiling of SARS-CoV-2-Infected Calu-3 Cells Reveals Immune-Related Signaling Pathways.

The COVID-19 disease, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), emerged in late 2019 and rapidly spread worldwide, becoming a pandemic that infected millions of people and caused significant deaths. COVID-19 continues to be a major threat, and there is a need to deepen our understanding of the virus and its mechanisms of infection. To study the cellular responses to SARS-CoV-2 infection, we performed an RNA sequencing of infected vs. uninfected Calu-3 cells. Total RNA was extracted from infected (0.5 MOI) and control Calu-3 cells and converted to cDNA. Sequencing was performed, and the obtained reads were quality-analyzed and pre-processed. Differential expression was assessed with the EdgeR package, and functional enrichment was performed in EnrichR for Gene Ontology, KEGG pathways, and WikiPathways. A total of 1040 differentially expressed genes were found in infected vs. uninfected Calu-3 cells, of which 695 were up-regulated and 345 were down-regulated. Functional enrichment analyses revealed the predominant up-regulation of genes related to innate immune response, response to virus, inflammation, cell proliferation, and apoptosis. These transcriptional changes following SARS-CoV-2 infection may reflect a cellular response to the infection and help to elucidate COVID-19 pathogenesis, in addition to revealing potential biomarkers and drug targets.

Subcutaneous, Oral, and Intranasal Immunization of BALB/c Mice with Leishmania infantum K39 Antigen Induces Non-Protective Humoral Immune Response.

Visceral leishmaniasis is a high-burden disease caused by parasites of the Leishmania genus. The K39 kinesin is a highly antigenic protein of Leishmania infantum, but little is known about the immune response elicited by this antigen. We evaluated the humoral immune response of female BALB/c mice (n = 6) immunized with the rK39-HFBI construct, formed by the fusion of the K39 antigen to a hydrophobin partner. The rK39-HFBI construct was administered through subcutaneous, oral, and intranasal routes using saponin as an adjuvant. We analyzed the kinetics of IgG, IgG1, and IgG2a production. The groups were then challenged by an intravenous infection with L. infantum promastigote cells. The rK39-HFBI antigen-induced high levels of total IgG (p < 0.05) in all groups, but only the subcutaneous route was associated with increased production of IgG1 and IgG2a 42 days after immunization (p < 0.05), suggesting a potential secondary immune response following the booster dose. There was no reduction in the splenic parasite load; thus, the rK39-HFBI failed to protect the mice against infection under the tested conditions. The results presented here demonstrate that the high antigenicity of the K39 antigen does not contribute to a protective immune response against visceral leishmaniasis.

Quantum Biochemistry and MM-PBSA Description of the ZIKV NS2B-NS3 Protease: Insights into the Binding Interactions beyond the Catalytic Triad Pocket.

The Zika virus protease NS2B-NS3 has a binding site formed with the participation of a H51-D75-S135 triad presenting two forms, active and inactive. Studies suggest that the inactive conformation is a good target for the design of inhibitors. In this paper, we evaluated the co-crystallized structures of the protease with the inhibitors benzoic acid (5YOD) and benzimidazole-1-ylmethanol (5H4I). We applied a protocol consisting of two steps: first, classical molecular mechanics energy minimization followed by classical molecular dynamics were performed, obtaining stabilized molecular geometries; second, the optimized/relaxed geometries were used in quantum biochemistry and molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) calculations to estimate the ligand interactions with each amino acid residue of the binding pocket. We show that the quantum-level results identified essential residues for the stabilization of the 5YOD and 5H4I complexes after classical energy minimization, matching previously published experimental data. The same success, however, was not observed for the MM-PBSA simulations. The application of quantum biochemistry methods seems to be more promising for the design of novel inhibitors acting on NS2B-NS3.

Orally hypoglycemic activity of an insulin mimetic glycoprotein isolated from Cnidoscolus quercifolius Pohl. (Euphorbiaceae) seeds, Cq-IMP.

The genus Cnidoscolus (Euphorbiaceae) is widely distributed in tropical areas. In the Northeast of Brazil, the species C. quercifolius is endemic and has been used in traditional medicine. In this study, a novel protein was isolated from C. quercifolius seeds and characterized by its molecular weight, primary structure, isoelectric point (pI), and carbohydrate content. The hypoglycemic activity of this protein was investigated by in vitro assay with the RIN-5F glucose-responsive cell line and in vivo test using alloxan-induced diabetic mice models. In addition, safe use of the protein was also investigated by cytotoxicity, hemagglutinating, and immunogenicity assays. The protein which was named Cq-IMP (Cnidoscolus quercifolius - Insulin Mimetic Protein) showed a single 11.18 KDa glycopolypeptide chain (16.4% of carbohydrates, m/m), pI of 8.0 and N-terminal sequence (TKDPELKQcKKQQKKqQQYDDDDKK) with similarity around 46-62% to sucrose binding protein-like and vicilin-like protein that was confirmed by mass spectrometry tryptic peptides analysis. Besides that, Cq-IMP presented anti-insulin antibody cross-reactivity as hypoglycemic activity in both in vitro and in vivo models. Additionally, it did not present any toxicity by methods tested. In conclusion, Cq-IMP is an insulin-mimetic protein, with a potent hypoglycemic activity and no toxicity showing great potential for therapeutic applications and drug development.

Role of membrane sterol and redox system in the anti-candida activity reported for Mo-CBP2, a protein from Moringa oleifera seeds.

Plant proteins are emerging as an alternative to conventional treatments against candidiasis. The aim of this study was to better understand the mechanism of action of Mo-CBP2 against Candida spp, evaluating redox system activity, lipid peroxidation, DNA degradation, cytochrome c release, medium acidification, and membrane interaction. Anti-candida activity of Mo-CBP2 decreased in the presence of ergosterol, which was not observed with antioxidant agents. C. albicans treated with Mo-CBP2 also had catalase and peroxidase activities inhibited, while superoxide dismutase was increased. Mo-CBP2 increased the lipid peroxidation, but it did not alter the ergosterol profile in live cells. External medium acidification was strongly inhibited, and cytochrome c release and DNA degradation were detected. Mo-CBP2 interacts with cell membrane constituents, changes redox system enzymes in C. albicans and causes lipid peroxidation by ROS overproduction. DNA degradation and cytochrome c release suggest apoptotic or DNAse activity. Lipid peroxidation and H+-ATPases inhibition may induce the process of apoptosis. Finally, Mo-CBP2 did not have a cytotoxic effect in mammalian Vero cells. This study highlights the biotechnological potential of Mo-CBP2 as a promising molecule with low toxicity and potent activity. Further studies should be performed to better understand its mode of action and toxicity.

Detection of serum and salivary IgE and IgG1 immunoglobulins specific for diagnosis of food allergy.

Given the growing incidence and prevalence of life-threatening food allergies, health concerns have raised new perspectives for in vivo and in vitro diagnostic methodologies, pointing to saliva as a promising material, already used to diagnose other pathologies. Based on the above considerations, this study aimed to verify the possible use of saliva for the detection of IgE and IgG1 in the diagnosis of food allergy. This was a randomized, cross-sectional clinical study with a quantitative approach, developed at a hospital referral center in allergy in the state of Ceará, from January to July 2015. The sample consisted of 36 children of both sexes, aged between 1 and 60 months, with a diagnosis of cow's milk protein allergy (CMPA) by the RAST test. Children hospitalized or under immunosuppressive drugs were excluded from the study. Serum and saliva samples of the participants were collected and subsequently subjected to the indirect immunoenzymatic assay (ELISA) for the detection of specific serum and salivary immunoglobulins for food: corn, papaya, cow's milk, egg white, wheat, soybeans, peanuts, nuts, kiwi, cacao, fish, shrimp, bananas and tomatoes. For comparison of serum and saliva results, the T-test of independent samples and Mann-Whitney were adopted, for samples with normal and non-normal distribution respectively. A confidence interval of 95% was adopted for significant results. It was observed that 100% (n = 36) of the participants presented cow's milk allergy through the indirect ELISA, detecting IgE or IgG1 in serum and saliva. When serum IgE and IgG1 concentrations were compared, there was no statistical difference (p > 0.05) in 12 of the 14 foods evaluated. The same amount (n = 12) of non-significant differences (p > 0.05) was observed in the comparison of the 14 foods under IgE and IgG1 contractions in saliva. In the verification of the average values of IgE present in the serum and saliva of the foods, only cow's milk, fish and papaya showed statistically significant differences (p < 0.05). Of the total food evaluated, only the average levels of IgG1 present in serum and saliva showed a significant value (p < 0.05) in banana and tomato. These findings indicate that the detection of IgE and IgG1 in saliva proves to be as efficient as in the serum. The use of the salivary technique for use in the diagnosis of food allergy is suggested.

Proteomic analysis to identify candidate biomarkers associated with type 1 diabetes.

PURPOSE: Type 1 diabetes mellitus (DM1) is one of the most common chronic diseases observed during childhood. The incidence of DM1 is increasing worldwide, and there is currently no way to prevent or delay the onset or to cure the disease. Most diseases, including diabetes, stem from abnormalities in the functioning of proteins, and some studies have reported the expression of protein variation to be involved in the development of DM1. Thus, the aim of this study was to investigate the differential expression of serum proteins in patients with DM1. MATERIALS AND METHODS: Serum of patients with DM1 (n=30) and healthy controls (n=30) was collected. A proteomic approach was used with depletion of albumin and immunoglobulin G chromatography on serum samples followed by data-independent, label-free mass spectrometric analysis. RESULTS: A total of eight serum proteins were identified as being differentially expressed and involved in the immune system, lipid metabolism, and pathways of coagulation. DM1 was associated with the upregulation of six proteins: alpha-2-macroglobulin, apolipoprotein A-II, ß2 glycoprotein I, Ig alpha-2 chain C region, alpha-1-microglobulin, and prothrombin. A total of two proteins were downregulated, including pregnancy zone protein and complement C4. CONCLUSION: To the best of our knowledge, these findings show differential expression of proteins revealing new proteins that may be involved in the development and progression of diabetes.

Proteome of the rete testis fluid from tropically-adapted Morada Nova rams.

The rete testis has a close relationship with sperm development and may have other functions besides serving as an intercalated channel. The aim of this study was to identify and characterize the proteins of rete testis fluid (RTF) from tropically-adapted Morada Nova rams. Testicles obtained from six Morada Nova rams were dissected and the head of the epididymis was separated to access the efferent ducts. Rete testis fluid was obtained by gentle massage of the testis. The fluid was centrifuged to remove cell debris and sperm. RTF samples (containing 400µg protein) were separated by 2-D SDS-PAGE and gels, analyzed using PDQuest software (Bio Rad, USA). Proteins were identified using tandem mass spectrometry. Gene ontology and protein network were analyzed using the software tool for searching annotations of proteins (STRAP) and STRING database. Gels had, on average, 227±13.5 spots and 51% of the proteins were found above 40kDa, corresponding to 65% of the intensity of all spots detected. Based on gene ontology analysis, the most common biological processes associated with RTF proteins were regulation (24.3%) and cellular process (23.3%). Binding (27.3%) and catalytic activity (19.3%) corresponded to the most frequent molecular functions. Albumin, clusterin, serotransferrin, immunoglobulin gamma-1 chain and alpha-2-HS-glycoprotein were the most abundant proteins in the ram rete testis fluid. In conclusion, proteins identified in the ram rete testis fluid are linked to several physiological processes associated with sperm protection and spermatogenesis.