In Silico Analysis of Protein-Protein Interactions of Putative Endoplasmic Reticulum Metallopeptidase 1 in Schizosaccharomyces pombe.

Ermp1 is a putative metalloprotease from Schizosaccharomyces pombe and a member of the Fxna peptidases. Although their function is unknown, orthologous proteins from rats and humans have been associated with the maturation of ovarian follicles and increased ER stress. This study focuses on proposing the first prediction of PPI by comparison of the interologues between humans and yeasts, as well as the molecular docking and dynamics of the M28 domain of Ermp1 with possible target proteins. As results, 45 proteins are proposed that could interact with the metalloprotease. Most of these proteins are related to the transport of Ca2+ and the metabolism of amino acids and proteins. Docking and molecular dynamics suggest that the M28 domain of Ermp1 could hydrolyze leucine and methionine residues of Amk2, Ypt5 and Pex12. These results could support future experimental investigations of other Fxna peptidases, such as human ERMP1.

Comparative Review of the State of the Art in Research on the Porcine Epidemic Diarrhea Virus and SARS-CoV-2, Scope of Knowledge between Coronaviruses.

This review presents comparative information corresponding to the progress in knowledge of some aspects of infection by the porcine epidemic diarrhea virus (PEDV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronaviruses. PEDV is an alphacoronavirus of great economic importance due to the million-dollar losses it generates in the pig industry. PEDV has many similarities to the SARS-CoV-2 betacoronavirus that causes COVID-19 disease. This review presents possible scenarios for SARS-CoV-2 based on the collected literature on PEDV and the tools or strategies currently developed for SARS-CoV-2 that would be useful in PEDV research. The speed of the study of SARS-CoV-2 and the generation of strategies to control the pandemic was possible due to the knowledge derived from infections caused by other human coronaviruses such as severe acute respiratory syndrome (SARS) and middle east respiratory syndrome (MERS). Therefore, from the information obtained from several coronaviruses, the current and future behavior of SARS-CoV-2 could be inferred and, with the large amount of information on the virus that causes COVID-19, the study of PEDV could be improved and probably that of new emerging and re-emerging coronaviruses.

ERK activation modulates invasiveness and Reactive Oxygen Species (ROS) production in triple negative breast cancer cell lines.

Triple negative breast cancer (TNBC) is the breast cancer subtype with the worst prognosis and still lacks a targeted therapy. In this study, we found increased ERK phosphorylation in TNBC cell lines and an important role for ERK in sustaining the migration of TNBC cells. Although ROS have been suggested to have an important role in sustaining MAPK signaling, antioxidant treatment increased ERK phosphorylation, probably suggesting increased invasive potential. Interestingly, treatment with PD0325901 (PD), a MEK inhibitor, decreased ROS levels in TNBC cells and decreased mitochondrial fragmentation in the MDAMB231 cell line. Our data supports an important role for MEK/ERK in TNBC, sustaining cellular migration, regulating mitochondrial dynamics and ROS production in this breast cancer subtype.

Immunoinformatics approach for predicting epitopes in HN and F proteins of Porcine rubulavirus.

Porcine rubulavirus (PRV), which belongs to the family Paramyxoviridae, causes blue eye disease in pigs, characterized by encephalitis and reproductive failure in newborn and adult pigs, respectively. There is no effective treatment against PRV and no information on the effectiveness of the available vaccines. Continuous outbreaks have occurred in Mexico since the early 1980s, which have caused serious economic losses to pig producers. Vaccination can be used to control this disease. Searching for effective antigen candidates against PRV, we first sequenced the PAC1 F protein, then we used various immunoinformatics tools to predict antigenic determinants of B-cells and T-cells against the two glycoproteins of the virus (HN and F proteins). Finally, we used AutoDock Vina to determine the binding energies. We obtained the F gene sequence of a PRV strain collected in the early 1990s in Mexico and compared its amino acid profile with previous and more recent strains, obtaining an identity similarity of 97.78 to 99.26%. For the F proteins, seven linear B-cell epitopes, six conformational B-cell epitopes and twenty-nine T-cell MHC class I epitopes were predicted. For the HN proteins, sixteen linear B-cell epitopes, seven conformational B-cell epitopes and thirty-four T-cell MHC class I epitopes were predicted. The ATRSETDYY and AAYTTTTCF epitopes of the HN protein might be important for neutralizing the viral infection. We determined the in silico binding energy between the predicted epitopes on the F and HN proteins and swine MHC-I molecules. The binding energy of these epitopes ranged from -5.8 to -7.8 kcal/mol. The present study aimed to assess the use of HN and F proteins as antigens, either as recombinant proteins or as a series of peptides that could activate different responses of the immune system. This may help identify relevant immunogens, saving time and costs in the development of new vaccines or diagnostic tools.

Effectiveness of PCR primers for the detection of occult hepatitis B virus infection in Mexican patients.

BACKGROUND: Occult hepatitis B infection (OBI) is defined as the presence of hepatitis B virus (HVB) DNA in the liver of HBsAg negative individuals with or without detectable viral DNA in serum. OBI is a diagnostic challenge as it is characterized by a very low viral load, intermittently detectable through time. Individuals with OBI can develop chronic hepatic disease, including liver cirrhosis and hepatocellular carcinoma. The aim of this work was to produce tools to improve OBI detection of the HVB genotypes prevalent in Mexico. METHODS: We designed and tested primers to detect OBI in serum samples by nested and real-time PCR. Conserved sites in the viral genome were determined by alignment of the most frequent HBV genotypes in Mexico (H, G/H, F and D) and primers spanning the entire viral genome were designed for first round and nested PCR. Primers were tested in serum samples of 45 patients not co-infected with hepatitis C virus or with HIV, out of a group of 116 HBsAg (-)/anti-HBc (+) individuals. Primers were also tested in a control group with chronic HBV. Nested PCR products obtained from HBsAg (-)/anti-HBc (+) were sequenced and used to design primers for real-time PCR (SYBR Green). RESULTS: The most effective primer pairs to detect HBV products by nested PCR targeted ORF regions: PreS2/P, S/P, X/PreC, and C; while by real-time PCR they targeted ORF regions PreS2/P, S/P, X, and C. Out of the 45 HBsAg (-)/anti-HBc (+) patients tested, the viral genome was detected in 28 (62.2%) and 34 (75.5%), with nPCR and real-time PCR respectively. CONCLUSION: Primers designed for real-time PCR detected up to 75.5% of suspected OBI Mexican patients, with or without liver disease, which represents an improvement from previous PCR strategies.

Antiviral resistance markers in influenza virus sequences in Mexico, 2000-2017.

BACKGROUND: Influenza causes high rates of morbidity and mortality. Genetic variability of influenza viruses generates resistance to antivirals, which are of two types, since they act on two different viral targets: adamantanes, which block the M2 ion channel, and the neuraminidase (NA) inhibitors. METHODS: In Mexico, the available studies on the antiviral resistance of circulating influenza strains are scarce, so this work undertook an analysis of the Mexican sequences reported in public gene banks to perform a systematic analysis of the antiviral resistance markers on both M2 and NA. In all, 284 M2 sequences and 423 NA sequences were retrieved from three genetic databases (sequences from 2000 to 2017 were considered). RESULTS: The resistance markers to M2 blockers were present in 100% of H1N1 pdm2009, 83.6% of H3N2, and 5.8% of seasonal H1N1 sequences. Two resistance markers conferring resistance to NA inhibitors were present in seasonal H1N1 sequences, H275Y (50.0%) and N70S (33.3%). None of these viruses had both resistance markers, which are associated with oseltamivir resistance. The more frequent resistance marker in H1N1 pdm2009 NA sequences was H275Y, present in 3.6%, while S247N was present in 0.30%. Only one of the resistance-associated markers (Q136K) in NA (1.5%) was present in the analyzed H3N2 sequences, while sequences of influenza B virus did not present resistance markers to NA inhibitors. Some influenza A H1N1 pdm2009 sequences (1.8%) presented resistance markers to both M2 and NA. CONCLUSION: Based on the present analysis, 7.1% of the all serotypes of influenza virus A sequences analyzed in Mexico from 2000 to 2017 have mutations conferring resistance to NA inhibitors. Because of this, and the limited availability of influenza drugs, it is necessary to increase the epidemiological surveillance, including molecular analysis, which will provide data such as the presence of changes associated with antiviral resistance.

Taraxacum officinale and Urtica dioica extracts inhibit dengue virus serotype 2 replication in vitro.

BACKGROUND: Urtica dioica, Taraxacum officinale, Calea integrifolia and Caesalpinia pulcherrima are widely used all over the world for treatment of different illnesses. In Mexico, these plants are traditionally used to alleviate or counteract rheumatism and inflammatory muscle diseases. In the present study we evaluated the activity of aqueous and methanolic extracts of these four plants, on the replication of dengue virus serotype 2 (DENV2). METHODS: Extraction process was carried out in a Soxtherm® system at 60, 85 and 120 °C; a chemical fractionation in silica gel chromatography was performed and compounds present in the active fractions were identified by HPLC-DAD-ESI/MSn. The cytotoxic concentration and the inhibitory effect of extracts or fractions on the DENV2 replication were analyzed in the BHK-21 cell line (plaque forming assay). The half maximal inhibitory concentration (IC50) and the selectivity index (SI) were calculated for the extracts and fractions. RESULTS: The methanolic extracts at 60 °C of T. officinale and U. dioica showed the higher inhibitory effects on DENV2 replication. After the chemical fractionation, the higher activity fraction was found for U. dioica and T. officinale, presenting IC50 values of 165.7 ± 3.85 and 126.1 ± 2.80 µg/ml, respectively; SI values were 5.59 and 6.01 for each fraction. The compounds present in T. officinale, were luteolin and caffeoylquinic acids derivatives and quercertin diclycosides. The compounds in the active fraction of U. dioica, were, chlorogenic acid, quercertin derivatives and flavonol glycosides (quercetin and kaempferol). CONCLUSIONS: Two fractions from U. dioica and T. officinale methanolic extracts with anti-dengue activity were found. The compounds present in both fractions were identified, several recognized molecules have demonstrated activity against other viral species. Subsequent biological analysis of the molecules, alone or in combination, contained in the extracts will be carried out to develop therapeutics against DENV2.

Interferon lambda inhibits dengue virus replication in epithelial cells.

BACKGROUND: In viral disease, infection is controlled at the cellular level by type I interferon (IFN-I), but dengue virus (DENV) has the ability to inhibit this response. Type III interferon, also known as lambda IFN (IFN-III or IFN-λ), is a complementary pathway to the antiviral response by IFN-I. This work analyzed the IFN-λ (IFN-III) mediated antiviral response against DENV serotype 2 (DENV-2) infection. METHODS: Dengue fever patients were sampled to determine their IFN-λ levels by ELISA. To study the IFN-λ response during DENV infection we selected the epithelial cell line C33-A, and we demonstrated that it is permissive to DENV-2 infection. The effect of IFN-λ on virus replication was determined in these cells, in parallel to the expression of IFN-stimulated genes (ISGs), and Suppressor of Cytokine Signaling (SOCS), genes measured by RT-qPCR. RESULTS: We found increased (~1.8 times) serological IFN-λ in dengue fever patients compared to healthy blood donors. IFN-λ inhibited DENV-2 replication in a dose-dependent manner in vitro. The reduction of viral titer corresponded with increased ISG mRNA levels (MX1 and OAS1), with the highest inhibition occurring at ISG's peak expression. Presence of IFN-negative regulators, SOCS1 and SOCS3, during DENV-2 infection was associated with reduced IFN-λ1 expression. CONCLUSIONS: Evidence described here suggests that IFN-λ is a good candidate inhibitor of viral replication in dengue infection. Mechanisms for the cellular and organismal interplay between DENV and IFN- λ need to be further studied as they could provide insights into strategies to treat this disease. Furthermore, we report a novel epithelial model to study dengue infection in vitro.

Modulation of apoptosis by V protein mumps virus.

BACKGROUND: The Urabe AM9 vaccine strain of mumps virus contains two variants of V protein: VWT (of HN-A1081 viral population) and VGly (of HN-G1081). The V protein is a promoting factor of viral replication by blocking the IFN antiviral pathway. FINDINGS: We studied the relationship between V protein variants and IFN-α2b-induced apoptosis. V proteins decrease activation of the extrinsic IFN-α2b-induced apoptotic pathway monitored by the caspase 8 activity, being the effect greater with the VWT protein. Both V proteins decrease the activity of caspase 9 of the intrinsic apoptotic pathway. In a system without IFN, the VWT and VGly proteins expression promotes activation of caspases 3 and 7. However, when the cellular system was stimulated with IFN-α, this activity decreased partially. TUNEL assay shows that for treatment with IFN-α and ibuprofen of cervical adenocarcinoma cells there is nuclear DNA fragmentation but the V protein expression reduces this process. CONCLUSIONS: The reduction in the levels of caspases and DNA fragmentation, suggesting that V protein, particularly VWT protein of Urabe AM9 vaccine strain, modulates apoptosis. In addition, the VWT protein shows a protective role for cell proliferation in the presence of antiproliferative signals.

Interaction of mumps virus V protein variants with STAT1-STAT2 heterodimer: experimental and theoretical studies.

BACKGROUND: Mumps virus V protein has the ability to inhibit the interferon-mediated antiviral response by inducing degradation of STAT proteins. Two virus variants purified from Urabe AM9 mumps virus vaccine differ in their replication and transcription efficiency in cells primed with interferon. Virus susceptibility to IFN was associated with insertion of a non-coded glycine at position 156 in the V protein (VGly) of one virus variant, whereas resistance to IFN was associated with preservation of wild-type phenotype in the V protein (VWT) of the other variant. RESULTS: VWT and VGly variants of mumps virus were cloned and sequenced from Urabe AM9 vaccine strain. VGly differs from VWT protein because it possesses an amino acid change Gln103Pro (Pro103) and the Gly156 insertion. The effect of V protein variants on components of the interferon-stimulated gene factor 3 (ISGF3), STAT1 and STAT2 proteins were experimentally tested in cervical carcinoma cell lines. Expression of VWT protein decreased STAT1 phosphorylation, whereas VGly had no inhibitory effect on either STAT1 or STAT2 phosphorylation. For theoretical analysis of the interaction between V proteins and STAT proteins, 3D structural models of VWT and VGly were predicted by comparing with simian virus 5 (SV5) V protein structure in complex with STAT1-STAT2 heterodimer. In silico analysis showed that VWT-STAT1-STAT2 complex occurs through the V protein Trp-motif (W174, W178, W189) and Glu95 residue close to the Arg409 and Lys415 of the nuclear localization signal (NLS) of STAT2, leaving exposed STAT1 Lys residues (K85, K87, K296, K413, K525, K679, K685), which are susceptible to proteasome degradation. In contrast, the interaction between VGly and STAT1-STAT2 heterodimer occurs in a region far from the NLS of STAT2 without blocking of Lys residues in both STAT1 and STAT2. CONCLUSIONS: Our results suggest that VWT protein of Urabe AM9 strain of mumps virus may be more efficient than VGly to inactivate both the IFN signaling pathway and antiviral response due to differences in their finest molecular interaction with STAT proteins.

Biochemical characterization and structural prediction of a novel cytosolic leucyl aminopeptidase of the M17 family from Schizosaccharomyces pombe.

A new leucyl aminopeptidase activity has been identified in the fission yeast Schizosaccharomyces pombe. The enzyme, which has been purified and named leucyl aminopeptidase yspII (LAP yspII), had a molecular mass of 320 and 54 kDa by gel filtration and SDS/PAGE, respectively, suggesting a homohexameric structure. The enzyme cleaved synthetic aminoacyl-4-nitroanilides at an optimum of pH 8.5, and preferred leucine and methionine as N-terminal amino acids. A clear dependence on Mn2+ concentration for activity was found, and an apparent association constant of 0.33 mM was calculated for the metal ion. Bestatin behaved as a competitive inhibitor of LAP yspII (K(i) = 0.14 microM), while chelating agents such as chloroquine, EDTA and 1,10-phenanthroline also reduced enzyme activity. A MALDI-MS analysis, followed by sequencing of two of the resulting peptides, showed that LAP yspII undoubtedly corresponds to the putative aminopeptidase C13A11.05 identified in the S. pombe genome project. The protein exhibited nearly 40% sequence identity to fungal and mammalian aminopeptidases belonging to the M17 family of metallopeptidases. Catalytic residues (Lys292 and Arg366), as well as those involved in coordination with the cocatalytic metal ions (Lys280, Asp285, Asp303, Asp362 and Glu364) and those forming the hydrophobic pocket for substrate binding (Met300, Asn360, Ala363, Thr390, Leu391, Ala483 and Met486), were perfectly conserved among all known aminopeptidases. The S. pombe enzyme is predicted to be formed two clearly distinguished domains with a well conserved C-terminal catalytic domain showing a characteristic topology of eight beta-sheets surrounded by alpha-helical segments in the form of a saddle.