Molecular Aspects Concerning the Use of the SARS-CoV-2 Receptor Binding Domain as a Target for Preventive Vaccines.

The development of recombinant COVID-19 vaccines has resulted from scientific progress made at an unprecedented speed during 2020. The recombinant spike glycoprotein monomer, its trimer, and its recombinant receptor-binding domain (RBD) induce a potent anti-RBD neutralizing antibody response in animals. In COVID-19 convalescent sera, there is a good correlation between the antibody response and potent neutralization. In this review, we summarize with a critical view the molecular aspects associated with the interaction of SARS-CoV-2 RBD with its receptor in human cells, the angiotensin-converting enzyme 2 (ACE2), the epitopes involved in the neutralizing activity, and the impact of virus mutations thereof. Recent trends in RBD-based vaccines are analyzed, providing detailed insights into the role of antigen display and multivalence in the immune response of vaccines under development.

Ahora se habla más de lo frágiles que somos, y se hace más presente la pregunta acerca de cuáles son las verdaderas prioridades Reflexiones de la escritora española Belén Gopegui.

Belén Gopegui (Madrid, 1963) Licenciada en Derecho en la Universidad Autónoma de Madrid. Novelista y guionista española. Con su ópera prima La escala de los mapas, (1993) recibió varios premios y su tercera obra, La conquista del aire, fue adaptada al cine. Belén Gopegui fue descrita como la mejor de su generación por el escritor y ensayista español Francisco Umbral. Sus novelas han sido traducidas a varios idiomas. En 2019 se publica su conferencia Ella pisó la Luna, ellas pisaron la luna, un poderoso texto que reivindica a todas las mujeres cuyos logros no han visto la luz.

Bringing Cuban Biotech Research to Bear on COVID-19:All Hands and Minds on Deck.

This MEDICC Review roundtable gathers some of Cuba's top researchers in the fi elds of vaccines and biotechnology, all of whom work in institutions belonging to BioCubaFarma, the umbrella company of Cuban biotech and pharmaceutical R&D, production, distribution and export. Founded in 2012, the company is comprised of 34 enterprises with 61 lines of production and some 20,000 employees. A total of 765 of its products are registered in 53 countries and exported to an-other 50. Its scientists' research has resulted in 2640 patents in Cuba and globally.

Cuba's Pedro Kourí Tropical Medicine Institute:Battling COVID-19 One Study, One Test, One Patient at a Time.

This MEDICC Review roundtable brings you specialists from Havana's Pedro Kourí Tropical Medicine Institute (IPK), who are working directly with testing, research and patient care during the COVID-19 pandemic. Founded in 1937 by its namesake, the Institute has gained considerable worldwide prestige. Today, it is a PAHO-WHO Collaborating Center for the Study of Dengue and Its Vector, and for the Elimination of Tuberculosis. Its main role within Cuba's health system is as the national reference center for prevention, control, management and elimination of infectious diseases, including epidemics. Its 479 workers staff 32 departments, including laboratories, research and teach-ing facilities, a hospital and isolation center. The IPK's hospital treats later-stage AIDS patients, while the Institute is the nation-al reference center for attention to all HIV-positive patients and maintains the national HIV/AIDS registry, as well as registries for other infectious diseases. The institution was responsible for training the Cuban doctors who served in West Africa during the 2014-2016 Ebola outbreaks and for those going abroad to assist in the COVID-19 response today, and its professionals offer an internationally-recognized biennial course on dengue.

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation.

Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution.

Identification of Vimentin as a Potential Therapeutic Target against HIV Infection.

A combination of antiviral drugs known as antiretroviral therapy (ART) has shown effectiveness against the human immunodeficiency virus (HIV). ART has markedly decreased mortality and morbidity among HIV-infected patients, having even reduced HIV transmission. However, an important current disadvantage, resistance development, remains to be solved. Hope is focused on developing drugs against cellular targets. This strategy is expected to prevent the emergence of viral resistance. In this study, using a comparative proteomic approach in MT4 cells treated with an anti-HIV leukocyte extract, we identified vimentin, a molecule forming intermediate filaments in the cell, as a possible target against HIV infection. We demonstrated a strong reduction of an HIV-1 based lentivirus expressing the enhanced green fluorescent protein (eGFP) in vimentin knockdown cells, and a noteworthy decrease of HIV-1 capsid protein antigen (CAp24) in those cells using a multiround infectivity assay. Electron micrographs showed changes in the structure of intermediate filaments when MT4 cells were treated with an anti-HIV leukocyte extract. Changes in the structure of intermediate filaments were also observed in vimentin knockdown MT4 cells. A synthetic peptide derived from a cytoskeleton protein showed potent inhibitory activity on HIV-1 infection, and low cytotoxicity. Our data suggest that vimentin can be a suitable target to inhibit HIV-1.

Periplasmic proteins encoded by VCA0261-0260 and VC2216 genes together with copA and cueR products are required for copper tolerance but not for virulence in Vibrio cholerae.

The bacterial pathogen Vibrio cholerae requires colonizination of the human small intestine to cause cholera. The anaerobic and slightly acidic conditions predominating there enhance toxicity of low copper concentrations and create a selective environment for bacteria with evolved detoxifying mechanisms. We reported previously that the VCA0260, VCA0261 and VC2216 gene products were synthesized only in V. cholerae grown in microaerobiosis or anaerobiosis. Here we show that ORFs VCA0261 and VCA0260 are actually combined into a single gene encoding a 18.7 kDa protein. Bioinformatic analyses linked this protein and the VC2216 gene product to copper tolerance. Following the approach of predict-mutate and test, we describe for the first time, to our knowledge, the copper tolerance systems operating in V. cholerae. Copper susceptibility analyses of mutants in VCA0261-0260, VC2216 or in the putative copper-tolerance-related VC2215 (copA ATPase) and VC0974 (cueR), under aerobic and anaerobic growth, revealed that CopA represents the main tolerance system under both conditions. The VC2216-encoded periplasmic protein contributes to resistance only under anaerobiosis in a CopA-functional background. The locus tag VCA0261-0260 encodes a copper-inducible, CueR-dependent, periplasmic protein, which mediates tolerance in aerobiosis, but under anaerobiosis its role is only evident in CopA knock-out mutants. None of the genes involved in copper homeostasis were required for V. cholerae virulence or colonization in the mouse model. We conclude that copper tolerance in V. cholerae, which lacks orthologues of the periplasmic copper tolerance proteins CueO, CusCFBA and CueP, involves CopA and CueR proteins along with the periplasmic Cot (VCA0261-0260) and CopG (VC2216) V. cholerae homologues.

Peptide fractionation by SDS-free polyacrylamide gel electrophoresis for proteomic analysis via DF-PAGE.

Here we present a procedure for peptide fractionation by SDS-free polyacrylamide gel electrophoresis, based on discontinuous buffer systems. In the absence of SDS, peptide migration depends both on their molecular mass and on their net charge at the electrophoresis pH. By selecting the separation pH, peptide mobility is modulated. In the original discontinuous buffer system (Tris/glycine), peptides that migrate to the anode have pI values below 6.8 and distribute along the lane in a pI decreasing order, while at acidic pH, as that afforded by histidine/MOPS buffer system, peptides with pI below 5.5 are fractionated. Separation at acid pH is particularly useful for recovering phosphopeptides as well as other highly negatively charged peptides, as those containing sialic or sulfate substituents. Both separation conditions in Tris/glycine and in histidine/MOPS are applicable to proteomic studies, by dual-fractionation polyacrylamide gel electrophoresis (DF-PAGE). First, complex protein samples are separated via SDS-PAGE, and after in-gel proteolysis, peptides are loaded on a second SDS-free gel, where they are separated as described here.

Las Buenas Prácticas de Publicación, su evolución y el impacto esperado en salud pública / Good Publication Practice, its evolution and expected impact in Public Health

Los estándares internacionales para la publicación de información médica y biomédica se vuelven más exigentes. El concepto de Buenas Prácticas de Publicación se está ampliando más allá de los aspectos éticos y de estilo, para incluir aspectos relacionados con la transparencia y la confiabilidad de los resultados. Muchos nuevos requerimientos en el campo médico están recogidos en recomendaciones o guías compiladas por el grupo Enhancing the Quality and Transparency of Health Research, mientras que las guías sobre aspectos experimentales de publicaciones biomédicas están recogidas por el grupo denominado Minimum Information for Biological and Biomedical Investigation. Para suministrar la información requerida, los laboratorios de investigación básica y los grupos de ensayos clínicos que trabajan bajo los principios de prácticas de calidad, están en notable ventaja con respecto a aquellos que aún no han adoptado esta disciplina de trabajo. Una investigación puede ser realizada con recursos limitados. Pero, cualquiera que sean los recursos utilizados, la experimentación debe cumplir con criterios de calidad. Al requerir esta información, las nuevas exigencias para la aceptación de artículos están contribuyendo a la elevación de la calidad de la investigación médica y biomédica. En la medida en que los autores y los editores de las revistas médicas nacionales se mantengan informados sobre estas tendencias, podrán concretar acciones que repercutan positivamente en el rigor, la visibilidad y el impacto de los artículos publicados

International requirements for publishing medical and biomedical information are becoming more stringent. The original scope of Good Publication Practice is evolving, including not only ethics and style aspects but also transparency and reliability of scientific information at present. New requirements for medical publications are being structured in recommendations and guidelines compiled by the Enhancing the Quality and Transparency of Health Research group, whereas guidelines for publishing experimental research in biomedical sciences are compiled by the Minimum Information for Biological and Biomedical Investigation organization. For the provision of required information, the basic research laboratories and the clinical research groups working under the principles of Good Practice are significantly better prepared than those which have not yet adopted this work discipline. A research work may be performed with shortage of resources; however, regardless of the amount of resources, the experiment should comply with quality criteria. These new requirements are expected to increase the quality of medical and biomedical research. Authors and editors of national medical journals should be aware of this evolution in publishing standards, so that they could take actions that have a positive effect on the rigor, visibility and impact of the published articles

Las Buenas Prácticas de Publicación, su evolución y el impacto esperado en salud pública / Good Publication Practice, its evolution and expected impact in Public Health

Los estándares internacionales para la publicación de información médica y biomédica se vuelven más exigentes. El concepto de Buenas Prácticas de Publicación se está ampliando más allá de los aspectos éticos y de estilo, para incluir aspectos relacionados con la transparencia y la confiabilidad de los resultados. Muchos nuevos requerimientos en el campo médico están recogidos en recomendaciones o guías compiladas por el grupo Enhancing the Quality and Transparency of Health Research, mientras que las guías sobre aspectos experimentales de publicaciones biomédicas están recogidas por el grupo denominado Minimum Information for Biological and Biomedical Investigation. Para suministrar la información requerida, los laboratorios de investigación básica y los grupos de ensayos clínicos que trabajan bajo los principios de prácticas de calidad, están en notable ventaja con respecto a aquellos que aún no han adoptado esta disciplina de trabajo. Una investigación puede ser realizada con recursos limitados. Pero, cualquiera que sean los recursos utilizados, la experimentación debe cumplir con criterios de calidad. Al requerir esta información, las nuevas exigencias para la aceptación de artículos están contribuyendo a la elevación de la calidad de la investigación médica y biomédica. En la medida en que los autores y los editores de las revistas médicas nacionales se mantengan informados sobre estas tendencias, podrán concretar acciones que repercutan positivamente en el rigor, la visibilidad y el impacto de los artículos publicados(AU)

International requirements for publishing medical and biomedical information are becoming more stringent. The original scope of Good Publication Practice is evolving, including not only ethics and style aspects but also transparency and reliability of scientific information at present. New requirements for medical publications are being structured in recommendations and guidelines compiled by the Enhancing the Quality and Transparency of Health Research group, whereas guidelines for publishing experimental research in biomedical sciences are compiled by the Minimum Information for Biological and Biomedical Investigation organization. For the provision of required information, the basic research laboratories and the clinical research groups working under the principles of Good Practice are significantly better prepared than those which have not yet adopted this work discipline. A research work may be performed with shortage of resources; however, regardless of the amount of resources, the experiment should comply with quality criteria. These new requirements are expected to increase the quality of medical and biomedical research. Authors and editors of national medical journals should be aware of this evolution in publishing standards, so that they could take actions that have a positive effect on the rigor, visibility and impact of the published articles(AU)

Peptide fractionation by acid pH SDS-free electrophoresis.

SDS-free polyacrylamide gel electrophoresis is an effective alternative approach to peptide fractionation. Here we describe a discontinuous buffer system at acid pH that improves the separation of acidic peptides from tryptic digestion. MOPS and chloride act as trailing and leading ions, respectively, in this system, while histidine operates as counterion and buffers all solutions. In these electrophoretic conditions, peptides with pI below 5.5 migrate with low overall electrophoretic mobilities but high differences from one another, which allows for their efficient resolution. In silico analysis of several proteomes shows that the acid pH system allows a peptide simplification of 2.5-fold with respect to the total peptide mixture, and still a proteome coverage of about 95% is achievable. A straightforward method with a protocol including proteomic studies was achieved for SDS-PAGE of proteins, enzyme treatment and further peptide fractionation by SDS-free acid PAGE.

Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins.

The use of pore-forming toxins from sea anemones (actinoporins) in the construction of immunotoxins (ITs) against tumour cells is an alternative for cancer therapy. However, the main disadvantage of actinoporin-based ITs obtained so far has been the poor cellular specificity associated with the toxin's ability to bind and exert its activity in almost any cell membrane. Our final goal is the construction of tumour proteinase-activated ITs using a cysteine mutant at the membrane binding region of sticholysin-I (StI), a cytolysin isolated from the sea anemone Stichodactyla helianthus. The mutant and the ligand moiety would be linked by proteinase-sensitive peptides through the StI cysteine residue blocking the toxin binding region and hence the IT non-specific killing activity. To accomplish this objective the first step was to obtain the mutant StI W111C, and to evaluate the impact of mutating tryptophan 111 by cysteine on the toxin pore-forming capacity. After proteolysis of the cleavage sequence, a short peptide would remain attached to the toxin. The next step was to evaluate whether this mutant is able to form pores even with a residual peptide linked to cysteine 111. In this work we demonstrated that (i) StI W111C shows pore-forming capacity in a nanomolar range, although it is 8-fold less active than the wild-type recombinant StI, corroborating the previously reported importance of residue 111 for the binding of StI to membranes, and (ii) the mutant is able to form pores even with a residual seven-residue peptide linked to cysteine 111. In addition, it was demonstrated that binding of a large molecule to cysteine 111 renders an inactive toxin that is no longer able to bind to the membrane. These results validate the mutant StI W111C for its use in the construction of tumour proteinase-activated ITs.

Proteomic profile regulated by the anticancer peptide CIGB-300 in non-small cell lung cancer (NSCLC) cells.

CIGB-300 is a proapoptotic peptide-based drug that abrogates the CK2-mediated phosphorylation. This peptide has antineoplastic effect on lung cancer cells in vitro and in vivo. To understand the mechanisms involved on such anticancer activity, the NCI-H125 cell line proteomic profile after short-term incubation (45 min) with CIGB-300 was investigated. As determined by 2-DE or 2D-LC-MS/MS, 137 proteins changed their abundances more than 2-fold in response to the CIGB-300 treatment. The expression levels of proteins related to ribosome biogenesis, metastasis, cell survival and proliferation, apoptosis, and drug resistance were significantly modulated by the presence of CIGB-300. The protein translation process was the most affected (23% of the identified proteins). From the proteome analysis of the NCI-H125 cell line, novel potentialities for CIGB-300 as anticancer agent were evidenced.

Anaerobic growth promotes synthesis of colonization factors encoded at the Vibrio pathogenicity island in Vibrio cholerae El Tor.

Pathogenesis of the facultative anaerobe Vibrio cholerae takes place at the gut under low oxygen concentrations. To identify proteins which change their expression level in response to oxygen availability, proteomes of V. cholerae El Tor C7258 grown in aerobiosis, microaerobiosis and anaerobiosis were compared by two-dimensional electrophoresis. Twenty-six differentially expressed proteins were identified which are involved in several processes including iron acquisition, alanine metabolism, purine synthesis, energy metabolism and stress response. Moreover, two proteins implicated in exopolysaccharide synthesis and biofilm formation were produced at higher levels under microaerobiosis and anaerobiosis, which suggests a role of oxygen deprivation in biofilm development in V. cholerae. In addition, six proteins encoded at the Vibrio pathogenicity island attained the highest expression levels under anaerobiosis, and five of them are required for colonization: three correspond to toxin-coregulated pilus biogenesis components, one to soluble colonization factor TcpF and one to accessory colonization factor A. Thus, anaerobiosis promotes synthesis of colonization factors in V. cholerae El Tor, suggesting that it may be a key in vivo signal for early stages of the pathogenic process of V. cholerae.

Selectivity of bacterial proteome fractionation based on differential solubility: a mass spectrometry evaluation.

We investigate the selectivity achieved after differential solubilization of bacterial proteomes following two procedures, both based on successive extraction of proteins in solutions of increasing solubilizing power. Recently, these procedures have gained notable popularity and several commercial kits are now available. A total of 225 proteins in one case and 227 proteins in the other were identified by LC MSMS analysis; 146 of them were identified in both procedures. The proportions of proteins identified as present in only one fraction were 64 and 57%, respectively. The distribution of cytosolic, membrane, and ribosomal proteins among the successive extracts was analyzed in detail. The effect of (1) replacement of low-speed with high-speed centrifugation, (2) omission of detergents in urea solutions, (3) successive washes of pellets, and (4) reproducibility was evaluated. Proteins with positive grand averages of hydropathicity values and membrane proteins were found in all fractions. This study highlights the benefits and limitations of differential solubilization methods, focusing on practical aspects that may strongly influence their selectivity.

Proteomics based on peptide fractionation by SDS-free PAGE.

Here we demonstrate the usefulness of peptide fractionation by SDS-free polyacrylamide gel electrophoresis and its applicability to proteomics studies. In the absence of SDS, the driving force for the electrophoretic migration toward the anode is supplied by negatively charged acidic amino acid residues and other residues as phosphate, sulfate and sialic acid, while the resulting mobility depends on both the charge and the molecular mass of the peptides. A straightforward method was achieved for SDS-PAGE of proteins, enzyme digestion, peptide transfer and fractionation by SDS-free PAGE, which was named dual-fractionation polyacrylamide gel electrophoresis (DF-PAGE). This method increases the number of identified proteins 2.5-fold with respect to the proteins identified after direct analysis, and more than 80% of assigned peptides were found in unique SDS-free gel slices. A vast majority of identified peptides (93%) have p I values below 7.0, and 7% have p I values between 7.0 and 7.35. Peptide digests that were derived from complex protein mixtures were in consequence simplified as peptides that are positively charged are not recovered in the present conditions. The analysis of a membrane protein extract from Neisseria meningitidis by this approach allowed the identification of 97 proteins, including low-abundance components.

Outer membrane vesicles of the VA-MENGOC-BC vaccine against serogroup B of Neisseria meningitidis: Analysis of protein components by two-dimensional gel electrophoresis and mass spectrometry.

Neisseria meningitidis is a Gram-negative bacterium responsible for significant mortality worldwide. While effective polysaccharides-based vaccines exist against serogroups A, C, W135, and Y, no similar vaccine is suitable for children under 4 years against disease caused by serogroup B strains. Therefore, major vaccine efforts against this serogroup are based on outer membrane vesicles (OMVs), containing major outer membrane proteins. The OMV-based vaccine produced by the Finlay Institute in Cuba (VA-MENGOC-BC) contributed to the rapid decline of the epidemic in this Caribbean island. While the content of major proteins in this vaccine has been discussed, no detailed work of an outer membrane proteomic map of this, or any other, commercially available OMV-derived product has been published so far. Since OMVs exhibit a large bias toward a few major proteins and usually contain a high content of lipids, establishing the adequate conditions for high resolution, 2-DE of this kind of preparation was definitely a technical challenge. In this work, 2-DE and MS have been used to generate a proteomic map of this product, detailing the presence of 31 different proteins, and it allows the identification of new putative protective protein components it contains.

Proteomics of xenografted human breast cancer indicates novel targets related to tamoxifen resistance.

Tamoxifen is the most frequently used drug for hormone therapy of breast cancer patients, even though a high percentage of women are (or become) refractory to this treatment. The proteins involved in tamoxifen resistance of breast tumor cells as well as the mechanisms by which they interact, are still unknown. Some years ago, we established the xenograft breast tumor 3366, sensitive to tamoxifen and the 3366/TAM, resistant to tamoxifen, derived after two years of in vivo passages of the parental 3366 under tamoxifen treatment. Here, we compare the protein expression levels of both xenografts. 2-DE of proteins from total cell extracts showed very high reproducibility among tumors from each group (tamoxifen sensitive and tamoxifen resistant). The heuristic clustering analysis of these gels pooled them correctly in both groups. Twelve proteins were found up-regulated in the tamoxifen-resistant line, while nine were down-regulated. The proteins differentially expressed were identified by MS and sequence database analysis. Biological functions of these proteins are related to cell-cell adhesion and interaction, signal transduction, DNA and protein synthesis machinery, mitochondrial respiratory chain, oxidative stress processes and apoptosis. Three of the identified proteins (ALG-2 interacting protein and two GDP-dissociation inhibitors) could be directly involved in the resistance phenomenon.

Negative detection of biomolecules separated in polyacrylamide electrophoresis gels.

Here we describe the protocols for negative or reverse detection of proteins, nucleic acids and lipopolysaccharides separated in polyacrylamide electrophoresis gels. These protocols are based on the selective synthesis and precipitation of a white imidazole-zinc complex in the gel, which is absent from those zones where biomolecules are located. These methods are highly sensitive (1-10 ng of biomolecules per band), very cheap as they use inexpensive, common laboratory reagents (imidazole and a Zn II salt), rapid (less than 20 min after gel washing), robust and simple (two steps). Reverse-stained biomolecules are reversibly fixed in the gel. After brief incubation in a zinc chelating agent, biomolecules can be recovered from the gel with the same efficiency as from unstained gels. In consequence, they are procedures of choice for micropreparative applications. References covering typical applications are included.

An in-gel digestion procedure that facilitates the identification of highly hydrophobic proteins by electrospray ionization-mass spectrometry analysis.

A procedure is described for in-gel tryptic digestion of proteins that allows the direct analysis of eluted peptides in electrospray ionization (ESI) mass spectrometers without the need of a postdigestion desalting step. It is based on the following principles: (a) a thorough desalting of the protein in-gel before digestion that takes advantage of the excellent properties of acrylamide polymers for size exclusion separations, (b) exploiting the activity of trypsin in water, in the absence of inorganic buffers, and (c) a procedure for peptide extraction using solvents of proven efficacy with highly hydrophobic peptides. Quality of spectra and sequence coverage are equivalent to those obtained after digestion in ammonium bicarbonate for hydrophilic proteins detected with Coomassie blue, mass spectrometry-compatible silver or imidazole-zinc but are significantly superior for highly hydrophobic proteins, such as membrane proteins with several transmembrane domains. ATPase subunit 9 (GRAVY 1.446) is a membrane protein channel, lipid-binding protein for which both the conventional in-gel digestion protocol and in solution digestion failed. It was identified with very high sequence coverage. Sample handling after digestion is notably simplified as peptides are directly loaded into the ESI source without postdigestion processing, increasing the chances for the identification of hydrophobic peptides.