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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A comparative proteomics analysis conducted in MT4 cells showed that the expression levels of vimentin were downmodulated after treatment with a fraction of the dialyzable leukocyte extract which shows anti-HIV-1 activity. The relationship of this intermediate filaments (IF) forming protein with the viral replication cycle was then elucidated by establishing a knockdown cell line expressing low levels of vimentin. The early stages of HIV-1 replication were safely evaluated in this cell line, by using a third-generation lentiviral vector which expressed the green fluorescent protein. Using this challenge system as well as the replicative HIV infection assay, it was evidenced for the first time that a reduction in vimentin expression levels drastically affects HIV-1 replication. It was demonstrated by transmission electron microscopy and fluorescence microscopy techniques that a synthetic peptide derived from the keratin-10 protein (denominated CIGB-210) modifies vimentin IFs, also showing a potent inhibitory activity and remarkably low cell toxicity. This makes of CIGB-210 a promising therapeutic candidate against HIV/AIDS. Altogether, our results showed that vimentin is involved in the early stages of viral infection and could be a potential target for the development of novel drugs against HIV/AIDS. This work was granted with the Annual Award of the National Academy of Sciences of Cuba for the year 2016. [ABSTRACT FROM AUTHOR]

In order to increase the possibilities of detecting low abundance proteins in complex mixtures by proteomics techniques, it is essential to use protein or peptide separation methods to reduce the complexity of the biological sample. However, despite the wide use of several electrophoretic techniques, the usefulness of zone electrophoresis for the fractionation of complex peptide mixtures had not been previously investigated. In this work, a new method was established for proteomics studies, called Double Fractionation by PAGE (DF-PAGE). It combines protein fractionation by SDS-PAGE, in-gel enzymatic hydrolysis and peptide separation by SDS-free PAGE; the latter applied for the first time for fractionation and simplification of complex peptide mixtures. Then, it was necessary to design, for the case of DF-PAGE, a new batch buffer system for the selection of acid peptides (pI ≤ 5.5). DF-PAGE allowed the identification of a greater number of proteins than PAGE-SDS and isoelectric focusing in solution. Its application to the characterization of the active principle of the VA-MENGOC-BC® vaccine allowed the identification of 67 proteins previously undetected with traditional techniques. A series of proteins differentially modulated by the antitumor peptide CIGB-552 in the HT-29 cell line of colon adenocarcinoma were also identified by DF-PAGE, which contributed to the characterization of the molecular bases of the action of said peptide. This work granted the Annual Award of the National Academy of Sciences of Cuba for the year 2015. [ABSTRACT FROM AUTHOR]

The NS0 myeloma cell line is often used for the production of monoclonal antibodies (Mabs) and other recombinant proteins. The growth of these mammalian cells in a protein-free media has several advantages including cost, safety, consistency, efficiency and regulatory approval. However, the adaptation of the NS0 myeloma cell line so as to grow in a protein-free medium is poorly understood. In order to better understand this process, we applied proteomic techniques, specifically two-dimensional electrophoresis (2DE) and mass spectrometry (MS), to identify the key pathways involved in this adaptation. The analysis of changes in protein expression between the host myeloma cell line and two recombinant NS0 cell lines expressing different humanized Mabs has primarily revealed changes in proteins associated with carbohydrate metabolism, energy production, protein synthesis and folding, membrane transport and cell proliferation. Other factors that may be involved in the adaptation of NS0 myeloma cells to a protein-free medium are reported. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare the following competing financial interest(s): Y.V.-B., D.S.-M, S.F., L.R., B.S.-R., K.L., D.G.-R., D.G.R., and V.V.B. are co-inventors on provisional SARS-CoV-2 vaccine patents including results covered here., (© 2021 The Authors. Published by American Chemical Society.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Heavy metals are required as nutrients for essential functions in microorganisms. However, higher concentrations of these cations are generally toxic and may produce contrasting effects on living organisms. Enterobacter liquefaciens strain C-1, a bacterium isolated from the Moa mine in Cuba, is able to survive in the presence of high concentrations of heavy metals. The proteomes of Enterobacter liquefaciens strain C-1, grown under aerobic conditions in the presence and absence of Co (II) were compared using two-dimensional gel electrophoresis analysis in the isoelectric point range of 4-7 and the mass range of 15-120 kDa. Significant changes in the expression level (> two-fold) were detected for 13 spots: seven and six were up- and down-regulated, respectively. Because the genome of this bacterium is unknown, identification by peptide mass fingerprinting only succeeded in four cases and most of the cross-species identifications were supported by de novo sequencing of tryptic peptides followed by sequence alignment using the MS BLAST program. Twelve different proteins were identified, ten are involved in cellular antioxidant defence probably induced by the presence of Co (II). This is the first step towards understanding the role of proteins participating in the mechanism of resistance to heavy metals in this bacterium.