Post-translational modifications within tau paired helical filament nucleating motifs perturb microtubule interactions and oligomer formation.

Post-translationally modified tau is the primary component of tau neurofibrillary tangles, a pathological hallmark of Alzheimer's disease and other tauopathies. Post-translational modifications (PTMs) within the tau microtubule (MT)-binding domain (MBD), which encompasses two hexapeptide motifs that act as critical nucleating regions for tau aggregation, can potentially modulate tau aggregation as well as interactions with MTs and membranes. Here, we characterize the effects of a recently discovered tau PTM, lysine succinylation, on tau-tubulin interactions and compare these to the effects of two previously reported MBD modifications, lysine acetylation and tyrosine phosphorylation. As generation of site-specific PTMs in proteins is challenging, we used short synthetic peptides to quantify the effects on tubulin binding of three site-specific PTMs located within the PHF6∗ (paired helical filament [PHF] residues 275-280) and PHF6 (residues 306-311) hexapeptide motifs: K280 acetylation, Y310 phosphorylation, and K311 succinylation. We compared these effects to those observed for MBD PTM-mimetic point mutations K280Q, Y310E, and K311E. Finally, we evaluated the effects of these PTM-mimetic mutations on MBD membrane binding and membrane-induced fibril and oligomer formation. We found that all three PTMs perturb tau MT binding, with Y310 phosphorylation exerting the strongest effect. PTM-mimetic mutations partially recapitulated the effects of the PTMs on MT binding and also disrupted tau membrane binding and membrane-induced oligomer and fibril formation. These results imply that these PTMs, including the novel and Alzheimer's disease-specific succinylation of tau K311, may influence both the physiological and pathological interactions of tau and thus represent targets for therapeutic intervention.

Involvement of sulfates from cruzipain, a major antigen of Trypanosoma cruzi, in the interaction with immunomodulatory molecule Siglec-E.

In order to investigate the involvement of sulfated groups in the Trypanosoma cruzi host-parasite relationship, we studied the interaction between the major cysteine proteinase of T. cruzi, cruzipain (Cz), a sulfate-containing sialylated molecule and the sialic acid-binding immunoglobulin like lectin-E (Siglec-E). To this aim, ELISA, indirect immunofluorescence assays and flow cytometry, using mouse Siglec-E-Fc fusion molecules and glycoproteins of parasites, were performed. Competition assays verified that the lectins, Maackia amurensis II (Mal II) and Siglec-E-Fc, compete for the same binding sites. Taking into account that Mal II binding remains unaltered by sulfation, we established this lectin as sialylation degree control. Proteins of an enriched microsomal fraction showed the highest binding to Siglec-E as compared with those from the other parasite subcellular fractions. ELISA assays and the affinity purification of Cz by a Siglec-E column confirmed the interaction between both molecules. The significant decrease in binding of Siglec-E-Fc to Cz and to its C-terminal domain (C-T) after desulfation of these molecules suggests that sulfates contribute to the interaction between Siglec-E-Fc and these glycoproteins. Competitive ELISA assays confirmed the involvement of sulfated epitopes in the affinity between Siglec-E and Cz, probably modified by natural protein environment. Interestingly, data from flow cytometry of untreated and chlorate-treated parasites suggested that sulfates are not primary receptors, but enhance the binding of Siglec-E to trypomastigotic forms. Altogether, our findings support the notion that sulfate-containing sialylated glycoproteins interact with Siglec-E, an ortholog protein of human Siglec-9, and might modulate the immune response of the host, favoring parasitemia and persistence of the parasite.

Determination of the second virial coefficient of bovine serum albumin under varying pH and ionic strength by composition-gradient multi-angle static light scattering.

Composition-gradient multi-angle static light scattering (CG-MALS) is an emerging technique for the determination of intermolecular interactions via the second virial coefficient B22. With CG-MALS, detailed studies of the second virial coefficient can be carried out more accurately and effectively than with traditional methods. In addition, automated mixing, delivery and measurement enable high speed, continuous, fluctuation-free sample delivery and accurate results. Using CG-MALS we measure the second virial coefficient of bovine serum albumin (BSA) in aqueous solutions at various values of pH and ionic strength of a univalent salt (NaCl). The systematic variation of the second virial coefficient as a function of pH and NaCl strength reveals the net charge change and the isoelectric point of BSA under different solution conditions. The magnitude of the second virial coefficient decreases to 1.13 x 10(-5) ml*mol/g(2) near the isoelectric point of pH 4.6 and 25 mM NaCl. These results illuminate the role of fundamental long-range electrostatic and van der Waals forces in protein-protein interactions, specifically their dependence on pH and ionic strength.

Revisión de cirugía laparoscópica durante la pandemia por COVID-19 / Laparoscopic surgery during the COVID-19 pandemic a review

The SARS-CoV-2 pandemic has changed the management protocols of the different surgical areas and practices of health institutions around the world. The countries most affected by the disease have reported an alarming impact on the number of infected and deceased by COVID-19 among health workers. Personnel specialized in airway management have a greater risk of contagion when directly exposed to the aerosolization of the virus. This leads us to consider the probability of postponing or restricting procedures due to limited resources and/or due to patient conditions that increase the risk of death, given that the usual surgical techniques generate more aerosols. This narrative review article aims to analyze the risk of contamination of professionals in laparoscopic surgery of patients infected by COVID-19, proposing strategies to minimize the risk of exposure, identifying the necessary protection measures for health professionals, providing recommendations and adaptations of both the surgical technique and the organization of the operating room according to clinical and scientific evidence.

La pandemia por el SARS-CoV-2 ha cambiado los protocolos de manejo de las distintas áreas y prácticas quirúrgicas de las instituciones de salud en el mundo. Los países con mayor afectación de la enfermedad han reportado un impacto alarmante sobre el número de infectados y fallecidos por COVID-19 entre trabajadores de la salud. El personal especializado en el manejo de la vía aérea, posee un mayor riesgo de contagio al exponerse directamente a la aerosolización del virus. Esto conlleva a considerar la probabilidad de posponer o restringir procedimientos por limitación de los recursos y/o por condiciones del paciente que aumenten el riesgo de muerte, dado que las técnicas quirúrgicas habituales generan más aerosoles. Este artículo, de revisión narrativa, pretende analizar el riesgo de contaminación de los profesionales en cirugía laparoscópica de pacientes infectados por COVID-19, proponiendo estrategias para minimizar el riesgo de exposición, identificando las medidas necesarias de protección para los profesionales de la salud, aportando recomendaciones y adaptaciones tanto de la técnica quirúrgica como de la organización del quirófano según la evidencia clínica y científica

Effects of chlorate on the sulfation process of Trypanosoma cruzi glycoconjugates. Implication of parasite sulfates in cellular invasion.

Sulfation, a post-translational modification which plays a key role in various biological processes, is inhibited by competition with chlorate. In Trypanosoma cruzi, the agent of Chagas' disease, sulfated structures have been described as part of glycolipids and we have reported sulfated high-mannose type oligosaccharides in the C-T domain of the cruzipain (Cz) glycoprotein. However, sulfation pathways have not been described yet in this parasite. Herein, we studied the effect of chlorate treatment on T. cruzi with the aim to gain some knowledge about sulfation metabolism and the role of sulfated molecules in this parasite. In chlorate-treated epimastigotes, immunoblotting with anti-sulfates enriched Cz IgGs (AS-enriched IgGs) showed Cz undersulfation. Accordingly, a Cz mobility shift toward higher isoelectric points was observed in 2D-PAGE probed with anti-Cz antibodies. Ultrastructural membrane abnormalities and a significant decrease of dark lipid reservosomes were shown by electron microscopy and a significant decrease in sulfatide levels was confirmed by TLC/UV-MALDI-TOF-MS analysis. Altogether, these results suggest T. cruzi sulfation occurs via PAPS. Sulfated epitopes in trypomastigote and amastigote forms were evidenced using AS-enriched IgGs by immunoblotting. Their presence on trypomastigotes surface was demonstrated by flow cytometry and IF with Cz/dCz specific antibodies. Interestingly, the percentage of infected cardiac HL-1 cells decreased 40% when using chlorate-treated trypomastigotes, suggesting sulfates are involved in the invasion process. The same effect was observed when cells were pre-incubated with dCz, dC-T or an anti-high mannose receptor (HMR) antibody, suggesting Cz sulfates and HMR are also involved in the infection process by T. cruzi.

An anionic synthetic sugar containing 6-SO3 -NAcGlc mimics the sulfated cruzipain epitope that plays a central role in immune recognition.

Cruzipain (Cz), the major cysteine proteinase of Trypanosoma cruzi, is a glycoprotein that contains sulfated high-mannose-type oligosaccharides. We have previously determined that these sulfate groups are targets of specific immune responses. In order to evaluate the structural requirements for antibody recognition of Cz, a systematic structure-activity study of the chemical characteristics needed for antibody binding to the Cz sulfated epitope was performed by immunoassays. With this aim, different synthesized molecules were coupled to the proteins BSA and aprotinin and confronted with (a) mouse sera specific for Cz and its carboxy-terminal (C-T) domain, (b) antibodies raised in rabbits immunized with Cz and its C-terminal domain and (c) IgGs purified from human Chagas disease sera. Our results indicate that a glucosamine containing an esterifying sulfate group in position O-6 and an N-acetyl group was the preferred epitope for the immune recognition of sera specific for Cz and its C-T domain. Although to a minor extent, other anionic compounds bearing sulfate groups in different positions and number as well as different anionic charged groups including carboxylated or phosphorylated monosaccharides, disaccharides and oligosaccharides were recognized. In conclusion, we found that synthetic anionic sugar conjugates containing N-acetyl d-glucosamine-6-sulfate sodium salt (GlcNAc6S) competitively inhibit the binding of affinity purified rabbit anti-C-T IgG to the C-T extension of Cz. Extending these findings to the context of natural infection, immune assays performed with Chagas disease serum confirmed that the structure of synthetic GlcNAc6S mimics the N-glycan-linked sulfated epitope displayed in the C-T domain of Cz.