Oral administration of a recombinant modified RBD antigen of SARS-CoV-2 as a possible immunostimulant for the care of COVID-19.

BACKGROUND: Developing effective vaccines against SARS-CoV-2 that consider manufacturing limitations, equitable access, and acceptance is necessary for developing platforms to produce antigens that can be efficiently presented for generating neutralizing antibodies and as a model for new vaccines. RESULTS: This work presents the development of an applicable technology through the oral administration of the SARS-CoV-2 RBD antigen fused with a peptide to improve its antigenic presentation. We focused on the development and production of the recombinant receptor binding domain (RBD) produced in E. coli modified with the addition of amino acids extension designed to improve antigen presentation. The production was carried out in shake flask and bioreactor cultures, obtaining around 200 mg/L of the antigen. The peptide-fused RBD and peptide-free RBD proteins were characterized and compared using SDS-PAGE gel, high-performance chromatography, and circular dichroism. The peptide-fused RBD was formulated in an oil-in-water emulsion for oral mice immunization. The peptide-fused RBD, compared to RBD, induced robust IgG production in mice, capable of recognizing the recombinant RBD in Enzyme-linked immunosorbent assays. In addition, the peptide-fused RBD generated neutralizing antibodies in the sera of the dosed mice. The formulation showed no reactive episodes and no changes in temperature or vomiting. CONCLUSIONS: Our study demonstrated the effectiveness of the designed peptide added to the RBD to improve antigen immunostimulation by oral administration.

Deletion of the ATP20 gene in Ustilago maydis produces an unstable dimer of F1FO-ATP synthase associated with a decrease in mitochondrial ATP synthesis and a high H2O2 production.

The F1FO-ATP synthase uses the energy stored in the electrochemical proton gradient to synthesize ATP. This complex is found in the inner mitochondrial membrane as a monomer and dimer. The dimer shows higher ATPase activity than the monomer and is essential for cristae folding. The monomer-monomer interface is constituted by subunits a, i/j, e, g, and k. The role of the subunit g in a strict respiratory organism is unknown. A gene knockout was generated in Ustilago maydis to study the role of subunit g on mitochondrial metabolism and cristae architecture. Deletion of the ATP20 gene, encoding the g subunit, did not affect cell growth or glucose consumption, but biomass production was lower in the mutant strain (gΔ strain). Ultrastructure observations showed that mitochondrial size and cristae shape were similar in wild-type and gΔ strains. The mitochondrial membrane potential in both strains had a similar magnitude, but oxygen consumption was higher in the WT strain. ATP synthesis was 20 % lower in the gΔ strain. Additionally, the mutant strain expressed the alternative oxidase in the early stages of growth (exponential phase), probably as a response to ROS stress. Dimer from mutant strain was unstable to digitonin solubilization, avoiding its isolation and kinetic characterization. The isolated monomeric state activated by n-dodecyl-ß-D-maltopyranoside showed similar kinetic constants to the monomer from the WT strain. A decrease in mitochondrial ATP synthesis and the presence of the AOX during the exponential growth phase suggests that deletion of the g gene induces ROS stress.

Recombinant-phospholipase A2 production and architecture of inclusion bodies are affected by pH in Escherichia coli.

Aggregation of recombinant proteins into inclusion bodies (IBs) is the major drawback of heterologous expression in Escherichia coli. Here, we evaluated the effects of a pH shift after expression induction on recombinant phospholipase A2 production and its aggregation in IBs in E. coli Origami™, as compared to cultures with pH maintained at 7.5 or uncontrolled pH. Cultures shifted from 7.5 to pH 6.5 or 8.5 produced ∼15-25% less biomass as compared with those kept at 7.5 or without pH control. The cultures shifted to pH 8.5 showed a ∼50% higher yield of acetate per biomass, and the rPLA2 yield was improved 2.4-fold. Purified IBs formed at pH 8.5 containing ∼50% of rPLA2, were more susceptible to proteinase-K cleavage and bound less thioflavin-T, indicating lower amyloid content, with the concomitant enrichment of α-helical and random-coil secondary structures, as demonstrated by FTIR. Moreover, only one IB per cell was formed at pH 8.5; instead, more than two were observed under the other culture pH conditions. Nevertheless, under uncontrolled pH conditions, ∼300nm larger IBs were observed. Our work presents evidence of the usefulness of recombinant protein expression cultivated at pH 8.5 allowing the reduction of amyloid content in IBs.

Production of a recombinant phospholipase A2 in Escherichia coli using resonant acoustic mixing that improves oxygen transfer in shake flasks.

BACKGROUND: Shake flasks are widely used during the development of bioprocesses for recombinant proteins. Cultures of recombinant Escherichia coli with orbital mixing (OM) have an oxygen limitation negatively affecting biomass growth and recombinant-protein production. With the aim to improve mixing and aeration in shake flask cultures, we analyzed cultures subjected to OM and the novel resonant acoustic mixing (RAM) by applying acoustic energy to E. coli BL21-Gold (DE3): a producer of recombinant phospholipase A2 (rPLA2) from Micrurus laticollaris snake venom. RESULTS: Comparing OM with RAM (200 rpm vs. 7.5g) at the same initial volumetric oxygen transfer coefficient (kLa ≈ 80 h-1) ~69% less biomass was obtained with OM compared with RAM. We analyzed two more conditions increasing agitation until maximal speed (12.5 and 20g), and ~1.6- and ~1.4-fold greater biomass was obtained as compared with cultures at 7.5g. Moreover, the specific growth rate was statistically similar in all cultures carried out in RAM, but ~1.5-fold higher than that in cultures carried out under OM. Almost half of the glucose was consumed in OM, whereas between 80 and 100% of the glucose was consumed in RAM cultures, doubling biomass per glucose yields. Differential organic acid production was observed, but acetate production was prevented at the maximal RAM (20g). The amount of rPLA2 in both, OM and RAM cultures, represented 38 ± 5% of the insoluble protein. A smaller proportion of α-helices and ß-sheet of purified inclusion bodies (IBs) were appreciated by ATR-FTIR from cultures carried out under OM, than those from RAM. At maximal agitation by RAM, internal E. coli localization patterns of protein aggregation changed, as well as, IBs proteolytic degradation, in conjunction with the formation of small external vesicles, although these changes did not significantly affect the cell survival response. CONCLUSIONS: In moderate-cell-density recombinant E. coli BL21-Gold (DE3) cultures, the agitation increases in RAM (up to the maximum) was not enough to avoid the classical oxygen limitation that happens in OM shake flasks. However, RAM presents a decrease of oxygen limitation, resulting in a favorable effect on biomass growth and volumetric rPLA2 production. While under OM a higher recombinant protein yield was obtained.

Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli.

BACKGROUND: Inclusion bodies (IBs) are aggregated proteins that form clusters when protein is overexpressed in heterologous expression systems. IBs have been considered as non-usable proteins, but recently they are being used as functional materials, catalytic particles, drug delivery agents, immunogenic structures, and as a raw material in recombinant therapeutic protein purification. However, few studies have been made to understand how culture conditions affect the protein aggregation and the physicochemical characteristics that lead them to cluster. The objective of our research was to understand how pH affects the physicochemical properties of IBs formed by the recombinant sphingomyelinase-D of tick expressed in E. coli BL21-Gold (DE3) by evaluating two pH culture strategies. RESULTS: Uncontrolled pH culture conditions favored recombinant sphingomyelinase-D aggregation and IB formation. The IBs of sphingomyelinase-D produced under controlled pH at 7.5 and after 24 h were smaller (<500 nm) than those produced under uncontrolled pH conditions (>500 nm). Furthermore, the composition, conformation and ß-structure formation of the aggregates were different. Under controlled pH conditions in comparison to uncontrolled conditions, the produced IBs presented higher resistance to denaturants and proteinase-K degradation, presented ß-structure, but apparently as time passes the IBs become compacted and less sensitive to amyloid dye binding. CONCLUSIONS: The manipulation of the pH has an impact on IB formation and their physicochemical characteristics. Particularly, uncontrolled pH conditions favored the protein aggregation and sphingomyelinase-D IB formation. The evidence may lead to find methodologies for bioprocesses to obtain biomaterials with particular characteristics, extending the application possibilities of the inclusion bodies.

[Influenza-like illness. Therapeutic experience in family medicine]. / Enfermedad tipo influenza. Experiencia terapéutica en medicina familiar.

BACKGROUND: influenza is a highly contagious respiratory disease. Surveillance in Mexico is based on the detection of Influenza-Like Illness (ILI) and antiviral treatment should begin within 48 hours to avoid the main complication, pneumonia. The aim was to describe the experience of treatment of ILI in a family medicine unit. METHODS: a descriptive study included patients presented to the emergency room with ILI (38°C fever, headache and cough accompanied by other symptoms). We reviewed the reporting formats of Influenza. To follow up, we contacted them by telephone. Data are expressed as mean ± standard deviation. RESULTS: there were 537 patients attended with diagnosis of upper airway infection, 1.3 % met criteria for ILI. 85.7 % were men. The mean age was 18 ± 24.21 years. The patients were seen in a mean time of 19.14 hours after the symptoms have started; 100 % of the patients received treatment with oseltamivir and zanamivir; 14.3 % developed pneumonia. All the patients recovered without concomitant disease or complications. CONCLUSIONS: The use of a protocol in patients with influenza in a family medicine unit led an early diagnosis and treatment that favored the patients' health restoration.

Introducción: la influenza es una enfermedad respiratoria muy contagiosa. Se debe iniciar el tratamiento antiviral en las primeras 48 horas para evitar la neumonía. El objetivo de este informe es describir la experiencia en el tratamiento de la enfermedad tipo influenza en una unidad de medicina familiar. Métodos: estudio descriptivo de pacientes que acudieron a urgencias con enfermedad tipo influenza (fiebre de 38 °C, cefalea y tos, acompañadas de otro síntoma) y que fueron contactados por vía telefónica. Los datos se expresan en media ± desviación estándar. Resultados: de 537 consultas por infecciones respiratorias agudas, 1.3 % reunió los criterios de enfermedad tipo influenza; 85.7 % era del sexo masculino. El promedio de la edad fue de 18 ± 24.21 años. La atención inicial ocurrió a las 19.14 horas después de que se iniciaron los síntomas. Todos los pacientes recibieron oseltamivir y zanamivir; 14.3 % presentó neumonía. El 100 % se restableció. No hubo enfermedades concomitantes. Conclusiones: existe un protocolo para el manejo de la enfermedad tipo influenza que hace posible el diagnóstico y el tratamiento oportunos de los pacientes.

Identification of nucleoli in the early branching protist Giardia duodenalis.

Giardia duodenalis has been described as 'anucleolated'. In this work we analysed the subcellular distribution of several nucleolar markers in Giardia nuclei using silver and immunostaining techniques for electron and confocal laser microscopy as well as expression of epitope-tagged proteins in transgenic trophozoites. We identified anteronuclear fibrogranular structures corresponding to nucleolar organising regions with recruited ribonucleoprotein complexes, rRNA and epitope-tagged fibrillarin and rRNA-pseudouridine synthase (CBF5). Recombinant fibrillarin and CBF5 were targeted to this subcompartment. This study demonstrates the presence of nucleoli in G. duodenalis and provides a model to analyse minimal requirements for nucleolar assembly and maintenance in eukaryotic cells.

Association of the astrovirus structural protein VP90 with membranes plays a role in virus morphogenesis.

VP90, the capsid polyprotein precursor of human astrovirus Yuc8, is assembled into viral particles, and its processing at the carboxy terminus by cellular caspases, to yield VP70, has been correlated with the cell release of the virus. Here, we characterized the effect of the VP90-VP70 processing on the properties of these proteins, as well as on their intracellular distribution. VP90 was found in membrane-enriched fractions (mVP90), as well as in fractions enriched in cytosolic proteins (cVP90), while VP70 was found exclusively in the latter fractions. Upon trypsin activation, infectivity was detected in all VP90-containing fractions, confirming that both mVP90 and cVP90 are able to assemble into particles; however, the two forms of VP90 showed differential sensitivities to trypsin, especially at their carboxy termini, which in the case of mVP90 was shown to remain membrane associated after protease digestion. Structural protein oligomers were detected in purified VP70-containing viruses, as well as in membrane-enriched fractions, but they were less evident in cytosolic fractions. Ultrastructural studies of infected cells revealed different types of viral particles, some of which appeared to be associated with membranes. By immunoelectron microscopy, structural proteins were shown to form virus particles in clusters and to associate with the edges of vesicles induced during infection, which also appear to contain subviral particles inside. Nonstructural proteins and viral RNA colocalized with mVP90, but not with cVP90, suggesting that mVP90 might represent the form of the protein that is initially assembled into particles, at the sites where the virus genome is being replicated.

Infección puerperal

A pesar del conocimiento amplio de la morbilidad materna infecciosa, así como el uso frecuente de antimicrobianos profilácticos para disminuir la incidencia de endometriosis, dicha infección sigue siendo potencialmente complicación grave del parto. La infección pélvica posparto ha recibido muchos nombres a saber, endometritis, metritis, endometriosis y endomioparametris. De ellos, el más utilizado es el de endometriosis, para referirnos a la infección intrauterina posparto. El diagnostico se sospecha cuando una mujer posparto presenta fiebre en ausencia de otros sitios obvios de infección.

Epidemiología del uso de drogas en la población estudantil: tendencias en los últimos 10 años / Epidemiology of drug abuse in the student population: tendencies in the lost lo years

En este trabajo se presentan los datos más relevantes de 4 investigaciones que se llevaron a cabo en esta Institución de 1984 a la fecha, en el campo de la epidemiología de drogas. En la introducción se enfatiza la necesidad de considerar a la epidemiología como una estrategia global que implica realizar sistemáticamente estudios en diversos tipos de población (población general, población estudiantil, población en riesgo y población que asiste a centros de tratamiento), que permitan contextualizar e interpretar las cifras de prevalencia que arrojan los diversos estudios. Se discuten fundamentalmente las cifras de prevalencia obtenidas en 1976 y 1986, en muestras a nivel nacional de la población estudiantil que cursa los niveles de enseñanza media y media superior. Asimismo, se presentan resultados detallados sobre los patrones de consumo de drogas en la Muestra Nacional de Estudiantes de 1986, así como la relación del consumo de drogas con el autoreporte de eventos delictivos. Las cifras actuales de prevalencia indican que el consumo de drogas aumentó como sigue: la mariguana en 1.6%, los inhalantes en 3.5%, las anfetaminas en 1.5%, y la cocaína en 50%; y disminuyó muy ligeramente como sigue: los tranquilizantes y los alucinógenos en 1%, los sedantes en 3% y la heroína en 22%. Estas mismas tendencias a aumentar y a disminuir se observan en las mismas drogas entre la población de estudiantes norteamericanos durante el período de 1976 a 1985, pero con marcadas diferencias en su magnitud, pues tanto los porcentajes actuales de prevalencia como los porcentajes de cambio son mucho más elevados. En las conclusiones se discuten los hallazgos encontrados y se sugieren los temas de investigación para el futuro

Patrones de consumo de alcohol y normas relacionadas con dicho consumo, en una población de Michoacán México / Patterns of alcohol consumption and rules related to this in take in a population of Michoacán, México

En México coexisten altas tasas de abstención y de embriaguez, lo que repercute en tasas de problemas asociados superiores a las observadas en países en los que el alcohol disponible se distribuye homogéneamente en la población. En el presente trabajo se discute este fenómeno analizando prácticas de consumo y normas sociales en torno a esta conducta en un municipio rural del Estado de Michoacán. Resultados de diferentes estudios realizados en nuestro país señalan que las normas sociales influyen en la distribución del consumo en la población, favoreciendo por una parte la embriaguez en la población masculina y la abstención en la población femenina. Por otra parte la influencia internacional orientada hacia un cambio de normas y mayor igualdad entre los sexos puede afectar la costumbre probablemente ocasionando una disminución en las tasas de abstención por parte de las mujeres. Por este motivo se revisará el grado de aceptación tanto de hombres como de mujeres de sus propias costumbres y de las del sexo opuesto en torno a la abstención, al consumo moderado y a la embriaguez. Asimismo, se valorará la opinión de jóvenes en comparación con la de los adultos. La información se obtuvo mediante un muestro probabilístico bi-etápico de la población mayor de 15 años de ambos sexos que habitan viviendas normales en el Municipio de Tarimbaro, Michoacán. Se utilizó un cuestionario para ser administrado a través de una entrevista personal, llenado por encuestadores previamente capacitados en su manejo. Los resultados se compararán con los provenientes de un estudio realizado en población de nivel socioeconómico bajo en el Distrito Federal, obtenidos a través de encuestas de hogares en las que se utilizaron indicadores similares

Estudio epidemiológico sobre el uso de alcohol en población joven de 14 a 18 años / Epidemiological study of alcohol intake in a young population (14 to 18 years old)

En el presente estudio se reporta la prevalencia del consumo de alcohol en una población estudiantil de 14 a 18 años, de educación media y media superior del D.F. y Zona Metropolitana de México, estudiada por medio de una encuesta que contestaron 2912 estudiantes que asistian la las 52 escuelas seleccionadas por un muestreo bietápico, estratificado por conglomerados. Los resultado señalan que el 53% de la población estudiada consume alcohol por lo menos una vez al año y el 2.1% lo hace con patrón de alto-regular. Los hombres reportan consumir alcohol más frecuentemente que las mujeres; el consumo de alcohol es ligeramente más elevado en los estudiantes de 17 a 18 años. El consumo en general es mayor en la zona socioeconómica de ubicación de la escuela, media-alta (56.4%); y beben preferentemente en lugares donde has fiestas (32.3%). Dentro de los 88 estudiantes que reportaron haber tenido problemas legales, el 46.5% reportaron un consumo de alcohol levado en frecuencia y cantidad. La bebida de mayor consumo es la bebida destilada (28%), seguida de los vinos (23.3%) y de la cerveza (14.6%)