Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens.

We have developed a single process for producing two key COVID-19 vaccine antigens: SARS-CoV-2 receptor binding domain (RBD) monomer and dimer. These antigens are featured in various COVID-19 vaccine formats, including SOBERANA 01 and the licensed SOBERANA 02, and SOBERANA Plus. Our approach involves expressing RBD (319-541)-His6 in Chinese hamster ovary (CHO)-K1 cells, generating and characterizing oligoclones, and selecting the best RBD-producing clones. Critical parameters such as copper supplementation in the culture medium and cell viability influenced the yield of RBD dimer. The purification of RBD involved standard immobilized metal ion affinity chromatography (IMAC), ion exchange chromatography, and size exclusion chromatography. Our findings suggest that copper can improve IMAC performance. Efficient RBD production was achieved using small-scale bioreactor cell culture (2 L). The two RBD forms - monomeric and dimeric RBD - were also produced on a large scale (500 L). This study represents the first large-scale application of perfusion culture for the production of RBD antigens. We conducted a thorough analysis of the purified RBD antigens, which encompassed primary structure, protein integrity, N-glycosylation, size, purity, secondary and tertiary structures, isoform composition, hydrophobicity, and long-term stability. Additionally, we investigated RBD-ACE2 interactions, in vitro ACE2 recognition of RBD, and the immunogenicity of RBD antigens in mice. We have determined that both the monomeric and dimeric RBD antigens possess the necessary quality attributes for vaccine production. By enabling the customizable production of both RBD forms, this unified manufacturing process provides the required flexibility to adapt rapidly to the ever-changing demands of emerging SARS-CoV-2 variants and different COVID-19 vaccine platforms.

Conformational characterization of the mammalian-expressed SARS-CoV-2 recombinant receptor binding domain, a COVID-19 vaccine.

The COVID-19 pandemic has caused a large number of diseases worldwide. There are few vaccines to constrain this disease and the value of them is high. In this sense, the antigens of the vaccine platform Soberana, the receptor binding domain from SARS-CoV-2 Spike protein, both the monomeric (mRBD) and dimeric (dRBD) forms, have been developed. This study encompassed several analyses by different techniques like circular dichroism (CD), fluorescence spectroscopy (FS) and Gel Filtration- High Performance Liquid ChLC of mRBD and dRBD. Monomer and dimer exhibited similar far-UV CD spectral characteristics with 54% of ß-sheet content. Similar conformational features according to near-UV CD and FS studies were observed in both RBD. Stress stability studies by far-UV CD, FS, biological activity and GF-HPLC at 37 °C showed that mRBD is very stable. On the other hand, dRBD fluorescent emission showed a shift towards higher wavelengths as the incubation time increases, suggesting exposition of tryptophan residues, unlike what happens with mRBD. Biological activity outcome confirms these results. GF-HPLC profiles showed that in mRBD, the product of molecular stress are dimers and does not increase over time. However, dRBD showed dimer fragmentation as the main degradation species. This study reveals the usefulness of CD techniques for the analysis of degradation of RBD molecules as well as showed the difference in stability of both RBD molecules. Besides, our work provides useful insights into the production of a key protein used in diagnosis and therapeutics to fight COVID-19 pandemia.

Conformational characterization of the mammalian-expressed SARS-CoV-2 recombinant receptor binding domain, a COVID-19 vaccine

The COVID-19 pandemic has caused a large number of diseases worldwide. There are few vaccines to constrain this disease and the value of them is high. In this sense, the antigens of the vaccine platform Soberana, the receptor binding domain from SARS-CoV-2 Spike protein, both the monomeric (mRBD) and dimeric (dRBD) forms, have been developed. This study encompassed several analyses by different techniques like circular dichroism (CD), fluorescence spectroscopy (FS) and Gel Filtration- High Performance Liquid ChLC of mRBD and dRBD. Monomer and dimer exhibited similar far-UV CD spectral characteristics with 54% of ß-sheet content. Similar conformational features according to near-UV CD and FS studies were observed in both RBD. Stress stability studies by far-UV CD, FS, biological activity and GF-HPLC at 37 °C showed that mRBD is very stable. On the other hand, dRBD fluorescent emission showed a shift towards higher wavelengths as the incubation time increases, suggesting exposition of tryptophan residues, unlike what happens with mRBD. Biological activity outcome confirms these results. GF-HPLC profiles showed that in mRBD, the product of molecular stress are dimers and does not increase over time. However, dRBD showed dimer fragmentation as the main degradation species. This study reveals the usefulness of CD techniques for the analysis of degradation of RBD molecules as well as showed the difference in stability of both RBD molecules. Besides, our work provides useful insights into the production of a key protein used in diagnosis and therapeutics to fight COVID-19 pandemia.

Conformational characterization of a novel anti-HER2 candidate antibody.

Regulatory agencies establish that a broad physicochemical and biological characterization is necessary for the evaluation of comparability between a biosimilar candidate product and a reference commercial drug. Between them, conformational characterization of proteins is of vital importance to determine its folding and biological functions. In this work, the conformational features of a novel monoclonal antibody (called 5G4) were evaluated by means of circular dichroism spectroscopy and fluorescence. Secondary structure and thermal stability of mAbs were determined by circular dichroism in the far ultraviolet, while three-dimensional folding of proteins was analyzed by both circular dichroism in the near ultraviolet and intrinsic tryptophan fluorescence. In all experiments, Herceptin (Roche) was used as control. Both antibodies showed a composition of secondary structure predominantly of ß-sheets (55-56%) and thermal stability of ~ 75°C, suggesting structural similarity. The three-dimensional folding of proteins was also similar due to the absorption spectra of the aromatic residues and the emission wavelength maxima by fluorescence were comparable. The values of the fluorescence attenuation constant (Stern-Volmer constant) for increasing concentrations of acrylamide were also similar, suggesting a degree of exposure of tryptophan residues similar, although it was slightly decreased for Herceptin. Our data permit to consider that 5G4 monoclonal antibody showed similar conformational characteristics when compared with Herceptin.

Accuracy of auxiliary density functional theory hybrid calculations for activation and reaction enthalpies of pericyclic reactions.

Auxiliary density functional theory (ADFT) hybrid calculations are based on the variational fitting of the Coulomb and Fock potential and, therefore, are free of four-center electron repulsion integrals. So far, ADFT hybrid calculations have been validated successfully for standard enthalpies of formation. In this work the accuracy of ADFT hybrid calculations for the description of pericyclic reactions was quantitatively validated at the B3LYP/6-31G*/GEN-A2* level of theory. Our comparison with conventional Kohn-Sham density functional theory (DFT) results shows that the DFT and ADFT activation and reaction enthalpies are practically indistinguishable. A systematic study of various functionals (PBE, B3LYP, PBE0, CAMB3LYP, CAMPBE0 and HSE06) and basis sets (6-31G*, DZVP-GGA and aug-cc-pVXZ; X = D, T and Q) revealed that the ADFT HSE06/aug-cc-pVTZ/GEN-A2* level of theory yields best balanced accuracy for the activation and reaction enthalpies of the studied pericyclic reactions. With the successfully validate ADFT composite approach consisting of PBE/DZVP-GGA/GEN-A2* structure and transition state optimizations and single-point HSE06/aug-cc-pVTZ/GEN-A2* energy calculations, an accurate, reliable and efficient computational approach for the study of pericyclic reactions in systems at the nanometer scale is proposed.

Metodología de pre-procesamiento de datos adquiridos por MALDI-MSI en muestras de tejidos / Pre-processing methodology of acquired data by MALDI-MSI in tissue samples

En la última década, la Imagenología por Espectrometría de Masas MALDI (MALDI-MSI) ha demostrado su potencial en el campo de la proteómica. Debido a la alta dimensionalidad de los conjuntos de datos obtenidos en los experimentos de MADI-MSI y las variabilidades inherentes al proceso de adquisición, presentes en los mismos, se hace necesario llevar a cabo una etapa de pre-procesamiento, que reduzca estas distorsiones. La presente investigación propone una metodología de procesamiento de datos de MALDI-MSI sustentada en un conjunto de aplicaciones desarrolladas en MatLab, la Biblioteca Qt4, así como la herramienta de visualización DataCube Explorer. Entre los resultados se pueden destacar la obtención de cambios en las intensidades de los píxeles de las imágenes reconstruidas después de la introducción de ruido, así como el incremento de la Relación Señal-Ruido después de someter los espectros a los métodos de filtrado de Kaiser, Savitzky-Golay y Promedio deslizante, destacándose Kaiser sobre los demás, lo que puede traducirse como una disminución de los niveles de distorsión en los espectros de cada pixel. Se realizó la reconstrucción satisfactoria de la imagen patrón y su visualización con la herramienta DataCube Explorer(AU)

At last decade, MALDI Mass Spectrometry Imaging (MALDI-MSI) has demostrated being a powerful tool in proteomics field. Because of high dimensionality of the acquired datasets in MALDI-MSI experiments and the inherent variabilities to the acquisition process, present in them, it´s necessary carry out a pre-processing stage, which reduces these distortions. This paper proposes a processing methodology of data MALDI-MSI supported in applications developed in MATLAB, Qt4 Library, as well as the visualization tool Datacube Explorer. Among the results obtained, it can be highlighted the changes in the intensities of the pixels of the reconstructed images after introducing noise, as well as the increasing of Signal-Noise Ratio after applying the denoising methods Kaiser, Savitzky-Golay and "sliding average", highlighting Kaiser over the other techniques, which can be interpreted as a decreasing of the distortion levels in each pixel´s spectrum. The reconstruction of example image and its visualization with Datacube Explorer tool were satisfactory(AU)

Osteosarcoma en fibroadenoma de la mama femenina

Se describe la conducta seguida con una paciente que se detecta, en el autoexamen, nódulo de mama y acude a la consulta multidisciplinaria de biopsia aspirativa con aguja fina (BAAF). Con la sospecha diagnóstica, se orienta el estudio inmagenológico y la conducta adecuada. Se realizó el diagnóstico que se avala por inmunohistoquímica de un osteosarcoma de la mama originado del estroma con degeneración hialina de un fibroadenoma de la mama. Se revisa la literatura sobre el tema y se comenta sobre la utilidad de la consulta multidisciplinaria de BAAF.

The conduct followed by a female patient who detects herself a breast nodule during the self-examination and attends the multidisciplinary team’s office of fine needle aspiration biopsy (FNAB) is described. Based on the diagnostic suspicion, an imaging study is carried out and an adequate conduct is recommended. The diagnosis is made by immunohistochemistry of a breast osteosarcoma originated from the stroma with hyaline degeneration of a breast fibroadenoma. The literature on this topic is reviewed and comments are made on the usefulness of the multidisciplinary team’s office of FNAB.

Prevalencia de diabetes mellitus y eficiencia diagnóstica del programa de detección oportuna en una unidad de medicina familiar / Prevalence of diabetes mellitus and idagnostic efficiency in a screening program in a family medicine unit

El Instituto Mexicano del Seguro Social ha llevado a cabo en los últimos 25 años un Programa de Detección Oportuna de Diabetes, ante el incremento en su frecuencia y el hecho de que varios enfermos desconocen estarlo. Para evaluar su eficiencia y la prevalencia de diabetes estimada, se realizó un estudio transversal en una unidad de medicina familiar. La prevalencia global de sospecha fue del 2.0 por ciento y de diabetes del 1.0 por ciento (IC-95 por ciento 0.9-1.1), y ambas se incrementaron con la edad. La prevalencia de diabetes en aquellos sujetos hipertensos, obesos, mayores de 40 años y con antecedentes familiares fue del 7.9 por ciento (IC 95 por ciento 6.3-9.6) mayor que la de 0.3 por ciento (IC 95 por ciento 0.1-0.5) en aquellos con uno de estos factores de riesgo (X² tend 377; p; <0.001). La cobertura del programa en 1992 fue del 12 por ciento de la población blanco. El programa de detección oportuna sería más eficiente si se dirigiera a la población de alto riesgo, y si se vinculara con la atención médica

Displacement of Biomfhalaria glabrata by thiara granifera under natural conditions in the Dominican Republic

After a study of the population dynamics of Biomphalaria glabrata snails in several breeding places in the Dominican Republic, the snail Thiara granifera was introduced in some B. glabrata habitats. T. granifera became established in one point in one habitat in the townof Quisqueya, in the east of the country. Around this point of establishment 6 points were selected in order to observe the population dynamics of both species of snails and the chemical and biological characteristics at each point. Four of these points already harbored B. glabrata. One control point was selected also harboring B. glabrata. After 14 months of observations, the results showed that T. granifera was competing with and displacing B. glabrata. This competition does not seem to be competition for food or vital space. Rather, B. glabrata avoids the presence of T. granifera and moves away to new areas, and this is possibly due to a chemical substance(s) secreted by T. granifera or by physical contact with the large number of individuals of T. granifera

Incidencia y prevalencia de lesiones de rodilla en deportistas de alto rendimiento / Incidence and prevalence of lesions of the knee in high-yield sportsmen

Se hizo un resumen retrospectivo de 60 historias clínicas de pacientes deportistas de alto rendimiento con lesiones de rodillas, tratados en el Hospital Ortopédico Docente "Frank País". Se determinó una mayor incidencia de lesionados en el atletismo. Las lesiones más frecuentes de rodilla halladas en este estudio fueron en primer lugar las de menisco, seguidas de tendinitis de inserción y lesiones ligamentosas. El tratamiento quirúrgico se utilizó en un mayor número de pacientes y se observó con el mejor resultado

Desarticulación interescapulotorácica / Interscapulothoracic disarticulation

Se presentan 26 enfermos con tumores en la extremidad superior, primarios o secundarios, que por su extensión o como consecuencia de su reaparición ante el fracaso de otras terapéuticas se les realizó una desarticulación interescapulotoráxica. Se analizan las causas fundamentales que condujeron a estos pacientes a llegar a nuestro centro en esas condiciones y las ventajas del método quirúrgico que se empleó; se exponen los aspectos de la rehabilitación, se enfatiza la necesidad de establecer programas de divulgación tanto para la población, como para los estudiantes y profesionales de la salud, encaminados a disminuir la morbilidad y la mortalidad en esta enfermedad, en aras de elevar nuestras posibilidades de supervivencia y de realización de tratamientos o intervenciones menos mutilantes

Impacto de la migración sobre potencial de expansión de la esquistosomiasis intestinal en la Española: proyecto cooperativo entre los gobiernos de República Dominicana y Haití / Impact of the migration on the potential expantion of the squistosomiasis in \"La Hispaniola\"

Proyecto para determinar la dispersión general en la Española de Biomphalaria glabrata y B. havanensis, su susceptibilidad natural y de laboratorio a la cepa de Schistosoma mansoni dominicana y la influencia de las migraciones en la transmisión de la bilharziasis desde República Dominicana hasta Haití. Tendrá una duración de dos años a realizar en tres fases. En la primera descripción de las áreas seleccionadas para el estudio; búsqueda sistemática de moluscos Biomphalaria glabrata y B. havanensis e intensidad poblacional. En la segunda fase se procederá a estudiar la estructura y dinámica de la población con enfasis en patrones de migración. La tercera fase se iniciará paralelamente con la segunda y se determinará la prevalencia de S. mansoni en las áreas seleccionadas. Se usará el mismo diseño para datos de Biomphalaria havenenis de Haití, usando como control B. havanensis de República Dominicana