Diabetes Mellitus Type 1 has a Higher Impact on Corneal Endothelial Cell Density and Pachymetry than Diabetes Mellitus Type 2, Independent of Age: A Meta-Regression Model.

PURPOSE: Patients with diabetes mellitus (DM) often have keratopathy. However, the compromise of the corneal endothelium in type 1 DM (T1DM) and type 2 DM (T2DM) has so far not been well characterized. METHODS: We performed a systematic literature search to find articles on humans combining T1DM and/or T2DM and the corneal endothelium. The period was from inception to June 2020. The meta-regression evaluated the role of each type of DM on corneal endothelial cell density (CED) and pachymetry. The statistical models included age as a modulator to discriminate between the normal changes due to age and the effect of the disease and to determine the impact of the disease duration. RESULTS: The initial search identified 752 records, of which 17 were included in the meta-regression. Patients with T1DM had, on average, 193 cells/mm 2 lesser than control patients ( P < 0.00001). Patients with T2DM had 151 cells/mm 2 less compared with control patients ( P < 0.00001). The loss of corneal endothelial cells was expected because the aging was similar in patients with T1DM and T2DM and their control groups. Patients with T1DM and T2DM showed an increase in pachymetry versus control patients, and in both groups, it was associated with the duration of the disease. CONCLUSIONS: Both types of DM reduced CED and increased pachymetry. These differences were higher in patients with T1DM versus control patients than patients with T2DM versus control patients. In T1DM, CED reduction was not correlated with the time from diagnosis. In both groups, patients had CED reduction due to aging similar to that of their matched control patients.

In silico and in vitro analysis of cation-activated potassium channels in human corneal endothelial cells.

The corneal endothelium is the inner cell monolayer involved in the maintenance of corneal transparence by the generation of homeostatic dehydration. The glycosaminoglycans of the corneal stroma develop a continuous swelling pressure that should be counteracted by the corneal endothelial cells through active transport mechanisms to move the water to the anterior chamber. Protein transporters for sodium (Na+), potassium (K+), chloride (Cl-) and bicarbonate (HCO3-) are involved in this endothelial "pump function", however despite its physiological importance, the efflux mechanism is not completely understood. There is experimental evidence describing transendothelial diffusion of water in the absence of osmotic gradients. Therefore, it is important to get a deeper understanding of alternative models that drive the fluid transport across the endothelium such as the electrochemical gradients. Three transcriptomic datasets of the corneal endothelium were used in this study to analyze the expression of genes that encode proteins that participate in the transport and the reestablishment of the membrane potential across the semipermeable endothelium. Subsequently, the expression of the identified channels was validated in vitro both at mRNA and protein levels. The results of this study provide the first evidence of the expression of KCNN2, KCNN3 and KCNT2 genes in the corneal endothelium. Differences among the level of expression of KCNN2, KCNT2 and KCNN4 genes were found in a differentially expressed gene analysis of the dataset. Taken together these results underscore the potential importance of the ionic channels in the pathophysiology of corneal diseases. Moreover, we elucidate novel mechanisms that might be involved in the pivotal dehydrating function of the endothelium and in others physiologic functions of these cells using in silico pathways analysis.

Hypoxia-inducible factor HIF-1α modulates drugs resistance in colon cancer cells / Factor inducible por hipoxia HIF-1α modula la resistencia a drogas en células de cáncer de colon

Abstract Introduction: Drug resistance mechanisms may be associated with decreased cell death and its induction may depend on the response to oxidative stress caused by hypoxia. The correlation between hypoxia-inducible factor HIF-1α, the number of reactive oxygen species and their effect on cell survival has not yet been evaluated. Objective: The purpose of this study was to evaluate the effect of HIF-1α activity and reactive oxygen species (ROS) accumulation in apoptosis of colon cancer cells. Materials and methods: HT29 colon cancer cells were treated with Cobalt(II) chloride (CoCl2) or doxorubicin and the activity of HIF-1α was determined by ELISA assay. ROS were determined using fluorescence probe carboxy-H2DFFDA. Apoptosis was assessed by caspase-3 activation analysis, and PUMA and BAX mRNA levels by qRT-PCR. The reduction of the antiapoptotic effect due to hypoxia was attenuated by use of the endonuclease APE-1 (E3330) inhibitor. The endonuclease E3330 APE-1 inhibitor allowed evaluating the effect of ROS generated by doxorubicin and CoCl2 on apoptosis. Results: Chemical hypoxia in combination with doxorubicin is an oxidative stressor in HT29 cells and induces a reduction in the apoptotic process in a time-dependent manner. Conclusion: Resistance to hypoxia and doxorubicin-mediated cell death could be controlled by a mechanism related to the activity of HIF-1α and the amount of reactive oxygen species generated.

Resumen Introducción. Los mecanismos de resistencia a drogas podrían asociarse con disminución en la muerte celular y su inducción podría depender de la respuesta al estrés oxidativo que origina la hipoxia. La correlación entre factor inducible por hipoxia HIF-1α, cantidad de especies reactivas de oxígeno y su efecto sobre la supervivencia celular aún no ha sido evaluada. Objetivo. Evaluar el efecto de la inducción de la actividad de HIF-1α y la cantidad de especies reactivas de oxígeno sobre la apoptosis en células de cáncer de colon. Materiales y métodos. Células de cáncer de colon HT29 fueron tratadas con cloruro de cobalto (CoCl2) o doxorrubicina; la actividad de HIF-1α se evaluó por ELISA. Las especies reactivas de oxígeno fueron determinadas con sonda fluorescente carboxi-H2DFFDA. La apoptosis fue evaluada por la actividad de caspasa-3 y los niveles de mRNA de los genes proapoptóticos PUMA y BAX por qRT-PCR. El inhibidor de la endonucleasa APE-1 E3330 permitió evaluar el efecto de las especies reactivas de oxígeno generadas por doxorubicina y CoCl2 sobre la apoptosis. Resultados. La hipoxia química combinada + doxorubicina es estresor oxidativo en células HT29 e induce una reducción en el proceso apoptótico de manera tiempo dependiente. Conclusión. La resistencia a la muerte celular mediada por hipoxia y doxorubicina podría estar controlada por un mecanismo relacionado con la actividad de HIF-1α y la cantidad de especies reactivas de oxígeno generadas.

Differential regulation of AKT, MAPK and GSK3ß during C2-ceramide-induced neuronal death.

Evidence has implicated apoptosis as a mechanism underlying cell demise in diverse neurodegenerative diseases including Parkinson's disease (PD). Endogenous toxins and other stress signals activate the sphingomyelin pathway increasing the levels of ceramide, an important regulator of cell death. In the present paper we have analysed the contribution of PI3K/AKT-GSK3ß and MAPK (ERK and JNK) pathways to cell death in a catecholaminergic cell line following exposure to C(2)-ceramide. We also explored the potential neuroprotective action of insulin-like growth factor-1 (IGF-1) and neurotrophin-3 (NT3). We demonstrated that C(2)-ceramide-induced cell death is associated to an early decrease in phosphorylation (inhibition) of PI3K/AKT and ERK, followed by phosphorylation (activation) of JNK and de-phosphorylation (activation) of glycogen synthase kinase-3 beta (GSK3ß). NT3 and IGF-1 increased survival at early time points, but only IGF-1 is capable to attenuate C(2)-ceramide-mediated neuronal death, and this neuroprotection is associated to strong and permanent activation of AKT and inhibition of GSK3ß. In conclusion, C(2)-ceramide initiates a series of events including an early inactivation of PI3K/AKT and ERK pathways followed by activation of JNK and activation of GSK3ß and neuronal death, changes that are counteracted by IGF-1.

Desarrollo de la cristalografía estructural en el siglo XX. Su impacto en las ciencias biomédicas y las perspectivas en este campo / Structural Crystallography Development in the 20 th Century. Impact in the Biomedical Sciences and Perspectives in this Field

El desarrollo de la cristalografía estructural está estrechamente ligado a los avances en otras áreas de la ciencia y la tecnología: química, física y matemáticas, y a los adelantos en computación y robótica. Aunque el descubrimiento de los rayos X por Wilhelm Honrad Roentgen (1845-1923) tuvo lugar en 1895, el uso de la radiación en la determinación de la estructura de los cristales sólo se logró a partir del descubrimiento de Max von Laue (1876-1960), en 1912, según el cual un cristal expuesto a un haz de rayos X originaba sombras específicas. A partir de ese hecho, la estructura de cristales simples, como el cloruro de sodio y el diamante, fue determinada con el método de difracción de rayos X. Sin embargo, para moléculas complejas como las proteínas, que por sus medidas de peso molecular son catalogadas como macromoléculas, no fue fácil la determinación de su estructura tridimensional en esa época.

The development of structural crystallography is closely linked to advances in other areas of science and technology: chemistry, physics and mathematics, and to advances in computing and robotics. Although the discovery of X-rays by Wilhelm Honrad Roentgen (1845-1923) took place in 1895, the use of radiation in the determination of crystal structure was only achieved after the discovery by Max von Laue (1876-1960) in 1912 that a crystal exposed to an X-ray beam produced specific shadows. From that fact, the structure of single crystals, such as sodium chloride and diamond, was determined with the X-ray diffraction method. However, for complex molecules such as proteins, which are classified as macromolecules because of their molecular weight measurements, it was not easy to determine their three-dimensional structure at that time.

El primer mapa detallado de los genes de un hombre: un nuevo hito / The First Detailed Gene Map of a Man: A New Milestone

Los progresos obtenidos en el estudio sobre los aspectos moleculares de la célula han tenido un gran impacto en la medicina. Desde el descubrimiento del ADN como el material que contiene la información genética hecho por Osval Avery, en 1944, pasando por la descripción de James Dewey Watson y Francis Harry Compton Crick de la estructura del ADN en 1953, el desciframiento del código genético (esfuerzos de tres grupos de investigación: Marshall W. Nirenberg, 1961; Severo Ochoa, 1965, y Har Gobind Khorana, 1969), el desarrollo de métodos de secuenciación de los ácidos nucleicos (Sanger, 1977), la técnica de la amplificación del ADN por reacción en cadena de la polimerasa (PCR, por sus siglas en inglés: polymerase chain reaction) (Mullis, 1985) (8), el inicio del Proyecto Genoma Humano (1989), hasta el desciframiento del genoma humano; la aplicación de la biología molecular y la biotecnología ha permitido varios adelantos

The progress made in the study of the molecular aspects of the cell has had a great impact on medicine. From the discovery of DNA as the material containing genetic information by Osval Avery in 1944, through the description by James Dewey Watson and Francis Harry Compton Crick of the structure of DNA in 1953, the deciphering of the genetic code (efforts of three research groups: Marshall W. Nirenberg, 1961; Severo Ochoa, 1965, and Har Gobind Khorana, 1969), the discovery of the genetic code (efforts of three research groups: Marshall W. Nirenberg, 1961; Severo Ochoa, 1965, and Har Gobind Khorana, 1969), the development of nucleic acid sequencing methods (Sanger, 1977), the technique of DNA amplification by polymerase chain reaction (PCR) (M: polymerase chain reaction) (Mullis, 1985) (8), the beginning of the Human Genome Project (1989), until the deciphering of the human genome; the application of molecular biology and biotechnology has allowed several breakthroughs

Proteolytic hydrolysis and purification of the LRP/alfa-2-macroglobulin receptor domain from alpha-macroglobulins.

A new, easier and efficient purification method, using Sephacryl and DEAE-Sephacel, of the C-terminal fragment of two alpha-macroglobulins, alpha(2)-M and PZP, is presented. Two larger peptides were identified for each protein as the C-terminal fragment, with molecular weights of approximately 30 kDa and the N-terminal sequences were determined to be SSTQDTV for alpha(2)-M and VALHLS for PZP. The smaller peptides with molecular weights of 18 kDa correspond to a shorter C-terminal sequence of these proteins, and they were determined to be EEFPFA for alpha(2)-M and ALKVQTV for PZP, with no interfering sequences detected. The results confirmed the discriminatory capacity of the purification procedure and the purity of the fragments. This new methodology facilitates biological studies of alpha-macroglobulins, and will enable elucidation of the role the C-terminal region may exert to eliminate alpha-macroglobulin-proteinases complexes from the circulation by the LRP/receptor.

Muerte celular: blanco terapéutico en neurodegeneración y sepsis / Apoptosis: Therapeutic target in neurodegeneration and sepsis

La apoptosis celular se considera el principal mecanismo fisiopatológico asociado a la pérdida neuronal en las enfermedades neurodegenerativas. También durante la fase aguda de sepsis en que se presenta disfunción orgánica, se ha encontrado que existe un incremento en la tasa apoptótica del endotelio parenquimal y microvascular. De tal forma que las estrategias para prevenir la apoptosis (anti-apoptóticas) representan una valiosa herramienta para prevenir y/o retardar la aparición de la sintomatología en estos desórdenes, los cuales ocasionan una gran carga en morbi-mortalidad social y económica a nivel mundial. En la presente revisión se busca evidenciar que las estrategias anti-apoptóticas poseen un gran potencial terapéutico. En tal sentido, se revisarán algunas de estas potenciales terapias como los inhibidores de caspasas, la proteína C activada, la familia Bcl-2 y la vía de señalización mediada por PI3K/Akt.

Cellular apoptosis has been considered as the main physiological mechanism underlying neuronal demise associated to neurodegenerative diseases. Apoptosis has also been described in parenquimal and microvascular endothelium in the acute phase of sepsis during multi-organic dysfunction. Therefore, strategies aimed to prevent apoptosis (anti-apoptotic) represent a valuable tool for prevention and/or retardation of the appearance of clinical symptoms in these disorders, which generate a large morbilitymortality, social and economic burden worldwide. The present review is aim to show that antiapoptotic strategies hold a great therapeutic potential. In this sense, we will review some of these potential therapies such as caspase inhibitors, activated protein C, Bcl-2 family and the PI3K/Akt signalling pathway.

Identification and characterisation of a cDNA encoding a 17-kDa isoform of rat myelin basic protein.

The myelin basic proteins (MBPs) are the major proteins of the myelin membrane. Multiple MBP mRNAs and protein isoforms are generated by alternative RNA splicing. Here we describe the isolation and characterisation of a cDNA clone encoding a 17-kDa MBP isoform from the rat (Rattus norvegicus). The isoform is a 158-amino acid protein consisting of exons 1, 3, 4, 6 and 7 of the MBP gene. RT-PCR analysis of brain mRNA showed that transcripts encoding the 17-kDa isoform were expressed at higher levels early in post-natal development, up to 7 days post-partum.

Resistencia bacteriana: un problema actual / Bacterial resistance: a present problem

La resistencia bacteriana a la acción de los antibióticos utilizados en clínica es un problema prioritario de salud pública. El conocimiento de los mecanismos moleculares de resistencia, así como de las técnicas, tanto microbiológicas como moleculares para detectarla permiten un uso racional y adecuado de los antibióticos. En esta revisión se presentan las más recientes técnicas de laboratorio que permiten el estudio de la resistencia antimicrobiana desde la difusión en disco, hasta la reacción en cadena de la polimerasa. Se enfatiza la importancia de la interpretación de estas pruebas con el fin de beneficiar al paciente y prevenir en lo posible la diseminación de cepas resistentes.