Asociación entre enfermedad de hígado graso no alcohólico y aterosclerosis subclínica

Introduction: non-alcoholic fatty lever disease is linked to atherosclerosis due to the latest´s risk factors. There are also tendencies that affirm that there is a different factor on its pathogeny. The subclinical atherosclerosis is a preventive medicine concept, and looking for it can help finding clues for future irreversible damage.Objective: to determine the association between subclinical atherosclerosis in dyslipidemic patients with non-alcoholic fatty lever disease. Method: a descriptive, cross-sectional study was carried out in a population of 114 dyslipidemic patients with non-alcoholic fatty lever disease in the dyslipoproteinemias consultation at the Surgical Clinical Hospital ¨Hermanos Amejeiras¨ from 2016 to 2019.Results:  mean age was 52.9 ± 12.4 years, with male patients’ predominance. 62 patients showed thickening in the intima-media wider than 1mm in echocardiography, and just 8 patients had atheroma plaque in the carotid artery. The frequency of the altered lipidic profile was due to hypertriglyceridaemia and HDL cholesterol. Conclusions: no link was found between non-alcoholic fatty lever disease and subclinical atherosclerosis. Through the analysis, atherosclerosis was linked to variables as age and family history of ischemic heart disease. There is no association between subclinical atherosclerosis and the degree of progression in the non-alcoholic fatty lever disease given by the APRI index.Introducción: la enfermedad de hígado graso no alcohólico está asociada con la aterosclerosis como consecuencia de los factores de riesgo que la acompañan. Hay tendencias que apoyan que es un factor independiente en su patogenia. La aterosclerosis subclínica es un concepto de medicina preventiva y su búsqueda permite marcar la aparición futura de daños irreversibles. Objetivo: determinar la asociación de aterosclerosis subclínica en pacientes dislipidémicos con enfermedad de hígado graso no alcohólico. Método: se realizó un estudio descriptivo, transversal en 114 pacientes dislipidémicos con enfermedad de hígado graso no alcohólico de la consulta de dislipoproteinemias en el Hospital Clínico Quirúrgico “Hermanos Ameijeiras” en el período comprendido entre los años 2016 al 2019.Resultados: la media de edad fue de 52,9 ± 12,4 años con predominio del sexo masculino. En ecocardiografía, 62 pacientes con enfermedad de hígado graso no alcohólico presentaron aumento del grosor de la íntima media mayor de 1mm y solo 8 pacientes tenían placa de ateroma de la arteria carotídea. La frecuencia del perfil lipídico alterado fue a expensas de hipertrigliceridemia y de la HDL colesterol.Conclusiones: no existió asociación entre la enfermedad de hígado graso no alcohólico con aterosclerosis subclínica; mediante el análisis multivariado se encontró asociación franca de la aterosclerosis subclínica con variables como la edad y el antecedente familiar de cardiopatía isquémica. No hay asociación de aterosclerosis subclínica con el grado de progresión de enfermedad de hígado graso no alcohólico dado por el índice de APRI

Species-Specific Inactivation of Triosephosphate Isomerase from Trypanosoma brucei: Kinetic and Molecular Dynamics Studies

Human African Trypanosomiasis (HAT), a disease that provokes 2184 new cases a year in Sub-Saharan Africa, is caused by Trypanosoma brucei. Current treatments are limited, highly toxic, and parasite strains resistant to them are emerging. Therefore, there is an urgency to find new drugs against HAT. In this context, T. brucei depends on glycolysis as the unique source for ATP supply; therefore, the enzyme triosephosphate isomerase (TIM) is an attractive target for drug design. In the present work, three new benzimidazole derivatives were found as TbTIM inactivators (compounds 1, 2 and 3) with an I50 value of 84, 82 and 73 µM, respectively. Kinetic analyses indicated that the three molecules were selective when tested against human TIM (HsTIM) activity. Additionally, to study their binding mode in TbTIM, we performed a 100 ns molecular dynamics simulation of TbTIM-inactivator complexes. Simulations showed that the binding of compounds disturbs the structure of the protein, affecting the conformations of important domains such as loop 6 and loop 8. In addition, the physicochemical and drug-like parameters showed by the three compounds suggest a good oral absorption. In conclusion, these molecules will serve as a guide to design more potent inactivators that could be used to obtain new drugs against HAT

Biochemical Analysis of Two Single Mutants that Give Rise to a Polymorphic G6PD A-Double Mutant

Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme that plays a crucial role in the regulation of cellular energy and redox balance. Mutations in the gene encoding G6PD cause the most common enzymopathy that drives hereditary nonspherocytic hemolytic anemia. To gain insights into the effects of mutations in G6PD enzyme efficiency, we have investigated the biochemical, kinetic, and structural changes of three clinical G6PD variants, the single mutations G6PD A+ (Asn126AspD) and G6PD Nefza (Leu323Pro), and the double mutant G6PD A− (Asn126Asp + Leu323Pro). The mutants showed lower residual activity (≤50% of WT G6PD) and displayed important kinetic changes. Although all Class III mutants were located in different regions of the three-dimensional structure of the enzyme and were not close to the active site, these mutants had a deleterious effect over catalytic activity and structural stability. The results indicated that the G6PD Nefza mutation was mainly responsible for the functional and structural alterations observed in the double mutant G6PD A−. Moreover, our study suggests that the G6PD Nefza and G6PD A− mutations affect enzyme functions in a similar fashion to those reported for Class I mutations

RNAi-Mediated Specific Gene Silencing as a Tool for the Discovery of New Drug Targets in Giardia lamblia; Evaluation Using the NADH Oxidase Gene

The microaerophilic protozoan Giardia lamblia is the agent causing giardiasis, an intestinal parasitosis of worldwide distribution. Different pharmacotherapies have been employed against giardiasis; however, side effects in the host and reports of drug resistant strains generate the need to develop new strategies that identify novel biological targets for drug design. To support this requirement, we have designed and evaluated a vector containing a cassette for the synthesis of double-stranded RNA (dsRNA), which can silence expression of a target gene through the RNA interference (RNAi) pathway. Small silencing RNAs were detected and quantified in transformants expressing dsRNA by a stem-loop RT-qPCR approach. The results showed that, in transformants expressing dsRNA of 100–200 base pairs, the level of NADHox mRNA was reduced by around 30%, concomitant with a decrease in enzyme activity and a reduction in the number of trophozoites with respect to the wild type strain, indicating that NADHox is indeed an important enzyme for Giardia viability. These results suggest that it is possible to induce the G. lamblia RNAi machinery for attenuating the expression of genes encoding proteins of interest. We propose that our silencing strategy can be used to identify new potential drug targets, knocking down genes encoding different structural proteins and enzymes from a wide variety of metabolic pathways

The nuclear receptor FXR, but not LXR, up-regulates bile acid transporter expression in non-alcoholic fatty liver disease

Background. Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease. Patients with non-alcoholic steatohepatitis (NASH) have increased plasmatic and hepatic concentrations of bile acids (BA), suggesting that they can be associated with the progression of the disease. Hepatic nuclear receptors are known to modulate genes controlling BA metabolism; thus, in this work we aimed to compare the expression of liver nuclear receptors -farnesoid X (FXR), small heterodimer partner (SHP) and liver X alpha (LXRα) receptors- and BA transporters -sodium+/taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP)- in liver biopsy samples of patients with simple steatosis (SS) and NASH.Material and methods. Forty patients with biopsy-proven NALFD were enrolled between 2009 and 2012; liver biopsies were classified as SS (N = 20) or NASH (N = 20) according to the NAFLD activity score. Gene expression of nuclear FXR, LXRa, SHP, NTCP and BSEP was analyzed by real-time reverse transcription polymerase chain reaction and protein level was quantified by western blot.Results. Gene expression of FXR, SHP, NTCP and BSEP was significantly up-regulated in the NASH group in comparison with SS patients (P < 0.05). In contrast, protein level for FXR, SHP and NTCP was decreased in the NASH patients vs. the SS group (P < 0.05). Gene and protein profile of LXRa did not show differences between groups.Conclusions. The results suggest that liver nuclear receptors (FXR and SHP) and BA transporters (NTCP and BSEP) are associated with the progression of NAFLD

Structural and Functional Perturbation of <i>Giardia lamblia</i> Triosephosphate Isomerase by Modification of a Non-Catalytic, Non-Conserved Region

<div><p>Background</p><p>We have previously proposed triosephosphate isomerase of <i>Giardia lamblia</i> (GlTIM) as a target for rational drug design against giardiasis, one of the most common parasitic infections in humans. Since the enzyme exists in the parasite and the host, selective inhibition is a major challenge because essential regions that could be considered molecular targets are highly conserved. Previous biochemical evidence showed that chemical modification of the non-conserved non-catalytic cysteine 222 (C222) inactivates specifically GlTIM. The inactivation correlates with the physicochemical properties of the modifying agent: addition of a non-polar, small chemical group at C222 reduces the enzyme activity by one half, whereas negatively charged, large chemical groups cause full inactivation.</p><p>Results</p><p>In this work we used mutagenesis to extend our understanding of the functional and structural effects triggered by modification of C222. To this end, six GlTIM C222 mutants with side chains having diverse physicochemical characteristics were characterized. We found that the polarity, charge and volume of the side chain in the mutant amino acid differentially alter the activity, the affinity, the stability and the structure of the enzyme. The data show that mutagenesis of C222 mimics the effects of chemical modification. The crystallographic structure of C222D GlTIM shows the disruptive effects of introducing a negative charge at position 222: the mutation perturbs loop 7, a region of the enzyme whose interactions with the catalytic loop 6 are essential for TIM stability, ligand binding and catalysis. The amino acid sequence of TIM in phylogenetic diverse groups indicates that C222 and its surrounding residues are poorly conserved, supporting the proposal that this region is a good target for specific drug design.</p><p>Conclusions</p><p>The results demonstrate that it is possible to inhibit species-specifically a ubiquitous, structurally highly conserved enzyme by modification of a non-conserved, non-catalytic residue through long-range perturbation of essential regions.</p></div

Proteomics: a tool to develop novel diagnostic methods and unravel molecular mechanisms of pediatric diseases

Proteomics is the study of the expression of changes and post-translational modifications (PTM) of proteins along a metabolic condition either normal or pathological. In the field of health, proteomics allows obtaining valuable data for treatment, diagnosis or pathophysiological mechanisms of different illnesses. To illustrate the aforementioned, we describe two projects currently being performed at the Instituto Nacional de Pediatría: The immuno-proteomic study of cow milk allergy and the Proteomic study of childhood cataract. Cow's milk proteins (CMP) are the first antigens to which infants are exposed and generate allergy in some of them. In Mexico, the incidence of CMP allergy has been estimated at 5-7%. Clinical manifestations include both gastrointestinal and extra-gastrointestinal symptoms, making its diagnosis extremely difficult. An inappropriate diagnosis affects the development and growth of children. The goals of the study are to identify the main immune-reactive CMP in Mexican pediatric population and to design more accurate diagnostic tools for this disease. Childhood cataract is a major ocular disease representing one of the main causes of blindness in infants; in developing countries, this disease promotes up to 27% of cases related to visual loss. From this group, it has been estimated that close to 60% of children do not survive beyond two years after vision lost. PTM have been pointed out as the main cause of protein precipitation at the crystalline and, consequently, clouding of this tissue. The study of childhood cataract represents an outstanding opportunity to identify the PTM associated to the cataract-genesis process