RESUMEN
PURPOSE: Bariatric procedures reduce the capacity of the gut and alter the gastrointestinal transit time predisposing to micro-nutritional deficiencies. This study analyzed and compared the micro-nutritional parameters following laparoscopic sleeve gastrectomy (LSG) and Roux-en-Y gastric bypass (RYGB) in the Indian population. MATERIALS AND METHODS: This is a retrospective study of patients who underwent LSG or RYGB for morbid obesity at a tertiary care center between January 2015 and December 2016. The micronutrient parameters, namely, serum ferritin, vitamin B12, ionized calcium, vitamin D3, and parathormone (PTH) in the preoperative settings and subsequently at 1, 2, and 3 years were analyzed. RESULTS: A total of 390 patients were studied, of which 258 (66.15%) underwent LSG while 132 (33.85%) underwent RYGB. Baseline micronutrient parameters were comparable in the two groups. Anemia (58.1% vs. 59.1%), deficiencies of ferritin (31.7% vs. 34.3%), vitamin B12 (18.8% vs. 36.4%), ionized calcium (65.1% vs. 72.7%), vitamin D3 (95.3% vs. 90.9%), and secondary hyperparathyroidism (45.5% vs. 58.1%) were seen following LSG and RYGB at the end of 3 years, respectively. There was no significant difference found between LSG and RYGB in terms of micronutrient deficiencies studied, including rising in PTH at 1, 2, and 3 years. Vitamin D3 levels were significantly lower at 2 and 3 years following RYGB (p = 0.035 and p = 0.032, respectively). CONCLUSION: LSG and RYGB have comparable micronutrient deficiencies in the short- and mid-term except for vitamin D3, which is higher following RYGB. Long-term studies are needed to define optimum micronutrient supplement dosages for the Indian population.
Asunto(s)
Derivación Gástrica , Obesidad Mórbida , Suplementos Dietéticos , Gastrectomía , Humanos , Micronutrientes , Obesidad Mórbida/cirugía , Estudios RetrospectivosRESUMEN
Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe2+ recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Dioxigenasas/genética , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Proto-Oncogénicas/genética , 5-Metilcitosina/metabolismo , Animales , Ácido Ascórbico/farmacología , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Epigénesis Genética/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Medicina Regenerativa , Tretinoina/farmacología , Vitamina A/farmacologíaRESUMEN
The protein Lin28 and microRNA let-7 play critical roles in mammalian development and human disease. Lin28 inhibits let-7 biogenesis through direct interaction with let-7 precursors (pre-let-7). Accumulating evidence in vitro and in vivo suggests this interaction plays a dominant role in embryonic stem cell self-renewal and tumorigenesis. Thus the Lin28-let-7 interaction might be an attractive drug target, if not for the well-known difficulties in targeting protein-RNA interactions with drugs. The identification and development of suitable probe molecules to further elucidate therapeutic potential, as well as mechanistic details of this pathway will be valuable. We report the development and application of a biophysical high-throughput screening assay for the identification of small molecule inhibitors of the Lin28-pre-let-7 interaction. A library of pharmacologically active small molecules was screened and several small molecule inhibitors were identified and biochemically validated. Of these four validated inhibitors, two compounds successfully restored processing of pre-let-7g in the presence of Lin28, validating the concept. Thus, we have identified examples of small molecule inhibitors of the interaction between Lin28 and pre-let-7. This study provides a proof of concept for small molecule inhibitors that antagonise the effects of Lin28 and enhance processing of let-7 miRNA.
Asunto(s)
MicroARNs/metabolismo , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Secuencia de Bases , Evaluación Preclínica de Medicamentos , Polarización de Fluorescencia , MicroARNs/genética , Precursores del ARN/genéticaRESUMEN
RNA-protein interactions are vital throughout the HIV-1 life cycle for the successful production of infectious virus particles. One such essential RNA-protein interaction occurs between the full-length genomic viral RNA and the major structural protein of the virus. The initial interaction is between the Gag polyprotein and the viral RNA packaging signal (psi or Ψ), a highly conserved RNA structural element within the 5'-UTR of the HIV-1 genome, which has gained attention as a potential therapeutic target. Here, we report the application of a target-based assay to identify small molecules, which modulate the interaction between Gag and Ψ. We then demonstrate that one such molecule exhibits potent inhibitory activity in a viral replication assay. The mode of binding of the lead molecules to the RNA target was characterized by ¹H NMR spectroscopy.
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Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , ARN Lider Empalmado/efectos de los fármacos , ARN Viral/antagonistas & inhibidores , Ribonucleoproteínas/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Fármacos Anti-VIH/efectos adversos , Fármacos Anti-VIH/química , Sitios de Unión , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Células HEK293 , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Células HeLa , Humanos , Modelos Moleculares , Terapia Molecular Dirigida , Conformación de Ácido Nucleico/efectos de los fármacos , Desnaturalización de Ácido Nucleico/efectos de los fármacos , Concentración Osmolar , Compuestos de Quinolinio/efectos adversos , Compuestos de Quinolinio/química , Compuestos de Quinolinio/farmacología , ARN Viral/química , ARN Viral/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Bases de Schiff/efectos adversos , Bases de Schiff/química , Bases de Schiff/farmacología , Bibliotecas de Moléculas Pequeñas , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
This work presents a general method for determining single-molecule intramolecular dynamics in biomolecules by using a reporter fluorophore, whose fluorescence is quenched or partially quenched as a result of intramolecular motion, and a remote observer fluorophore. These fluorophores were excited independently with two different lasers, and the ratio of the two fluorophores' fluorescence was calculated. The time-varying ratio was then filtered to reduce contributions from molecules outside the overlapped laser volume and then correlated. The rates of opening and closing of a DNA hairpin were measured by using both fluorescence correlation spectroscopy and this method for comparison. We found at 50 pM, where molecules were studied one by one as they diffused through the probe volume, we obtained accurate opening and closing rates and could also measure dynamic heterogeneity. To demonstrate applicability to a more complex biological molecule we then probed intramolecular motions in the dimer of a human telomerase RNA fragment (hTR(380-444)), in the presence of an excess of monomer. The motion was found to occur on the time scale of 180-750 micros and slowed with increasing magnesium ion concentration. Blocking experiments using complementary oligonucleotides suggested that the motion involves substantial changes in dimer tertiary structure. This method appears to be a general method for selectively studying intramolecular motion in large biomolecules or complexes.
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Ácidos Nucleicos/química , Secuencia de Bases , Color , ADN/química , Dimerización , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Humanos , Técnicas In Vitro , Rayos Láser , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN/química , ARN/genética , Radiometría , Soluciones , Espectrometría de Fluorescencia/métodos , Telomerasa/genética , TermodinámicaRESUMEN
We have investigated the structure and unfolding kinetics of the human telomeric intramolecular G quadruplex by using single-molecule fluorescence resonance energy transfer. An exploration of conformational heterogeneity revealed two stable folded conformations, in both sodium- and potassium-containing buffers, with small differences between their enthalpies and entropies. Both folded conformations can be opened by the addition of a 21-base complementary DNA oligonucleotide. The unfolding of both substates occurs at the same rate, which showed dependence on the monovalent metal cation present. Temperature-dependence studies in 100 mM KCl gave an apparent activation enthalpy and entropy of 6.4 +/- 0.4 kcal.mol-1 and -52.3 +/- 1.4 cal.mol-1.K-1, respectively, indicating that the unfolding is entropically driven and can occur easily. In contrast, in 100 mM NaCl the respective values are 14.9 +/- 0.2 kcal.mol-1 and -23.0 +/- 0.8 cal.mol-1.K-1, suggesting a more significant enthalpic barrier. Molecular modeling suggests that the two species are likely to be the parallel and antiparallel quadruplex structures. The unfolding free energy barrier is estimated to be between 3 and 15 kBT based on Kramers' theory. We conclude that under near-physiological conditions these structures coexist and can interconvert on a minute time scale.