Anti-Inflammatory Responses Produced with Nippostrongylus brasiliensis-Derived Uridine via the Mitochondrial ATP-Sensitive Potassium Channel and Its Anti-Atherosclerosis Effect in an Apolipoprotein E Gene Knockout Mouse Model.

Atherosclerosis (AS) has become the leading cause of cardiovascular disease worldwide. Our previous study had observed that Nippostrongylus brasiliensis (Nb) infection or its derived products could inhibit AS development by inducing an anti-inflammatory response. We performed a metabolic analysis to screen Nb-derived metabolites with anti-inflammation activity and evaluated the AS-prevention effect. We observed that the metabolite uridine had higher expression levels in mice infected with the Nb and ES (excretory-secretory) products and could be selected as a key metabolite. ES and uridine interventions could reduce the pro-inflammatory responses and increase the anti-inflammatory responses in vitro and in vivo. The apolipoprotein E gene knockout (ApoE-/-) mice were fed with a high-fat diet for the AS modeling. Following the in vivo intervention, ES products or uridine significantly reduced serum and liver lipid levels, alleviated the formation of atherosclerosis, and reduced the pro-inflammatory responses in serum or plaques, while the anti-inflammatory responses showed opposite trends. After blocking with 5-HD (5-hydroxydecanoate sodium) in vitro, the mRNA levels of M2 markers were significantly reduced. When blocked with 5-HD in vivo, the degree of atherosclerosis was worsened, the pro-inflammatory responses were increased compared to the uridine group, while the anti-inflammatory responses decreased accordingly. Uridine, a key metabolite from Nippostrongylus brasiliensis, showed anti-inflammatory and anti-atherosclerotic effects in vitro and in vivo, which depend on the activation of the mitochondrial ATP-sensitive potassium channel.

Exploring the physicochemical properties of the integration of Tristearoyl uridine in Langmuir monolayers: An approach to cell membrane modeling for prodrugs.

Understanding the mechanisms by which drugs interact with cell membranes is crucial for unraveling the underlying biochemical and biophysical processes that occur on the surface of these membranes. Our research focused on studying the interaction between an ester-type derivative of tristearoyl uridine and model cell membranes composed of lipid monolayers at the air-water interface. For that, we selected a specific lipid to simulate nontumorigenic cell membranes, namely 1,2-dihexadecanoyl-sn-glycero-3-phospho-l-serine. We noted significant changes in the surface pressure-area isotherms, with a noticeable shift towards larger areas, which was lower than expected for ideal mixtures, indicating monolayer condensation. Furthermore, the viscoelastic properties of the interfacial film demonstrated an increase in both the elastic and viscous parameters for the mixed film. We also observed structural alterations using vibrational spectroscopy, which revealed an increase in the all-trans to gauche conformers ratio. This confirmed the stiffening effect of the prodrug on the lipid monolayer. In summary, this study indicates that this lipophilic prodrug significantly impacts the lipid monolayer's thermodynamic, rheological, electrical, and molecular characteristics. This information is crucial for understanding how the drug interacts with specific sites on the cellular membrane. It also has implications for drug delivery, as the drug's passage into the cytosol may involve traversing the lipid bilayer.

Physicochemical properties calculated using DFT method and changes of 5-methyluridine hemihydrate crystals at high temperatures.

5-methyluridine hemihydrate (5 mU) single crystals were synthesized by the slow solvent evaporation method. The physicochemical properties, such as frontier molecular orbitals, global reactivity indices and vibrational were computationally studied through density functional theory (DFT). In addition, structural, vibrational, and thermal properties were obtained by powder X-ray diffraction (PXRD), Raman spectroscopy, thermogravimetric (TG) analysis and differential scanning calorimetry (DSC). PXRD evaluated the structural behavior of 5 mU crystal in the temperature range of 300-460 K. The high-temperature PXRD results suggested that the crystal undergoes two dehydration processes, being a first occurring from the orthorhombic structure (P21212) to triclinic (P1), in which the water losses occurred around 380 K. A second dehydration triggers the change from the triclinic structure to monoclinic (P21) within the 420-435 K temperature range. Furthermore, after this temperature, the anhydrous 5 mU suffers a melting process near 460 K, which is remarkably characterized as an irreversible process. Raman spectroscopy was carried out to identify the vibrational modes linked to the water molecule and the noticeable changes in these bands due to high-temperature effects around 380 K and 410 K. Indeed, changes on Raman bands, such as intensity inversion, the disappearance of bands associated with the hydrogen bonds formed from the water molecules and uracil group, and the ribose group were observed. Finally, this study provided details on the structural and vibrational changes caused by the dehydration of 5 mU crystals and the importance of hydrogen bonds for understanding the intermolecular interactions of the 5 mU, a methylated nucleoside with important biological functions.

Computational Detection of Plant RNA Editing Events.

Computers are able to systematically exploit RNA-seq data allowing us to efficiently detect RNA editing sites in a genome-wide scale. This chapter introduces a very flexible computational framework for detecting RNA editing sites in plant organelles. This framework comprises three major steps: RNA-seq data processing, RNA read alignment, and RNA editing site detection. Each step is discussed in sufficient detail to be implemented by the reader. As a study case, the framework will be used with publicly available sequencing data to detect C-to-U RNA editing sites in the coding sequences of the mitochondrial genome of Nicotiana tabacum.

Core modified 8-17 DNAzymes with 2'-deoxy-2'-C-methylpyrimidine nucleosides.

The catalytic core of an 8-17 DNAzyme directed against STAT 3 was modified using (2'R) and (2'S) 2'-deoxy-2'-C-methyluridine and cytidine. While 2'-deoxy-2'-C-methyluridine significantly diminished the catalytic activity, 2'-deoxy-2'-C-methylcytidine replacement was better accepted, being the kact of modified DNAzymes at 8- and 11-positions comparable to the non-modified one. When 2'-O-methyl and phosphorothioate nucleotides were tested in the binding arms together with core modified DNAzymes the kcat was affected in a non predictable way, emphasizing the fact that both chemical substitutions should be considered globally. Finally, 2'-deoxy-2'-C-methyl modified DNAzymes stability was assayed finding that the double 2'-C-methyl modification in the catalytic core enhanced 70% the stability against a T47D cell lysate compared to a non-modified control.

CPBMF65, a synthetic human uridine phosphorylase-1 inhibitor, reduces HepG2 cell proliferation through cell cycle arrest and senescence.

Hepatocellular carcinoma (HCC) is the most prevalent type of tumor among primary liver tumors and is the second highest cause of cancer-related deaths worldwide. Current therapies are controversial, and more research is needed to identify effective treatments. A new synthetic compound, potassium 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-olate (CPBMF65), is a potent inhibitor of the human uridine phosphorylase-1 (hUP1) enzyme, which controls the cell concentration of uridine (Urd). Urd is a natural pyrimidine nucleoside involved in cellular processes, such as RNA synthesis. In addition, it is considered a promising biochemical modulator, as it may reduce the toxicity caused by chemotherapeutics without impairing its anti-tumor activity. Thus, the objective of this study is to evaluate the effects of CPBMF65 on the proliferation of the human hepatocellular carcinoma cell line (HepG2). Cell proliferation, cytotoxicity, apoptosis, senescence, autophagy, intracellular Urd levels, cell cycle arrest, and drug resistance were analyzed. Results demonstrate that, after incubation with CPBMF65, HepG2 cell proliferation decreased, mainly through cell cycle arrest and senescence, increasing the levels of intracellular Urd and maintaining cell proliferation reduced during chronic treatment. In conclusion, results show, for the first time, the ability of a hUP1 inhibitor (CPBMF65) to reduce HepG2 cell proliferation through cell cycle arrest and senescence.

New Insights in Purinergic Therapy: Novel Antagonists for Uridine 5'-Triphosphate-Activated P2Y Receptors from Brazilian Flora.

P2Y2 and P2Y4 receptors are physiologically activated by uridine 5'-triphosphate (UTP) and are widely expressed in many cell types in humans. P2Y2 plays an important role in inflammation and proliferation of tumor cells, which could be attenuated with the use of antagonists. However, little is known about the physiological functions related to P2Y4, due to the lack of selective ligands for these receptors. This can be solved through the search for novel compounds with antagonistic activity. The aim of this study was to discover new potential antagonist candidates for P2Y2 and P2Y4 receptors from natural products. We applied a calcium measurement methodology to identify new antagonist candidates for these receptors. First, we established optimal conditions for the calcium assay using J774.G8, a murine macrophage cell line, which expresses functional P2Y2 and P2Y4 receptors and then, we performed the screening of plant extracts at a cutoff concentration of 50 µg/mL. ATP and ionomycin, known intracellular calcium inductors, were used to stimulate cells. The calculated EC50 were 11 µM and 103 nM, respectively. These cells also responded to the UTP stimulation with an EC50 of 1.021 µM. Screening assays were performed and a total of 100 extracts from Brazilian plants were tested. Joannesia princeps Vell. (stem) and Peixotoa A. Juss (flower and leaf) extracts stood out due to their ability to inhibit UTP-induced responses without causing cytotoxicity, and presented an IC50 of 32.32, 14.99, and 12.98 µg/mL, respectively. Collectively, our results point to the discovery of potential antagonist candidates from Brazilian flora for UTP-activated receptors.

Anomalous interaction of tri-acyl ester derivatives of uridine nucleoside with a l-α-dimyristoylphosphatidylcholine biomembrane model: a differential scanning calorimetry study.

OBJECTIVES: Uridine was conjugated with fatty acids to improve the drug lipophilicity and the interaction with phospholipid bilayers. METHODS: The esterification reaction using carbodiimides compounds as coupling agents and a nucleophilic catalyst allowed us to synthesize tri-acyl ester derivatives of uridine with fatty acids. Analysis of molecular interactions between these tri-acyl ester derivatives and l-α-dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV) - as a mammalian cell membrane model - have been performed by differential scanning calorimetry (DSC). KEY FINDINGS: The DSC thermograms suggest that nucleoside and uridine triacetate softly interact with phospholipidic multilamellar vesicles which are predominantly located between the polar phase, whereas the tri-acyl ester derivatives with fatty acids (myristic and stearic acids) present a strongly interaction with the DMPC bilayer due to the nucleoside and aliphatic chains parts which are oriented towards the polar and lipophilic phases of the phospholipidic bilayer, respectively. However, the effects caused by the tri-myristoyl uridine and tri-stearoyl uridine are different. CONCLUSIONS: We show how the structural changes of uridine modulate the calorimetric behaviour of DMPC shedding light on their affinity with the phospholipidic biomembrane model.

Protein Phosphatase Sit4 Affects Lipid Droplet Synthesis and Soraphen A Resistance Independent of Its Role in Regulating Elongator Dependent tRNA Modification.

The protein phosphatase Sit4 has been shown to be required for lipogenesis and resistance against the acetyl-CoA carboxylase inhibitor soraphen A. Since Sit4 is also required for biosynthesis of Elongator dependent tRNA modifications such as 5-methoxycarbonylmethyluridine (mcm5U), we investigated the relevance of tRNA modifications in lipogenesis and soraphen A response. While sit4 and Elongator (elp3) mutants copy defects in mcm5U formation and stress sensitivity, they do not share soraphen A sensitivity and low lipid droplet (LD) phenotypes. In contrast to sit4, we found elp3 mutants to display partial soraphen A resistance and a high LD phenotype. Screening a collection of tRNA modification mutants additionally identified the tRNA pseudo-uridine synthase gene DEG1 to be required for soraphen A sensitivity. Since deg1 and elp3 share high LD and soraphen A resistance phenotypes, these are likely caused by translational defects. In support of this notion, we observe overexpression of tRNAGlnUUG suppresses lipolysis defects of deg1 mutants. Hence, the sit4 mutation results in a composite defect including tRNA modification deficiency and loss of Snf1 kinase dephosphorylation, which induce opposite effects on LD regulation. Importantly, however, the Snf1 kinase regulatory defects of the phosphatase mutant dominate over effects on LD regulation imposed by loss of the tRNA modification alone.

siRNA Modified with 2'-Deoxy-2'-C-methylpyrimidine Nucleosides.

(2'S)-2'-Deoxy-2'-C-methyluridine and (2'R)-2'-deoxy-2'-C-methyluridine were incorporated in the 3'-overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2'S)-2'-deoxy-2'-C-methylcytidine was incorporated in the 6-position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC50 values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3'-overhang region by (2'S)- or (2'R)-2'-deoxy-2'-C-methyluridine, reducing IC50 to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.

Synthesis and interaction of sterol-uridine conjugate with DMPC liposomes studied by differential scanning calorimetry.

Differential scanning calorimetry (DSC) is a thermoanalytical technique which provides information on the interaction between drugs and models of cell membranes. Studies on the calorimetric behavior of hydrated phospholipids within liposomes are employed to shed light on the changes in the physico-chemical properties when interacting with drugs. In this report, new potential anti-cancer drugs such as uridine and uridine derivatives (acetonide and its succinate), 3ß-5α,8α-endoperoxide-cholestan-6-en-3-ol (5,8-epidioxicholesterol) and conjugate (uridine acetonide-epidioxicholesterol succinate) have been synthesized. Steglich esterification method using coupling agents allowed to obtain the uridine acetonide-sterol conjugate. The study on the interaction between the drugs and dimiristoyl-phophatidilcholine (DMPC) liposomes has been conducted by the use of DSC. The analysis of the DSC curves indicated that the uridine and derivatives (acetonide and its succinate) present a very soft interaction with the DMPC liposomes, whereas the 5,8-epidioxicholesterol and the conjugate showed a strong effect on the thermotropic behavior. Our results suggested that the lipophilic character of uridine acetonide-sterol conjugate improves the affinity with the DMPC liposomes.

Novel hybrid DHPM-fatty acids: synthesis and activity against glioma cell growth in vitro.

We described the first synthesis of fatty acid 3,4-dihydropyrimidinones (DHPM-fatty acids) using the Biginelli multicomponent reaction. Antiproliferative activity on two glioma cell lines (C6 rat and U-138-MG human) was also reported. The novel DHPM-fatty acids reduced glioma cell viability relative to temozolomide. Hybrid oxo-monastrol-palmitic acid was the most potent, reducing U-138-MG human cell viability by ca. 50% at 10 µM. In addition, the DHPM-fatty acids showed a large safety range to neural cells, represented by the organotypic hippocampal culture. These results suggest that the increased lipophilicity of DHPM-fatty acids offer a promising approach to overcoming resistance to chemotherapy and may play an important role in the development of new antitumor drugs.

Human uridine phosphorylase-1 inhibitors: a new approach to ameliorate 5-fluorouracil-induced intestinal mucositis.

PURPOSE: 5-fluorouracil (5-FU) has been broadly used to treat solid tumors for more than 50 years. One of the major side effects of fluoropyrimidines therapy is oral and intestinal mucositis. Human uridine phosphorylase (hUP) inhibitors have been suggested as modulators of 5-FU toxicity. Therefore, the present study aimed to test the ability of hUP blockers in preventing mucositis induced by 5-FU. METHODS: We induced intestinal mucositis in Wistar rats with 5-FU, and the intestinal damage was evaluated in presence or absence of two hUP1 inhibitors previously characterized. We examined the loss of weight and diarrhea following the treatment, the villus integrity, uridine levels in plasma, and the neutrophil migration by MPO activity. RESULTS: We found that one of the compounds, 6-hydroxy-4-methyl-1H-pyridin-2-one-3-carbonitrile was efficient to promote intestinal mucosa protection and to inhibit the hUP1 enzyme, increasing the uridine levels in the plasma of animals. However, the loss of body weight, diarrhea intensity or neutrophil migration remained unaffected. CONCLUSION: Our results bring support to the hUP1 inhibitor strategy as a novel possibility of prevention and treatment of mucositis during the 5-FU chemotherapy, based on the approach of uridine accumulation in plasma and tissues.

Toxic effects of penoxsulam herbicide in two fish species reared in southern Brazil.

Toxic effects of penoxsulam herbicide on acetylcholinesterase, thiobarbituric acid-reactive substances and protein carbonyl were studied in silver catfish (Rhamdia sp.) and carp (Cyprinus carpio). Acetylcholinesterase activity was inhibited in both brain and muscle tissue, with the inhibition being greater in carp than in silver catfish. The levels of malondialdehyde (MDA), an indicator of lipid peroxidation, decreased in silver catfish brain tissue, but increased in the carp brain. MDA also increased significantly in muscle tissue of silver catfish. The levels of protein carbonyl, another measure of oxidative damage, increased in the brain of both fish species, and in the muscle of carp. However, silver catfish exhibited a decrease in muscle protein carbonyl. It appears that silver catfish may possess better mechanisms of defense against penoxsulam toxicity than carp.

Tratamento da dor neuropática: Eficácia, segurança e custo-efetividade de pregabalina ® e da associação vitamina B12, uridina e citidina (ETNA) / Treatment of diabetic neuropathic pain: Efficacy, safety and cost-effectiveness of pregabalin and the association of vitamin B12, uridine and cytidine / Tratamiento del dolor neuropático diabético: Eficacia, seguridad y coste-efectividad de pregabalina y de la asociación de la vitamina B12, uridina y citidina

TECNOLOGIAS: Medicamento Pregabalina e medicamento com Vitamina B12 associada à citidina e uridina (ETNA®). INDICAÇÃO: Tratamento da dor neuropática diabética. CARACTERIZAÇÃO DAS TECNOLOGIAS: A pregabalina é um anticonvulsivante indicado, entre outros, para o tratamento da dor neuropática em adultos. Seu mecanismo de ação é a redução do influxo de cálcio para regular a transmissão de mensagens excitatórias entre as células nervosas. O medicamento ETNA® é uma combinação de vitamina B12, e nucleotídeos uridina e citidina e é indicado para o tratamento de doenças dos nervos periféricos. PERGUNTA: As intervenções pregabalina e ETNA® são seguras, eficazes e custo-efetivas no tratamento da dor neuropática diabética, em relação às alternativas gabapentina e amitriptilina já disponíveis no SUS? BUSCA E ANÁLISE DE EVIDÊNCIAS CIENTÍFICAS: Foi realizada uma busca por revisões sistemáticas e estudos econômicos nas bases de dados The Cochrane Library (via Bireme), Medline (via Pubmed), LILACS e Centre for Reviews and Dissemination (CRD). Foi realizada também busca manual na internet e nas referências dos estudos encontrados. Foram selecionadas avaliações de tecnologias em saúde (ATS) em sites de agências internacionais e na Rede Brasileira de Avaliação de Tecnologias em Saúde (REBRATS). Foram selecionados estudos publicados em inglês, português ou espanhol. RESUMO DOS RESULTADOS DOS ESTUDOS SELECIONADOS: Foram incluídos nove estudos: quatro revisões sistemáticas, um ensaio clínico e quatro estudos econômicos. As revisões sistemáticas que consideraram a eficácia e segurança da pregabalina avaliaram como desfechos a redução na intensidade da dor, a taxa de resposta ao tratamento (≥50% na redução da dor), a impressão do paciente em relação à melhora e eventos adversos. Essas revisões apresentaram resultados a favor da pregabalina, porém em relação ao placebo. O ensaio clínico que avaliou a eficácia e segurança do medicamento ETNA® mostrou resultados a favor dessa intervenção, porém comparado somente à vitamina B12. Os resultados de custo-efetividade para a pregabalina consideraram como medida de efetividade o número de dias sem dor ou com dor leve, o número de dias com 30% e 50% de redução de dor e QALY (Anos de Visa Ajustados por Qualidade) ganhos. Apenas um estudo de custo-efetividade não favoreceu a pregabalina. RECOMENDAÇÕES: O tratamento da dor neuropática diabética é contemplado pelo Protocolo Clínico e Diretrizes Terapêuticas da Dor Crônica, o qual recomenda o medicamento amitriptilina em monoterapia (primeira escolha) ou a associação desta com gabapentina, em caso de falha terapêutica da monoterapia. As evidências avaliadas neste PTC permitem recomendar (fracamente) a pregabalina em substituição à gabapentina apenas em casos de falha terapêutica dos esquemas citados anteriormente, uma vez que nenhum estudo incluiu comparações diretas entre pregabalina e amitriptilina ou gabapentina, não comprovando haver superioridade terapêutica da tecnologia em relação aos medicamentos já disponíveis no SUS. Quanto ao ETNA®, a recomendação é contra o seu uso devido à escassez de estudos com evidências de qualidade suficiente para garantir a eficácia e segurança terapêutica dessa intervenção e justificar o gasto.(AU)

TECHNOLOGIES: Pregabalin and vitamin B12 associated with uridine and cytidine (ETNA®) . INDICATION: Treatment of diabetic neuropathic pain. TECHNOLOGIES CHARACTERIZATION: Pregabalin is an anticonvulsant indicated for the treatment of neuropathic pain in adults. Its mechanism of action is the reduction of calcium influx to regulate transmission of excitatory messages between nerve cells. ETNA ® is a combination of vitamin B12, uridine and cytidine nucleotides and it is indicated for the treatment of peripheral nerves diseases. QUESTION: Are ETNA ® and pregabalin safe, effective and cost-effective options in the treatment of diabetic neuropathic pain, regarding the alternatives amitriptyline and gabapentin available at SUS? SEARCH AND ANALYSIS OF SCIENTIFIC EVIDENCE: a search for systematic reviews and economic studies was performed in the databases The Cochrane Library (via Bireme), Medline (via Pubmed), LILACS and Centre for Reviews and Dissemination (CRD). Manual search was also conducted on the internet and in the references of the studies found. Health Technology Assessments (HTA) have been selected in international agencies and in Brazilian Network for Health Technology Assessment (REBRATS). Studies published in English, Portuguese or Spanish were selected. SUMMARY OF RESULTS OF THE SELECTED STUDIES: Nine studies were included, four systematic reviews, one clinical trial and four economic studies. Systematic reviews considering efficacy and safety of pregabalin evaluated the following outcomes: reduction in pain intensity, treatment response rate (≥50% on pain reduction), the Patient Global Impression of Change and adverse events. These reviews showed results in favor of pregabalin compared to placebo. The ETNA® Clinical trial showed results in favor of the intervention but compared to vitamin B12 alone. The results of cost-effectiveness for pregabalin considered as a measure of effectiveness the number of days with no pain or mild pain, the number of days with 30% and 50% reduction in pain and QALY gains. Only one cost-effectiveness study has not favored pregabalin. RECOMMENDATIONS: The treatment for neuropathic pain is contemplated in the therapeutic guideline for chronic disease, which recommends the use of amitriptyline in monotherapy as first choice or its association with gabapentin in cases of therapeutic failure with the monotherapy. The evidences appraised here allow recommending (weakly) the use of pregabalin in replacement of gabapentin only in cases of therapeutic failure of the schemes mentioned above, since no study included direct comparisons between pregabalin and amitriptyline or gabapentin, making it impossible to support the therapeutic superiority of pregabalin compared to other drugs already used in Brazil. The recommendation is against ETNA® use due to lack of studies with sufficient evidence of quality to ensure the efficacy and safety of this intervention and justify the expense.(AU)

TECNOLOGÍAS: Pregabalina y vitamina B12 asociado a uridina y citidina (ETNA®). INDICACIÓN: Tratamiento de la neuropatía diabética. CARACTERIZACIÓN DE LAS TECNOLOGÍAS: La pregabalina es un anticonvulsivo indicado, entre otras cosas, para el tratamiento de la neuropatía diabética en adultos. Su mecanismo de acción es la reducción de la afluencia de calcio para regular la transmisión de mensajes excitatorios entre las células nerviosas. ETNA ® es una combinación de vitamina B12 y de nucleótidos de uridina y citidina y está indicado para el tratamiento de enfermedades de los nervios periféricos. PREGUNTA: ¿Las intervenciones pregabalina y ETNA ® son seguros, eficaces y coste-efectiva en el tratamiento de la neuropatía diabética, en comparación con las alternativas gabapentina y amitriptylina ya disponible en el SUS? BÚSQUEDA Y ANÁLISIS DE LA EVIDENCIA CIENTÍFICA: una búsqueda de revisiones sistemáticas y estudios económicos se realizó en las bases de datos de la Cochrane Library (vía Bireme), MEDLINE (vía PubMed), LILACS y Centre for Reviews and Dissemination (CRD). Búsqueda manual también se llevó a cabo. Comentarios de las tecnologías de la salud han sido seleccionadas en las agencias internacionales y en la Red Brasileña de Evaluación de Tecnologías Sanitarias (REBRATS). Se seleccionaron los estudios publicados en inglés, portugués o español. RESUMEN DE LOS RESULTADOS DE LOS ESTUDIOS SELECCIONADOS: Se incluyeron nueve estudios, cuatro revisiones sistemáticas, un ensayo clínico y cuatro estudios económicos. Revisiones sistemáticas de eficacia e seguridad de pregabalina encontraron como resultados la reducción de la intensidad del dolor, la tasa de respuesta al tratamiento, la impresión del paciente con respecto a la mejora y los eventos adversos. Estos exámenes mostraron resultados a favor de la pregabalina, pero en relación con el placebo. El ensayo clínico que evaluó la eficacia de la droga ETNA ® mostró resultados a favor de la intervención, pero en comparación con la vitamina B12 en monoterapia. Los resultados de coste-efectividad de pregabalina han considerado como una medida de efectividad el número de días sin dolor o dolor leve , el número de días con 30 % y 50% de reducción en el dolor y AVAC (años de vida ajustado por calidad) . Sólo un estudio de costo-efectividad no ha favorecido la pregabalina. RECOMENDACIONES: El tratamiento de la neuropatía diabética se contempla en las guías clínicas y terapéuticas del dolor crónico, que recomienda el uso de amitriptylina en monoterapia (primera elección) o asociado con la gabapentina, en caso de fracaso terapéutico con la monoterapia. Las evidencias evaluadas aquí permiten recomendar (débilmente) el uso de pregabalina en el reemplazo de la gabapentina sólo en casos de fracaso terapéutico de los sistemas antes mencionados, ya que ningún estudio incluyó comparaciones directas entre pregabalina y amitriptilina o gabapentina, no probando superioridad terapéutica de la pregabalina en comparación con otros fármacos ya utilizados en Brasil. En cuanto a ETNA, la recomendación es contra su uso debido a la falta de estudios con pruebas de calidad suficiente para garantizar la eficacia y seguridad de esta intervención y justificar el gasto.(AU)

Quantitative proteomic analysis of the Hfq-regulon in Sinorhizobium meliloti 2011.

Riboregulation stands for RNA-based control of gene expression. In bacteria, small non-coding RNAs (sRNAs) are a major class of riboregulatory elements, most of which act at the post-transcriptional level by base-pairing target mRNA genes. The RNA chaperone Hfq facilitates antisense interactions between target mRNAs and regulatory sRNAs, thus influencing mRNA stability and/or translation rate. In the α-proteobacterium Sinorhizobium meliloti strain 2011, the identification and detection of multiple sRNAs genes and the broadly pleitropic phenotype associated to the absence of a functional Hfq protein both support the existence of riboregulatory circuits controlling gene expression to ensure the fitness of this bacterium in both free living and symbiotic conditions. In order to identify target mRNAs subject to Hfq-dependent riboregulation, we have compared the proteome of an hfq mutant and the wild type S. meliloti by quantitative proteomics following protein labelling with (15)N. Among 2139 univocally identified proteins, a total of 195 proteins showed a differential abundance between the Hfq mutant and the wild type strain; 65 proteins accumulated ≥2-fold whereas 130 were downregulated (≤0.5-fold) in the absence of Hfq. This profound proteomic impact implies a major role for Hfq on regulation of diverse physiological processes in S. meliloti, from transport of small molecules to homeostasis of iron and nitrogen. Changes in the cellular levels of proteins involved in transport of nucleotides, peptides and amino acids, and in iron homeostasis, were confirmed with phenotypic assays. These results represent the first quantitative proteomic analysis in S. meliloti. The comparative analysis of the hfq mutant proteome allowed identification of novel strongly Hfq-regulated genes in S. meliloti.

Trichomonas vaginalis ribosomal RNA: identification and characterisation of the transcription promoter and terminator sequences.

Trichomonas vaginalis is a parasitic protozoan of both medical and biological relevance. Transcriptional studies in this organism have focused mainly on type II pol promoters, whereas the elements necessary for transcription by polI or polIII have not been investigated. Here, with the aid of a transient transcription system, we characterised the rDNA intergenic region, defining both the promoter and the terminator sequences required for transcription. We defined the promoter as a compact region of approximately 180 bp. We also identified a potential upstream control element (UCE) that was located 80 bp upstream of the transcription start point (TSP). A transcription termination element was identified within a 34 bp region that was located immediately downstream of the 28S coding sequence. The function of this element depends upon polarity and the presence of both a stretch of uridine residues (U's) and a hairpin structure in the transcript. Our observations provide a strong basis for the study of DNA recognition by the polI transcriptional machinery in this early divergent organism.

A rational approach to the regioselective deacetylation of 2',3',5'-tri-O-acetyluridine by Novozym 435 catalysed alcoholysis.

To give a rational explanation for the behaviour of 2',3',5'-tri-O-acetyluridine (TAU) catalysed alcoholysis using Novozym 435, the commercial biocatalyst with immobilized Candida antarctica lipase B (CALB), a set of experiments analyzing the role of the alcohol/substrate (A/S) molar ratio, alcohol/biocatalyst (A/B) and substrate/biocatalyst (S/B) mass ratios were carried out. At a A/S=120 and a S/B=6.16, 2',3'-di-O-acetyluridine (DAU) was obtained in 92% at 22h. The observed trend towards the exclusive formation of DAU at very high alcohol amounts can be explained on the basis of the change of substrate orientation from normal to inverse. The simple molecular modelling analysis supports that key O/H atoms from TAU and the resulting intermediates display the adequate distances to generate productive binding only when the inverse coordination of TAU is present through the 5'-moiety of TAU, at high ethanol concentrations. At these conditions a possible allosteric-like effect of ethanol, combined with water in an H-network in the catalytic triad and in its neighbourhood, could explain the high selectivity towards the production of DAU at selected conditions.

Crystal structure determination and dynamic studies of Mycobacterium tuberculosis Cytidine deaminase in complex with products.

Cytidine deaminase (CDA) is a key enzyme in the pyrimidine salvage pathway. It is involved in the hydrolytic deamination of cytidine or 2'-deoxycytidine to uridine or 2'-deoxyuridine, respectively. Here we report the crystal structures of Mycobacterium tuberculosis CDA (MtCDA) in complex with uridine (2.4 Å resolution) and deoxyuridine (1.9 Å resolution). Molecular dynamics (MD) simulation was performed to analyze the physically relevant motions involved in the protein-ligand recognition process, showing that structural flexibility of some protein regions are important to product binding. In addition, MD simulations allowed the analysis of the stability of tetrameric MtCDA structure. These findings open-up the possibility to use MtCDA as a target in future studies aiming to the rational design of new inhibitor of MtCDA-catalyzed chemical reaction with potential anti-proliferative activity on cell growth of M. tuberculosis, the major causative agent of tuberculosis.