Efeito de géis fluoretados suplementados com trimetafosfato de sódio nanoparticulado sobre a remineralização do esmalte dental in situ / Effect of fluoride gels supplemented with nanosized sodium trimetaphosphate on enamel remineralization in situ

O objetivo do presente estudo foi avaliar o efeito de géis fluoretados suplementados com nanopartículas de Trimetafosfato de Sódio (TMP) sobre a remineralização de lesões de cárie artificiais in situ. Blocos de esmalte dental bovino (n=160) foram aleatoriamente divididos entre os grupos de estudo após análise de dureza de superfície (DS) e indução de lesões de subsuperfície. Os géis testados foram: Placebo (sem flúor ou TMP ­ controle negativo), 9000 µg F/g (9000F ­ controle positivo), 4500 µg F/g + 5% TMP microparticulado (4500 5%TMPmicro) e 4500 µg F/g + 5% TMP nanoparticulado (4500 5%TMPnano). Dez voluntários utilizaram dispositivos palatinos contendo 4 blocos de esmalte durante 3 dias, após uma única aplicação dos géis, seguindo um protocolo duplo-cego e cruzado. Dois blocos de esmalte foram removidos imediatamente após a aplicação dos géis, para determinar a concentração de fluoreto de cálcio (CaF2) formado. Após cada fase, determinou-se a porcentagem de recuperação de dureza de superfície (%RDS) e CaF2 retido no esmalte. Os dados foram submetidos ANOVA de medidas repetidas e teste de Student-Newman-Keuls (p< 0.05). A maior %RDS foi observada para o gel 4500 5%TMPnano, seguido por 4500 5%TMPmicro, 9000F e Placebo, com diferenças significativas entre os grupos. Em relação ao CaF2 formado, a maior concentração foi observada para o grupo 9000F. Não foram observadas diferenças significativas entre os grupos 9000F, 4500 5%TMPmicro e 4500 5%TMPnano para concentrações de CaF2 retido. Conclui-se que a adição de TMP a géis fluoretados melhorou significativamente a remineralização de lesões de cárie in situ. O uso de TMP em escala nanométrica potencializou ainda mais este efeito(AU)

The present study aimed to evaluate the effect of fluoride gels supplemented with nano-sized sodium trimetaphosphate (TMP) on the remineralization of artificial caries lesions in situ. Bovine enamel blocks (n=160) were randomly distributed among study groups after surface microhardness (SH) analysis and induction of subsurface lesions. Test groups included: Placebo (without F and TMP ­ negative control), 9000 µg F/g (9000F ­ positive control), 4500 µg F/g + 5% micrometric TMP (4500 5%+ TMPmicro) and 4500 µg F/g + 5% nano-sized TMP (4500 + 5%TMPnano). Ten volunteers used palatal devices containing 4 enamel blocks during 3 days, after a single application of gels, following a double-blind and crossover protocol. Two enamel blocks were removed immediately after topical application of F to determine calcium fluoride (CaF2) formed on enamel. After each phase, the samples were analyzed by percentage of surface hardness recovery (%SHR) and CaF2 retained on enamel. Data were analyzed by repeated-measures ANOVA and Student-NewmanKeuls test (p< 0.05). The highest %SHR was observed for 4500 5%TMPnano gel, following by 4500 5%TPMmicro, 9000F, and Placebo, with significant differences among all groups. Regarding CaF2 formed, the highest concentration was observed in the 9000F group. No significant differences were observed among 9000F, 4500 5%TMPmicro and 4500 5%TMPnano groups for concentrations of CaF2 retained. It was concluded that the addition of TMP to gels improved the remineralization of caries lesions in situ. The use of nano-sized TMP further enhanced this effect(AU)

Pretreatment HIV-1 drug resistance in Argentina: results from a surveillance study performed according to WHO-proposed new methodology in 2014-15.

BACKGROUND: In Argentina, current national guidelines recommend starting with NNRTI-based regimens. Recently, there have been some local reports regarding concerning levels of NNRTI-transmitted resistance, but surveillance has never been carried out at a national level. OBJECTIVES: To determine the prevalence of HIV drug resistance in people starting ART in Argentina using a WHO-proposed methodology. METHODS: This was a cross-sectional, nationally representative study. Twenty-five antiretroviral-dispensing sites throughout the country were randomly chosen to enrol at least 330 persons starting ART, to generate a point prevalence estimate of resistance-associated mutations (RAMs) with a 5% CI (for the total population and for those without antiretroviral exposure). All consecutive patients older than 18 years starting or restarting ART in the chosen clinics were eligible. Samples were processed with Trugene and analysed using the Stanford algorithm. RESULTS: Between August 2014 and March 2015, we obtained 330 samples from people starting ART. The mean ±â€ŠSD age was 35 ±â€Š11 years, 63.4% were male, 16.6% had prior antiretroviral exposure and the median (IQR) CD4 count was 275 cells/mm3 (106-461). The prevalence of RAMs found was 14% (±4%) for the whole population (3% NRTI-RAMs; 11% NNRTI-RAMs and 2% PI-RAMs) and 13% (±4%) for those without prior antiretroviral exposure (3%, 10% and 2%, respectively). The most common mutation was K103N. CONCLUSIONS: This surveillance study showed concerning levels of HIV drug resistance in Argentina, especially to NNRTIs. Due to this finding, Argentina's Ministry of Health guidelines will change, recommending performing a resistance test for everyone before starting ART. If this is taken up properly, it also might function as a continuing surveillance tool.

Thymidine radical formation via one-electron transfer oxidation photoinduced by pterin: Mechanism and products characterization.

UV-A radiation (320-400nm), recognized as a class I carcinogen, induces damage to the DNA molecule and its components through different mechanisms. Pterin derivatives are involved in various biological functions, including enzymatic processes, and it has been demonstrated that oxidized pterins may act as photosensitizers. In particular, they accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder. We have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to photosensitize the degradation of the pyrimidine nucleotide thymidine 5'-monophosphate (dTMP) in aqueous solutions under UV-A irradiation. Although thymine is less reactive than purine nucleobases, our results showed that Ptr is able to photoinduce the degradation of dTMP and that the process is initiated by an electron transfer from the nucleotide to the triplet excited state of Ptr. In the presence of molecular oxygen, the photochemical process leads to the oxidation of dTMP, whereas Ptr is not consumed. In the absence of oxygen, both compounds are consumed to yield a product in which the pterin moiety is covalently linked to the thymine. This compound retains some of the spectroscopic properties of Ptr, such as absorbance in the UV-A region and fluorescence properties.

Tetramethylpyrazine protects Schwann cells from ischemia-like injury and increases cell survival in cold ischemic rat nerves

Tetramethylpyrazine (TMP), a major active ingredient of Ligusticum wallichi Franchat extract (a Chinese herb), exhibits neuroprotective properties in ischemia. In this study, we assessed its protective effects on Schwann cells (SCs) by culturing them in the presence of oxygen glucose deprivation (OGD) conditions and measuring cell survival in cold ischemic rat nerves. In the OGD-induced ischemic injury model of SCs, we demonstrated that TMP treatment not only reduced OGD-induced cell viability losses, cell death, and apoptosis of SCs in a dose-dependent manner, and inhibited LDH release, but also suppressed OGD-induced downregulation of Bcl-2 and upregulation of Bax and caspase-3, as well as inhibited the consequent activation of caspase-3. In the cold ischemic nerve model, we found that prolonged cold ischemic exposure for four weeks was markedly associated with the absence of SCs, a decrease in cell viability, and apoptosis in preserved nerve segments incubated in University of Wisconsin solution (UWS) alone. However, TMP attenuated nerve segment damage by preserving SCs and antagonizing the decrease in nerve fiber viability and increase in TUNEL-positive cells in a dose-dependent manner. Collectively, our results indicate that TMP not only provides protective effects in an ischemia-like injury model of cultured rat SCs by regulating Bcl-2, Bax, and caspase-3, but also increases cell survival and suppresses apoptosis in the cold ischemic nerve model after prolonged ischemic exposure for four weeks. Therefore, TMP may be a novel and effective therapeutic strategy for preventing peripheral nervous system ischemic diseases and improving peripheral nerve storage.

Tetrametilpirazina (TMP), o principal componente do extrato de Ligusticum wallichi Franchat (erva chinesa), apresenta propriedades neuroprotetoras na isquemia. Nesse estudo, avaliamos seus efeitos protetores nas células de Schwann (SC), cultivando-as na presença de condições de depleção de oxigênio da glicose (OGD) e medindo a sobrevivência dos nervos de ratos isquêmicos pelo resfriamento. No modelo de lesão isquêmica em SC induzida por OGD, demonstramos que o tratamento com TMP não somente reduziu as perdas de viabilidade celular induzida por OGD, a morte celular, a apoptose de SC dose-dependente e inibiu a liberação de LDH, mas, também, suprimiu a infra-regulação do Vcl-2 e a supra-regulação de Bax e caspase-3, e inibiu a consequente ativação da caspase-3. No modelo de nervo isquêmico por resfriamento, observamos que a exposição prolongada ao resfriamento por quatro semanas estava, marcadamente, associada com a ausência de SC, com o decréscimo da viabilidade celular e a apoptose em segmentos de nervo incubados na solução da Universidade de Wisconsin apenas. Entretanto, a TMP atenuou o dano no segmento do nervo preservando SC e antagonizando a diminuição da viabilidade da fibra nervosa e o aumento das células TUNEL-positiva de modo dose-dependente. De forma conjunta, nossos resultados indicam que o TMP não só fornece efeitos protetores em um modelo de dano semelhante à isquemia de SC de ratos cultivados pela regulação de BCl-2, Bax e caspase 3, mas, também, aumenta a sobrevivência celular e suprime a apoptose no modelo de isquemia por resfriamento por exposição prolongada por quatro semanas. Então, TMP pode ser uma estratégia terapêutica eficaz para prevenir doenças isquêmicas do sistema nervoso periférico e melhora a armazenagem do nervo periférico.

Influence of antidepressant drugs on Ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) from salivary glands of rats.

Xerostomia is commonly caused by antidepressant drugs and ATP can influence the saliva production. Adenosine is the product of extracellular hydrolysis of adenine nucleotides in submandibular gland cells, which occurs by the action of ectonucleotidases. In this study, we have evaluated the effect of three different antidepressants in ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP1-3) activities in cultured cells from salivary glands. Rats received imipramine (10mg/ml), fluoxetine (20mg/ml) or moclobemide (30mg/ml) by oral gavage. The drugs were administered once a day for 14 days. Our results have shown that the hydrolysis of p-nitrophenyl-5'-thymidine monophosphate increased in all treatments. These effects were not consequence of transcriptional control of E-NPP1-3 genes. The results reported here can highlight the importance of ectonucleotidases in the most common side effect caused by antidepressant therapy.

Planejamento racional de novos tuberculostáticos com base na estrutura das enzimas enoil redutase e timidina monofosfato quinase do Mycobacterium tuberculosis / Rational design of new antituberculosis agents based on the structures of Mycobacterium tuberculosis enzymes enoyl reductase and thymidylate kinase

Nos últimos quinze anos, a tuberculose (TB) ressurgiu tanto em países em desenvolvimento quanto naqueles desenvolvidos. Em 1993, a Organização Mundial da Saúde (OMS) declarou a TB como emergência global de saúde pública. Atualmente, um terço da população mundial está infectada pelo Mycobacterium tuberculosis e mais de 10% destes indivíduos desenvolverão a doença ativa. Em 2006, estimaram-se 9 milhões de novos casos de TB em todo o mundo. No Brasil, aproximadamente 95.000 novos casos são registrados anualmente, com incidência de 50 casos em cada 100.000 habitantes. Tendo em vista o quadro alarmante da TB no mundo e, em especial no Brasil, e considerando os índices elevados de resistência do microrganismo aos fármacos convencionalmente utilizados na terapêutica, há necessidade urgente do desenvolvimento de novos tuberculostáticos. Além disso, a busca por novos alvos de ação se faz necessária, já que os antimicobacterianos utilizados na terapia anti-TB têm como alvo apenas pequeno número de enzimas relacionadas a funções essenciais do microrganismo. A biossíntese bacteriana de ácidos graxos tem despertado atenção especial como alvo atraente no desenvolvimento de novos agentes antibacterianos. Diferenças bioquímicas e funcionais fazem com que as enzimas envolvidas em tal processo sejam alvos potencialmente atraentes para o desenvolvimento de novos agentes antibacterianos/antimicobacterianos. As enoil-acp redutases são enzimas determinantes na etapa de alongamento de ácidos graxos, produtos intermediários na biossíntese dos principais constituintes da parede celular micobacteriana, os ácidos micólicos...

Biochemical characterization of ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP, E.C. 3.1.4.1) from rat heart left ventricle.

In the present study we investigate the biochemical properties of the members of NPP family in synaptosomes prepared from rat heart left ventricles. Using p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) as substrate for E-NPPs in rat cardiac synaptosomes, we observed an alkaline pH dependence, divalent cation dependence and the K ( M ) value corresponded to 91.42 +/- 13.97 microM and the maximal velocity (V ( max )) value calculated was 63.79 +/- 3.59 nmol p-nitrophenol released/min/mg of protein (mean +/- SD, n = 4). Levamisole (1 mM), was ineffective as inhibitor of p-Nph-5'-TMP hydrolysis in pH 8.9 (optimum pH for the enzyme characterized). Suramin (0.25 mM) strongly reduced the hydrolysis of p-Nph-5'-TMP by about 46%. Sodium azide (10 and 20 mM) and gadolinium chloride (0.3 and 0.5 mM), E-NTPases inhibitors, had no effects on p-Nph-5'-TMP hydrolysis. RT-PCR analysis of left ventricle demonstrated the expression of NPP2 and NPP3 enzymes, but excluded the presence of NPP1 member. By quantitative real-time PCR we identified the NPP3 as the enzyme with the highest expression in rat left ventricle. The demonstration of the presence of the E-NPP family in cardiac system, suggest that these enzymes could contribute with the fine-tuning control of the nucleotide levels at the nerve terminal endings of left ventricles that are involved in several cardiac pathologies.

The effects of angiotensin II and genetic hypertension upon extracellular nucleotide hydrolysis by rat platelet ectoenzymes.

The extracellular nucleotides, ATP and ADP, as well as adenosine have been implicated in a great number of physiological functions. ADP is one of the major platelet recruiting factors, whereas ATP is considered to be a competitive inhibitor of ADP-induced platelet aggregation and adenosine is able to induce vasodilatation and to inhibit platelet aggregation. The di- and triphosphate nucleosides can be hydrolyzed by members of several families of ectonucleotidases, including ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) that, together with an ecto-5'-nucleotidase, catalyze adenosine formation. The renin-angiotensin system is the most important regulator of renal and cardiovascular functions and angiotensin II induces, physiologically, platelet activation. The aim of this study was to clarify the effects of ANGII and genetic hypertension upon extracellular nucleotide hydrolysis by rat platelet ectoenzymes. ANGII, in all tested doses (5, 50, 500 and 5000 pmol), was able to increase ATP (21, 31, 44 and 27%, respectively), ADP (22, 28, 78 and 37%, respectively) and AMP (40, 64, 60 and 64%, respectively) hydrolysis by rat platelets. Furthermore, losartan, a specific antagonist of the AT1 angiotensin-receptor, prevented the nucleotide hydrolysis effects. Additionally, an increase in AMP (about 144%) hydrolysis and a decrease in p-Nph-5'TMP (about 27%) hydrolysis were observed in platelets from spontaneously hypertensive rats (SHR) when compared to Wistar normotensive rats. We, herein, present data to demonstrate interactions between rat platelet angiotensinergic and adenosinergic systems that could contribute to the understanding and treatment of cardiovascular diseases such as hypertension, thrombosis and arteriosclerosis.

Ecto-nucleotide pyrophosphatase/phosphodiesterase as part of a multiple system for nucleotide hydrolysis by platelets from rats: kinetic characterization and biochemical properties.

In this study, we describe an ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) activity in rat platelets. Using p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP) as a substrate for E-NPP, we demonstrate an enzyme activity that shares the major biochemical properties described for E-NPPs: alkaline pH dependence, divalent cation dependence and blockade of activity by metal ion chelator. K(m) and V(max) values for p-Nph-5'-TMP hydrolysis were found to be 106 +/- 18 microM and 3.44 +/- 0.18 nmol p-nitrophenol/min/mg (mean +/- SD, n = 5). We hypothesize that an E-NPP is co-localized with an ecto-nucleoside triphosphate diphosphohydrolase and an ecto-5'-nucleotidase on the platelet surface, as part of a multiple system for nucleotide hydrolysis, since they can act under distinct physiological conditions and can be differently regulated. Thus, 0.25 mM suramin inhibited p-Nph-5'-TMP, ATP and ADP hydrolysis, while 0.5 mM AMP decreased only p-Nph-5'-TMP hydrolysis. Besides, 5.0, 10 and 20 mM sodium azide just inhibited ATP and ADP hydrolysis. Angiotensin II (5.0 and 10 nM) affected only ADP hydrolysis. Gadolinium chloride (0.2 and 0.5 mM) strongly inhibited the ATP and ADP hydrolysis. The E-NPP described here represents a novel insight into the control of platelet purinergic signaling.