RESUMO

ABSTRACTThe removal of organic matter and nitrogen from domestic sewage was evaluated using a system composed of two sequential reactors: an anaerobic reactor (ANR) with suspended sludge and an aerobic (AER) reactor with suspended and adhered sludge to polyurethane foams. Nitrogen removal consisted of AER operating at low dissolved oxygen (DO) concentrations; this favoured the simultaneous nitrification and denitrification (SND) process. The concentration of COD and N were 440 mgO2.L-1 and 37 mgTN.L-1, respectively. The operation was divided into three phases (P), lasting 51, 53, and 46 days, respectively. The initial DO concentrations applied in the AER were: 3.0 (PI) and 1.5 mg.L-1 (PII and PIII). In PIII, the AER effluent was recirculated to the ANR at a ratio of 0.25. Kinetic assays were performed to determine the nitrification and denitrification rates of the biomasses (ANR and AER in PIII). Changes in the microbial community were evaluated throughout phases PI to PIII by massive sequencing. In PIII, the best results obtained for chemical oxygen demand (COD) and total nitrogen (TN-N) removal efficiencies, were close to 94% and 65%, respectively. Under these conditions, system effluent concentrations below 30 mg COD.L-1 and 15 mg TN-N.L-1 were verified. The nitritation and nitration rates were 10.5 and 6.5 mg N.g VSS-1.h-1, while the denitrification via nitrite and nitrate were 6.8 and 5.8 mg N.g VSS-1.h-1, respectively. A mixotrophic community was prevalent, with Rhodococcus, Nitrosomonas, Pseudomnas, and Porphyromonas being dominant or co-dominant in most of the samples, confirming the SND process in the AER sludge.

RESUMO

PII proteins are signal transduction proteins that belong to a widely distributed family of proteins involved in the modulation of different metabolisms in bacteria. These proteins are homotrimers carrying a flexible loop, named T-loop, which changes its conformation due to the recognition of diverse key metabolites, ADP, ATP, and 2-oxoglutarate. PII proteins interact with different partners to primarily regulate a set of nitrogen pathways. In some organisms, PII proteins can also control carbon metabolism by interacting with the biotin carboxyl carrier protein (BCCP), a key component of the acetyl-CoA carboxylase (ACC) enzyme complex, inhibiting its activity with the consequent reduction of fatty acid biosynthesis. Most bacteria contain at least two PII proteins, named GlnB and GlnK, with different regulatory roles. In mycobacteria, only one PII protein was identified, and the three-dimensional structure was solved, however, its physiological role is unknown. In this study we purified the Mycobacterium tuberculosis (M. tb) PII protein, named GlnB, and showed that it weakly interacts with the AccA3 protein, the α subunit shared by the three different, and essential, Acyl-CoA carboxylase complexes (ACCase 4, 5, and 6) present in M. tb. A M. smegmatis deletion mutant, ∆MsPII, exhibited a growth deficiency on nitrate and nitrite as unique nitrogen sources, and accumulated nitrite in the culture supernatant. In addition, M. tb PII protein was able to interact with the C-terminal domain of the ammonium transporter Amt establishing the ancestral role for this PII protein as a GlnK functioning protein.

RESUMO

Impaired nitric oxide (NO) formation may be associated with endothelial dysfunction and increased cardiovascular disease risk in preeclampsia (PE). Functional single-nucleotide polymorphisms (SNPs) of nitric oxide synthase 3 (NOS3) (rs3918226) and guanylate cyclase 1, soluble, alpha 3 (GUCY1A3) (rs7692387) increase susceptibility to the adverse consequences due to inadequate generation of NO by the endothelium. However, no previous study has examined whether these SNPs affect NO formation in healthy pregnancy and in gestational hypertension (GH) and PE. Here, we compared the alleles and genotypes of NOS3 (rs3918226) and GUCY1A3 (rs7692387) SNPs in normotensive pregnant women (NP, n = 153), in GH (n = 96) and PE (n = 163), and examined whether these SNPs affect plasma nitrite concentrations (a marker of NO formation) in these groups. We further examined whether the interaction among SNP genotypes is associated with GH and PE. Genotypes were determined using TaqMan allele discrimination assays, and plasma nitrite concentrations were determined by an ozone-based chemiluminescence assay. Multifactor dimensionality reduction was used to examine the interactions among SNP genotypes. Regarding NOS3 rs3918226, the CT genotype (p = 0.046) and T allele (p = 0.020) were more frequent in NP than in GH, and GH patients carrying the CT+TT genotypes showed lower nitrite concentrations than NP carrying the CT+TT genotypes (p < 0.05). Regarding GUCY1A3 rs7692387, the GA genotype (p = 0.013) and A allele (p = 0.016) were more frequent in PE than in NP, and NP women carrying the GG genotype showed higher nitrite concentrations than GH or PE patients carrying the GG genotype (p < 0.05). However, we found no significant interactions among genotypes for these functional SNPs to be associated with GH or PE. Our novel findings suggest that NOS3 rs3918226 and GUCY1A3 rs7692387 may affect NO formation and association with hypertensive disorders of pregnancy.

RESUMO

Nitrite is a nitric oxide (NO) metabolite, which may be bioactivated to generate NO in vivo and supplement endogenous NO formation, especially in cardiovascular and metabolic diseases. However, it is not known whether treatment with oral nitrite results in the accumulation of NO metabolites in different organs. Moreover, treatment with omeprazole, an inhibitor of gastric acid secretion, severely affects the gastric formation of S-nitrosothiols induced with oral nitrite treatment. However, no previous study has examined whether omeprazole affects the nitrite-induced accumulation of NO metabolites in different organs. This study examined in rats the effects of oral sodium nitrite treatment (15 mg/kg via gavage for 1 or 7 days) associated with omeprazole (10 mg/kg or vehicle) on nitrite and nitrate and nitrosylated species (RXNO) concentrations (measured using ozone-based chemiluminescence methods) assessed in the plasma, aorta, heart, liver, brain, and muscle. While our results showed that NO metabolite accumulation in different organs is not uniform, we found that the skeletal muscle, the heart, and the liver accumulate NO metabolites, particularly RXNO. This response was significantly attenuated by omeprazole in the heart and in the skeletal muscle. Together, these findings may indicate that the skeletal muscle, the heart, and the liver are major reservoir sites for NO metabolites after oral nitrite treatment, with major increases in nitrosylated species.

RESUMO

This study evaluated the use of the S-nitrosothiols, S-nitroso-N-acetylcysteine (NAC-SNO) and S-nitroso-N-acetylcysteine ethyl ester (NACET-SNO), at different concentrations (25-300 mg nitrite equivalent - NEq/kg) as sodium nitrite substitutes in restructured cooked hams. The pH value and instrumental cured color were not affected by the type or amount of curing agent used. Products with 25 and 50 mg/kg ingoing nitrite had lower thiobarbituric acid-reactive substance values than those with equimolar amounts of S-nitrosothiols. Products with >150 mg NEq/kg of S-nitrosothiols had residual nitrite similar to 50 mg/kg nitrite, and this resulted in the same volatile compound profile as nitrite added in equimolar amounts. A 300 mg NEq/kg of S-nitrosothiols was required to obtain a similar and minimally stable cured pink color perception as sliced samples with 50-150 mg/kg added nitrite. The results obtained reinforce the great potential of both alternative curing agents in the complete replacement of nitrite by equimolar amounts in restructured cooked products; however, differences in cured color stability should be considered.

Assuntos

RESUMO

Abstract The present study involves the chemical and bacteriological analysis of water from different sources i.e., bore, wells, bottle, and tap, from Peshawar, Mardan, Swat and Kohat districts of Khyber Pakhtunkhwa (KP) province, Pakistan. From each district, 50 water samples (10 samples from each source), regardless of urban and rural status, were collected from these sources and analysed for sulphates, nitrates, nitrites, chlorides, total soluble solids and coliforms (E. coli). Results indicated that majority of the water sources had unacceptable E. coli count i.e.> 34 CFU/100mL. E. coli positive samples were high in Mardan District, followed by Kohat, Swat and Peshawar district. Besides this, the some water sources were also chemically contaminated by different inorganic fertilizers (nitrates/nitrites of sodium, potassium) but under safe levels whereas agricultural and industrial wastes (chloride and sulphate compounds) were in unsafe range. Among all districts, the water quality was found comparatively more deteriorated in Kohat and Mardan districts than Peshawar and Swat districts. Such chemically and bacteriologically unfit water sources for drinking and can cause human health problems.

Resumo O presente estudo envolve a análise química e bacteriológica de água de diferentes fontes, ou seja, furo, poços, garrafa e torneira, dos distritos de Peshawar, Mardan, Swat e Kohat da província de Khyber Pakhtunkhwa (KP), Paquistão. De cada distrito, 50 amostras de água (10 amostras de cada fonte), independentemente do status urbano e rural, foram coletadas dessas fontes e analisadas para sulfatos, nitratos, nitritos, cloretos, sólidos solúveis totais e coliformes (E. coli). Os resultados indicaram que a maioria das fontes de água tinha uma contagem inaceitável de E. coli, ou seja, > 34 UFC / 100 mL. As amostras positivas para E. coli foram elevadas no distrito de Mardan, seguido por Kohat, Swat e distrito de Peshawar. Além disso, algumas fontes de água também foram contaminadas quimicamente por diferentes fertilizantes inorgânicos (nitratos/nitritos de sódio, potássio), mas em níveis seguros, enquanto os resíduos agrícolas e industriais (compostos de cloreto e sulfato) estavam em níveis inseguros. Entre todos os distritos, a qualidade da água foi considerada comparativamente mais deteriorada nos distritos de Kohat e Mardan do que nos distritos de Peshawar e Swat. Essas fontes de água química e bacteriologicamente impróprias para beber podem causar problemas à saúde humana.

RESUMO

Abstract The present study involves the chemical and bacteriological analysis of water from different sources i.e., bore, wells, bottle, and tap, from Peshawar, Mardan, Swat and Kohat districts of Khyber Pakhtunkhwa (KP) province, Pakistan. From each district, 50 water samples (10 samples from each source), regardless of urban and rural status, were collected from these sources and analysed for sulphates, nitrates, nitrites, chlorides, total soluble solids and coliforms (E. coli). Results indicated that majority of the water sources had unacceptable E. coli count i.e.> 34 CFU/100mL. E. coli positive samples were high in Mardan District, followed by Kohat, Swat and Peshawar district. Besides this, the some water sources were also chemically contaminated by different inorganic fertilizers (nitrates/nitrites of sodium, potassium) but under safe levels whereas agricultural and industrial wastes (chloride and sulphate compounds) were in unsafe range. Among all districts, the water quality was found comparatively more deteriorated in Kohat and Mardan districts than Peshawar and Swat districts. Such chemically and bacteriologically unfit water sources for drinking and can cause human health problems.

Resumo O presente estudo envolve a análise química e bacteriológica de água de diferentes fontes, ou seja, furo, poços, garrafa e torneira, dos distritos de Peshawar, Mardan, Swat e Kohat da província de Khyber Pakhtunkhwa (KP), Paquistão. De cada distrito, 50 amostras de água (10 amostras de cada fonte), independentemente do status urbano e rural, foram coletadas dessas fontes e analisadas para sulfatos, nitratos, nitritos, cloretos, sólidos solúveis totais e coliformes (E. coli). Os resultados indicaram que a maioria das fontes de água tinha uma contagem inaceitável de E. coli, ou seja, > 34 UFC / 100 mL. As amostras positivas para E. coli foram elevadas no distrito de Mardan, seguido por Kohat, Swat e distrito de Peshawar. Além disso, algumas fontes de água também foram contaminadas quimicamente por diferentes fertilizantes inorgânicos (nitratos/nitritos de sódio, potássio), mas em níveis seguros, enquanto os resíduos agrícolas e industriais (compostos de cloreto e sulfato) estavam em níveis inseguros. Entre todos os distritos, a qualidade da água foi considerada comparativamente mais deteriorada nos distritos de Kohat e Mardan do que nos distritos de Peshawar e Swat. Essas fontes de água química e bacteriologicamente impróprias para beber podem causar problemas à saúde humana.

Assuntos

RESUMO

Antibiotics in wastewater treatment plants can alter the physiological activity and the structure of microbial communities through toxic and inhibitory effects. Physiological adaptation, kinetic, and population dynamics behavior of a nitrifying sludge was evaluated in a sequential batch reactor (SBR) fed with 14.4 mg/L of ampicillin (AMP). The addition of AMP did not affect ammonium consumption (100 mg NH4+-N/L) but provoked nitrite accumulation (0.90 mg NO2--N formed/mg NH4+-N consumed) and an inhibition of up to 67% on the nitrite oxidizing process. After 30 cycles under AMP feeding, the sludge recovered its nitrite oxidizing activity with a high nitrate yield (YNO3-) of 0.87 ± 0.10 mg NO3--N formed/mg NH4+-N consumed, carrying out again a stable and complete nitrifying process. Increases in specific rate of nitrate production (qNO3-) showed the physiological adaptation of the nitrite oxidizing bacteria to AMP inhibition. Ampicillin was totally removed since the first cycle of addition. Exposure to AMP had effects on the abundance of bacterial populations, promoting adaptation of the nitrifying sludge to the presence of the antibiotic and its consumption. Nitrosomonas and Nitrosospira always remained within the dominant genera, keeping the ammonium oxidizing process stable while an increase in Nitrospira abundance was observed, recovering the stability of the nitrite oxidizing process. Burkholderia, Pseudomonas, and Thauera might be some of the heterotrophic bacteria involved in AMP consumption.

RESUMO

Preeclampsia is a maternal hypertension disorder associated with vascular dysfunction and fetal and placental growth restrictions. Placental ischemia is suggested as the primary trigger of preeclampsia-associated impairments of both endothelium-derived nitric oxide (NO) and the vascular activity of extracellular matrix metalloproteinase-2 (MMP-2). Reduced uteroplacental perfusion pressure (RUPP) is a placental ischemia model of preeclampsia. Reduction of sodium nitrite to NO may occur during ischemic conditions. However, sodium nitrite effects in the RUPP model of preeclampsia have not yet been investigated. Pregnant rats were divided into four groups: normotensive pregnant rats (Norm-Preg), pregnant rats treated with sodium nitrite (Preg + Nitrite), preeclamptic rats (RUPP), and preeclamptic rats treated with sodium nitrite (RUPP + Nitrite). Maternal blood pressure and fetal and placental parameters were recorded. Vascular function, circulating NO metabolites, and the gelatinolytic activity of vascular MMP-2 were also examined. Sodium nitrite attenuates increased blood pressure, prevents fetal and placental weight loss, counteracts vascular hyper-reactivity, and partially restores NO metabolites and MMP-2 activity. In conclusion, sodium nitrite reduction to NO may occur during RUPP-induced placental ischemia, thereby attenuating increased blood pressure, fetal and placental growth restriction, and vascular hyper-reactivity associated with preeclampsia and possibly restoring NO and MMP-2 activity, which underlie the blood pressure-lowering effects.

Assuntos

RESUMO

Sodium and potassium nitrates and nitrites are preservatives widely used in meat products and some cheese. They are important toxicologically but there is a lack of data on the exposure of the Brazilian population to these additives. This study aimed to verify the frequency of the use of nitrates and nitrites in processed foods in Brazil and to estimate their theoretical intake by the Brazilian population. A database was built of supermarket products containing nitrates, nitrites, and antioxidants. The Theoretical Maximum Daily Intake (TMDI) and TMDI balanced by the prevalence of food consumption (TMDI BPFC) were determined using consumption data from the Household Budget Surveys (2008/2009 and 2017/2018). The TMDI for nitrates and nitrites was lower than the Acceptable Daily Intake (ADI) for all population groups. Considering the prevalence of food consumption (consumers only), the TMDI BPFC values were lower than the ADI for nitrates (between 0.4 and 0.9 times the ADI) but very high values were obtained for nitrites (between 10 and 24 times the ADI). Our results suggest that the Brazilian population, especially some population groups, may be consuming unsafe amounts of nitrite. As a consequence, their health may be at risk.

Assuntos

RESUMO

This study investigated the effect of a 50% reduction in sodium nitrite and the addition of nisin (200 mg/kg) and different concentrations (0, 0.5%, 0.75%, and 1%) of jabuticaba peel extract (JPE) on the main attributes affected by this chemical additive in Bologna-type sausages. The modified treatments showed approximately 50% lower residual nitrite than the control throughout the storage (60 days at 4 °C). The proposed reformulation did not affect the color (L*, a*, and b*), and the ΔE values (< 2) demonstrated high color stability during storage. Physicochemical (TBARS and volatile compounds) and sensory analyses performed to evaluate oxidative stability indicated that JPE exhibited antioxidant activity comparable to sodium nitrite. The microbiological quality of the reformulated products was similar to the control, but further studies should be conducted to assess the effect of this reformulation strategy on the growth of pathogenic microorganisms impacted by nitrite.

Assuntos

RESUMO

Canine Visceral leishmaniasis (CanL) poses a severe public health threat in several countries. Disease progression depends on the degree of immune response suppression. MicroRNAs (miRs) modulate mRNA translation into proteins and regulate various cellular functions and pathways associated with immune responses. MiR-21 and miR-148a can alter the parasite load and M1 macrophages are the principal cells in dogs' leishmanicidal activity. A previous study found increased miR-21 and miR-148a in splenic leukocytes (SL) of dogs with CanL using microarray analysis and in silico analysis identified PTEN pathway targets. PTEN is involved in the immune regulation of macrophages. We measured PTEN and the production of reactive oxygen species (ROS) and nitric oxide (NO) before and after transfection SLs of dogs with CanL with mimic and inhibition of miR-21 and miR-148a. PTEN levels increased, NO and ROS decreased in SLs from dogs with CanL. Inhibition of miRNA-21 resulted in PTEN increase; in contrast, PTEN decreased after miR-148a inhibition. Nitrite (NO2) levels increased after transfection with miR-21 inhibitor but were decreased with miR-148a inhibitor. The increase in miR-21 promoted a reduction in ROS and NO levels, but miR-148a inhibition increased NO and reduced ROS. These findings suggest that miR-21 and miR-148a can participate in immune response in CanL, affecting PTEN, NO, and ROS levels.

RESUMO

Nitric oxide (NO) is an important mediator responsible for vasodilation in pulmonary hypertension (PH) in humans. Based on human literature, it is suggested that in dogs there is also NO production decrease in lung tissue in the presence of PH with hypoxia. Therefore, the aim of this research was to determine the indirect plasmatic NO concentration in dogs with PH secondary or not to the left-side heart disease (LHD) and also with low, intermediate and high probability of PH to characterize the NO involvement on PH in dogs. Blood samples were collected from 35 dogs with probability of PH to NO measurement. NO concentration was estimated by the nitrite/nitrate concentration, and it was significantly different (p=0.002) in dogs with PH secondary to LHD (median=14 µM, range 11.19-16.59) and not secondary to LHD (median=25.88µM, range 15.08-36.71). However, this was not significant for the probability of low, intermediate, and high PH, although there was a tendency for NO concentration to be higher in dogs with high PH. The results of this study demonstrate that there is release of NO in dogs with PH, as well as that its dosage could differentiate dogs with PH secondary to LHD from dogs with non-secondary PH.

O óxido nítrico (ON) é um importante vasodilatador na hipertensão pulmonar (HP) em seres humanos. Baseado na literatura humana, sugere-se que em cães também ocorra a diminuição da produção de ON no tecido pulmonar na presença de HP frente à hipóxia. Dessa forma, o objetivo desta pesquisa foi determinar a concentração indireta do ON plasmático em cães com HP secundária ou não à doença do lado esquerdo do coração (LHD), bem como caracterizar os achados ecocardiográficos de diagnóstico e a probabilidade de HP (baixa, intermediária e alta). Para isso, foram coletadas amostras de sangue de 35 cães com probabilidade de HP para mensuração de ON. A concentração do ON foi estimada pela concentração de nitrito/nitrato, e esta foi significativamente diferente (P=0,002) nos cães com HP secundária à LHD (mediana=14µM, intervalo 11,19-16,59) e não secundária à LHD (mediana=25,88µM, intervalo 15,08-36,71), porém não significativa para a probabilidade de HP baixa, intermediária e alta, embora houvesse uma tendência a ser maior a concentração de ON nos cães com probabilidade alta. Os resultados deste estudo demonstram que há liberação do ON em cães com HP, bem como que sua dosagem conseguiu diferenciar cães com HP secundária à LHD de cães com HP não secundária.

Assuntos

RESUMO

Leishmaniasis is a neglected tropical infectious disease with thousands of cases annually; it is of great concern to global health, particularly the most severe form, visceral leishmaniasis. Visceral leishmaniasis treatments are minimal and have severe adverse effects. As guanidine-bearing compounds have shown antimicrobial activity, we analyzed the cytotoxic effects of several guanidine-bearing compounds on Leishmania infantum in their promastigote and amastigote forms in vitro, their cytotoxicity in human cells, and their impact on reactive nitrogen species production. LQOFG-2, LQOFG-6, and LQOFG-7 had IC50 values of 12.7, 24.4, and 23.6 µM, respectively, in promastigotes. These compounds exhibited cytotoxicity in axenic amastigotes at 26.1, 21.1, and 18.6 µM, respectively. The compounds showed no apparent cytotoxicity in cells from healthy donors. To identify mechanisms of action, we evaluated cell death processes by annexin V and propidium iodide staining and nitrite production. Guanidine-containing compounds caused a significant percentage of death by apoptosis in amastigotes. Independent of L. infantum infection, LQOFG-7 increased nitrite production in peripheral blood mononuclear cells, which suggests a potential mechanism of action for this compound. Therefore, these data suggest that guanidine derivatives are potential anti-microbial molecules, and further research is needed to fully understand their mechanism of action, especially in anti-leishmanial studies.

RESUMO

This paper describes the design and construction of dual microfluidic paper-based analytical devices (dual-µPADs) as a lab-on-paper platform involving a "do-it-yourself" fabrication protocol. The device comprises a colorimetric and electrochemical module to obtain a dual-mode signal readout sensing strategy. A 3D pen polymeric resin was used to prepare graphite carbon-based electrodes and hydrophobic barriers on paper substrates. The proposed carbon-based ink was employed to manufacture electrodes on paper based on a stencil-printing approach, which were further characterized by electrochemical and morphological analyses. The analytical performance of the dual-µPADs was simultaneously evaluated for lactate, pH, nitrite, and salivary amylase (sAA) analysis. To demonstrate the proof-of-concept, saliva samples collected from both healthy individuals and those with periodontitis were successfully tested to demonstrate the feasibility of the proposed devices. Samples collected from individuals previously diagnosed with periodontitis showed high levels of nitrite and sAA (> 94 µmol L-1 and > 610 U mL-1) in comparison with healthy individuals (≤ 16 µmol L-1 and 545 U mL-1). Moreover, periodontitis saliva resulted in acid solution and almost null lactate levels. Notably, this protocol supplies a simple way to manufacture dual-µPADs, a versatile platform for sensitive detecting of biomarkers in saliva playing a crucial role towards the point-of-care diagnosis of periodontal disease.

Assuntos

RESUMO

Urinary tract infection (UTI) is one of the most common bacterial infections among humans. Urine culture is the gold standard diagnostic method for UTI; however, the dipstick test for nitrite is a widely used method signalling the presence of urinary nitrate-reducing bacteria. Unlike the gold standard, the dipstick test is easy to perform, while it is also less time-consuming and less expensive, and produces a result in a few minutes. This study investigates the sensitivity of the dipstick test for nitrite compared with the Griess test in urine samples from UTI caused by Enterobacterales species. We used the Griess test, which is the gold standard in nitrite measurement, to determine the sensitivity of the nitrite dipstick test. Semiquantitative urine culture was performed using standard procedures, and Enterobacterales identification was performed by manual conventional biochemical tests. In the first sample selection, 3 % (8/267) of urine samples suspected of UTI, analysed from March to April 2016, were nitrite-negative by dipstick test but positive for Enterobacterales in the urine culture. In the second sample selection, 5 % (2/44) of urine samples from October to December 2022 were also nitrite-negative but showed urine Enterobacterales isolation. All nitrite-negative dipstick results were consistent with the Griess test. Escherichia coli was the most prevalent bacterium, followed by Klebsiella pneumoniae, independent of sample selection. The dipstick test is a safe alternative for investigating nitrite in urine samples. We believe that the cause of nitrite-negative results is a lack of dietary nitrate, dilution of urine and exogenous interference (e.g. ascorbic acid). These findings support the idea that standard urine culture is necessary to rule out UTI.

Assuntos

RESUMO

A copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide in the denitrifier Sinorhizobium meliloti 2011 (SmNirK), a microorganism used as bioinoculant in alfalfa seeds. Wild type SmNirK is a homotrimer that contains two copper centers per monomer, one of type 1 (T1) and other of type 2 (T2). T2 is at the interface of two monomers in a distorted square pyramidal coordination bonded to a water molecule and three histidine side chains, H171 and H136 from one monomer and H342 from the other. We report the molecular, catalytic, and spectroscopic properties of the SmNirK variant H342G, in which the interfacial H342 T2 ligand is substituted for glycine. The molecular properties of H342G are similar to those of wild type SmNirK. Fluorescence-based thermal shift assays and FTIR studies showed that the structural effect of the mutation is only marginal. However, the kinetic reaction with the physiological electron donor was significantly affected, which showed a âˆ¼ 100-fold lower turnover number compared to the wild type enzyme. UV-Vis, EPR and FTIR studies complemented with computational calculations indicated that the drop in enzyme activity are mainly due to the void generated in the protein substrate channel by the point mutation. The main structural changes involve the filling of the void with water molecules, the direct coordination to T2 copper ion of the second sphere aspartic acid ligand, a key residue in catalysis and nitrite sensing in NirK, and to the loss of the 3 N-O coordination of T2.

Assuntos

RESUMO

Nitrite is a ubiquitous pollutant in modern society. Developing new strategies for its determination is very important, and electroanalytical methods present outstanding performance on this task. However, the use of bare electrodes is not recommended because of their predisposition to poisoning and passivation. We herein report a procedure to overcome these limitations on carbon fiber microelectrodes through pulsed amperometry. A three-pulse amperometry approach was used to reduce the current decay from 47% (after 20 min under constant potential) to virtually 0%. Repeatability and reproducibility were found to have an RSD lower than 0.5% and 7%, respectively. Tap water and synthetic inorganic saliva samples were fortified with nitrite, and the results obtained with the proposed sensor were in good agreement with the amount added.

Assuntos

RESUMO

A sensor device based on doped-carbon quantum dots is proposed herein for detection of nitrite in meat products by fluorescence quenching. For the sensing platform, carbon quantum dots doped with boron and functionalized with nitrogen (B,N-Cdot) were synthesized with an excellent 44.3% quantum yield via a one-step hydrothermal route using citric acid, boric acid, and branched polyethylenimine as carbon, boron, and nitrogen sources, respectively. After investigation of their chemical structure and fluorescent properties, the B,N-Cdot at aqueous suspensions showed high selectivity for NO2- in a linear range from 20 to 50 mmol L-1 under optimum conditions at pH 7.4 and a 340 nm excitation. Furthermore, the prepared B,N-Cdots successfully detected NO2- in a real meat sample with recovery of 91.4-104% within the analyzed range. In this manner, a B,N-Cdot/PVA nanocomposite film with blue emission under excitation at 360 nm was prepared, and a first assay detection of NO2- in meat products was tested using a smartphone application. The potential application of the newly developed sensing device containing a highly fluorescent probe should aid in the development of a rapid and inexpensive strategy for NO2- detection.

Assuntos

RESUMO

Aim: This work examined whether ARG1 (rs2781659, rs2781667, rs2246012 and rs17599586) and ARG2 (rs3742879 and rs10483801) single-nucleotide polymorphisms (SNPs) are associated with antihypertensive therapy responsiveness in preeclampsia (PE) and their effects on arginase isoforms and nitrite concentrations in responsive and nonresponsive patients. Methods: SNP genotypes were determined by TaqMan assays. Plasma arginase levels were measured by ELISA and nitrite concentrations were measured using an ozone-based chemiluminescence assay. Results: The G allele for ARG2 rs3742879 (A>G) was less frequent in nonresponsive compared with responsive patients (15.5% vs 24.7%, respectively) and the G carriers of the nonresponsive subgroup had lower arginase 2 (9.2 ± 7.5 ng/ml vs 19.1 ± 17.3 ng/ml) and higher nitrite concentrations (110.2 ± 52.8 nM vs 78.5 ± 37.9 nM) than carriers of the AA genotype (all p < 0.05). Conclusion: ARG2 SNP rs3742879 is associated with diminished arginase 2 levels and increased nitric oxide formation in nonresponsive PE patients.

Assuntos