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Growing health and environmental concerns have increased demand for all-natural products, with a focus on clean labelling. Sodium nitrite is the most widely used additive in the meat industry because it imparts the typical cured flavour and colour to meat products and, most importantly, their microbiological safety. However, due to health concerns, the European Commission is proposing revised regulations to reduce nitrate and nitrite levels in meat products. As a result, the meat industry is actively seeking alternatives. This study explored the production of four cooked hams utilising nitrate-rich vegetable sources combined with two different nitrate-reducing commercial food cultures, alongside a control ham prepared with sodium nitrite (150 ppm). Microbiological, physico-chemical (pH, water activity, nitrate and nitrite concentration, lipid profile, lipid oxidation) and sensory (texture and colour profile) characterisation of the products was carried out. Challenge tests for Listeria monocytogenes, Clostridium sporogenes and Clostridium perfringens have been performed to assess the growth of pathogens, if present in the products. Results revealed comparable microbiological and physico-chemical profiles across ham formulations, with minor differences observed in colour parameters for sample C. The sensory analysis showed that for the pilot ham formulations A and D, there were no significant differences in consumer perception compared to the control ham. In the challenge tests, L. monocytogenes levels were similar in both control and tested hams. There were no significant differences in C. sporogenes and C. perfringens counts at any temperature or between test and control samples. These results indicate that this technology has a potential future in the cured meat sector, as regulators mandate the reduction of added synthetic chemicals and consumers seek healthier and more natural ingredients in their daily diets., Competing Interests: Declaration of competing interest N.K. is an employee of the company Primor Charcutaria Prima - S.A.:, which provided raw materials and collaborated on the manuscript conceptualisation., (Copyright © 2024. Published by Elsevier Ltd.)

The effects of cauliflower treated with atmospheric cold plasma (ACP), as a natural nitrite source, on the curing of ground ham and nitrosamine formation were investigated. Ground ham was prepared using sodium nitrite and ACP-treated cauliflower powder (PTCP) to achieve initial nitrite concentrations of 60 and 100 mg/kg, respectively. ACP treatment generated nitrite in cauliflower but significantly reduced the antioxidant activity (P < 0.05). As a nitrite source, PTCP had similar effects as sodium nitrite in the development of cured color in ground ham, with a comparable residual nitrite content (P ≥ 0.05). Three nitrosamines, N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), and N-nitrosopyrrolidine (NPYR), were detected in ground ham. NPYR formation was significantly lower in ground ham treated with PTCP at an initial nitrite concentration of 100 mg/kg (P < 0.05). Therefore, the use of a natural nitrite source manufactured through ACP treatment can prospectively achieve suitable curing efficiency while simultaneously suppressing nitrosamine formation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The mitigation of nitrosamine formation in drug products has been studied and approaches such as using formulations with pH modifiers and antioxidants have been shown to decrease the formation of nitrosamines. However, more studies are needed to explore the effectivness of mitigation strategies with different drug models and formulations. The primary objective of this work was to assess the role of different antioxidants and pH modifiers in tablet formulations to mitigate the formation of NDMA, prepared in-house, using metformin hydrochloride as a model drug. A study design for manufacturing metformin hydrochloride formulations was created to evaluate potential mitigation stratigies. The formulations were prepared by wet granulation that included a sodium nitrite spike and various antioxidants such as ascorbic acid, caffeic acid and ferulic acid at various concentrations that may inhibit nitrosamine formation. The study design also included pH modifiers such as hydrochloric acid and sodium carbonate. The metformin hydrochloride formulations were placed under stability conditions that included humidity, temperature and time over a six month period. NDMA inhibition was found to be most effective in formulations with basic pH, followed by the addition of tested antioxidants with 0.1% concentrations in the formulations. All tested antioxidants showed complete mitigation in formulations with 0.5% and 1% concentrations. In summary, basic pH and the inclusion of antioxidants exhibited the potential to mitigate the formation of NDMA in metformin hydrochloride tablets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

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., Competing Interests: Declaration of Competing Interest The author has no conflicts of interest for disclosing to the paper entitled “Jabuticaba peel extract and nisin: A promising combination for reducing sodium nitrite in Bologna-type sausages”., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

This study aimed to evaluate a pomegranate peel extract (PPE), selected for its level of phytochemical compounds and antioxidant activities, as a nitrite substitute in dry sausages, as well as its effect on lipid and protein oxidative changes and instrumental colour during the drying period (28 days). Of the extraction solvents screened, water: acetone 3:7 (v/v) was the most effective extraction solvent, yielding extracts with the highest content of phenolic compounds, flavonoids and condensed tannins and antioxidant activities (ABTS, DPPH and FRAP). Four batches of dry sausages were produced with different incoming amounts of sodium nitrite (NaNO 2 ) and PPE: 1) 150 ppm NaNO 2 ; 2) 0 ppm NaNO 2 ; 3) 0 ppm NaNO 2  + 1% PPE (v/w) and 4) 0 ppm NaNO 2  + 2% PPE (v/w). Nitrite removal increased lipid oxidation in uncured dry sausages, while nitrite and PPE caused lower TBA-RS values in cured and PPE treated sausages. During drying, both nitrite and PPE addition significantly decreased carbonyl and thiol contents compared to the uncured dry sausages. A dose-response was found for PPE, with lower carbonyl and thiol concentrations, the higher the level of PPE added. PPE significantly modified instrumental colour coordinates L*a*b* producing significant total colour changes compared to cured dry sausages., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ramon Cava Lopez reports financial support and administrative support were provided by FEDER and JUNTA DE EXTREMADURA., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study was conducted to evaluate the effects of nitrite-rich (celery powder; CP) and pigment-rich (purple sweet potato powder, PSP; red beet powder, RB) substitutes for synthetic sodium nitrite (CON) on the quality characteristics of emulsion-type pork sausages during four weeks of cold storage. Natural substitutes decreased the pH, lightness, and textural properties of pork sausages during storage (P < 0.05). Pigment-rich substitutes showed a decreased antioxidant effect after two weeks of storage when compared to the nitrite-rich groups (CON and CP; P < 0.05). Pigment-rich substitutes also accelerated the discoloration of pork sausages by increasing yellowness (RB and PSP) and decreasing redness (PSP) during storage (P < 0.05). However, these two pigment-rich substitutes showed different trends in redness (higher in PSP and lower in RB) and yellowness (higher in RB and lower in PSP) when compared to the nitrite-rich groups (CON and CP). Different types (nitrite-rich and pigment-rich) of natural substitutes for sodium nitrite had different effects on the quality characteristics of emulsion-type pork sausages throughout the four weeks of storage evaluated in this study., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

We report here a robust and practical strategy for chemical protein synthesis using an o -nitrobenzyl group as a temporary protective group for an N-terminal cysteine residue of intermediate hydrazide fragments. By reinvestigating the photoremoval of an o -nitrobenzyl group, we establish a robust and reliable strategy for its quantitative photodeprotection. The o -nitrobenzyl group is completely stable to oxidative NaNO 2 treatment and has been applied to the convergent chemical synthesis of programmed death ligand 1 fragment, providing a practical avenue for hydrazide-based native chemical ligation.

The objective of this study was to investigate the effects of different concentrations of sodium nitrite on the quality and protein oxidation of salted meat during 21 days of curing. The salted meat was treated with sodium nitrite at 50, 100, and 150 mg/kg for curing, and salted meat without sodium nitrite was used as a control. The results showed that in salted meat added with sodium nitrite, the carbonyl group, disulfide bond, dityrosine, surface hydrophobicity, and the transformation rate from α-helix to β-sheet were all significantly reduced, whereas the sulfhydryl group content of myofibrillar proteins was significantly increased compared to the control. Meanwhile, the total volatile basic nitrogen and aerobic plate content were significantly decreased, while both the pH and a* value were significantly increased with an increase in nitrite concentration compared to the control group. Importantly, this phenomenon was also observed in salted meat treated with low doses of sodium nitrite (50 mg/kg). In conclusion, the quality of salted beef can be improved by adding low-dose sodium nitrite to inhibit protein oxidation during the curing process. PRACTICAL APPLICATION: A low dose of sodium nitrite inhibited the rate of α-helix to β-sheet transformation of myofibrillar proteins in salted meat, reducing the exposure of hydrophobic groups and decreasing the production of protein oxidation products and TVB-N to improve the quality of salted meat. These results provided a theoretical basis and technical guidance for the application of low-dose sodium nitrite in meat processing enterprises., (© 2022 Institute of Food Technologists®.)

Methemoglobin (metHb) arises from the oxidation of ferrous hemoglobin (HbFe2+, Hb) to ferric hemoglobin (HbFe3+, metHb), which is unable to bind gaseous ligands such as oxygen (O2) and carbon monoxide (CO), and binds to nitric oxide (NO) significantly slower compared to Hb. Therefore, metHb does not elicit vasoconstriction and systemic hypertension in vivo due to its extremely slow NO scavenging rate in comparison to cell-free Hb, but will induce oxidative tissue injury, demonstrating the potential of using metHb as a control material when studying the toxicity of cell-free Hb. Hence, the goal of this work was to develop a novel manufacturing strategy for production of metHb that is amenable to scale-up. In this study, small scale (e.g. 1 mL reaction volume) screening experiments were initially conducted to determine the optimal molar ratio of Hb to the oxidization agents hydrogen peroxide (H2O2) or sodium nitrite (NaNO2) to achieve the highest conversion of Hb into metHb. A spectral deconvolution program was employed to determine the molar fraction of various species (hemichrome, metHb, oxyHb, metHb-[Formula: see text], and NaNO2) in solution during the oxidation reaction. From this analysis, either a 1:1 or 1:5 molar ratio was identified as optimal molar ratios of Hb:NaNO2 (heme basis) that yielded the highest conversion of Hb into metHb with negligible amounts of side products. Hence in order to reduce the reaction time, a 1:5 molar ratio was chosen for large scale (i.e. 1.5 L reaction volume) synthesis of bovine metHb (metbHb) and human metHb (methHb). The biophysical properties of metHb were then characterized to elucidate the potential of using the synthesized metHb as a non-O2 carrying control material. The haptoglobin binding kinetics of metHb were found to be similar to Hb. Additionally, the synthesized metHb was stable in phosphate buffered saline (PBS, 50 mM, pH 7.4) at 4°C for approximately one week, indicating the high stability of the material., Competing Interests: The authors have declared that no competing interests exist.

Food preservatives such as NaNO 2 , which are widely used in human food products, undoubtedly affect, to some extent, human organs and health. For this reason, there is a need to reduce the hazards of these chemical preservatives, by replacing them with safe natural bio-preservatives, or adding them to synthetic ones, which provides synergistic and additive effects. The Citrus genus provides a rich source of such bio-preservatives, in addition to the availability of the genus and the low price of citrus fruit crops. In this study, we identify the most abundant flavonoids in citrus fruits (hesperidin) from the polar extract of mandarin peels (agro-waste) by using spectroscopic techniques, as well as limonene from the non-polar portion using GC techniques. Then, we explore the synergistic and additive effects of hesperidin from total mandarin extract with widely used NaNO 2 to create a chemical preservative in food products. The results are promising and show a significant synergistic and additive activity. The combination of mandarin peel extract with NaNO 2 had synergistic antibacterial activity against B . cereus , Staph . aureus , E . coli , and P . aeruginosa , while hesperidin showed a synergistic effect against B . cereus and P . aeruginosa and an additive effect against Staph . aureus and E . coli . These results refer to the ability of reducing the concentration of NaNO 2 and replacing it with a safe natural bio-preservative such as hesperidin from total mandarin extract. Moreover, this led to gaining benefits from their biological and nutritive values.

The present study evaluated the use of the probiotic Lacticaseibacillus paracasei DTA-83 as a nitrite-reducing agent to produce potentially probiotic or postbiotic pre-converted nitrite from celery. The results obtained were compared to those achieved by direct addition of sodium nitrite for the typical reddish color formation in cooked pork sausages and the inhibitory potential against the growth of target microorganisms, including the clostridia group. Regarding the sausages color, similar findings were observed when comparing the use of pre-converted nitrite from celery produced by L. paracasei DTA-83 and the direct addition of sodium nitrite. Additionally, it presented an inhibitory effect against Salmonella spp., which was not observed with the direct addition of nitrite, revealing a potential strategy to control salmonellosis in the matrix. However, a non-equivalent preservative effect against Clostridium perfringens (INCQS 215) was determined. The results highlight a promising alternative to produce probiotic or postbiotic meat ingredients; however, further studies should be conducted to investigate doses that achieve microbial control., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The effect of supercritical fluid extract of tomato pomace (TP) and essential oil of organic peppermint (PM) on pH, color, residual nitrite content, lipid oxidation (TBARS value) and total plate count (TPC) of cooked pork sausages produced with 50 mg/kg of sodium nitrite was investigated. Five batches were produced: T1: 100 mg/kg of sodium nitrite; T2: 50 mg of sodium nitrite; T3: 50 mg of sodium nitrite and 0.150 µL/g TP; T4: 50 mg of sodium nitrite, 0.075 µL/g TP and 0.075 µL/g PM; T5: 50 mg of sodium nitrite and 0.150 µL/g PM. The lowest residual nitrite content and TBARS value were observed in treatment T4. The inclusion of TP increased redness of cooked pork sausages. TPC was the lowest in treatment T5. The results of this study showed that the addition of TP and PM enhanced quality of cooked sausages produced with reduced level of sodium nitrite., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

R-2-(4-Hydroxyphenoxy)propionic acid (R-HPPA) is a pivotal intermediate for the synthesis of aryloxyphenoxypropionate (APP) herbicide. To rapidly screen microbial isolates with the capacity of hydroxylating R-2-phenoxypropionic acid to R-HPPA from various environmental samples, a convenient and safe 96-well microplate assay method with sodium nitrite (NaNO 2 ) as chromogenic reagent was proposed and optimized. The optimum assay conditions were as follows: the detection wavelength was 420 nm, the concentration of NaNO 2 solution was 6.0 g/L, color reaction temperature was 60 °C, the pH of the NaNO 2 solution was 2.4, and the reaction time was 40 min. With the aid of this method, screening for microorganisms with C-4-specific hydroxylation activity of R-PPA was conducted. As a result, 23 strains among 3744 single colonies isolated from various samples exhibited the hydroxylation activity. Among these strains, the highest bioconversion rate was achieved by Penicillium oxalicum A5 and Aspergillus versicolor A12, respectively. After 72-h cultivation in shake flask, their conversion rates of R-HPPA from 10 g/L R-PPA reached 21.18% and 40.24%, respectively. The established method was effective in rapid screening of microbes capable of biosynthesizing R-HPPA through hydroxylation of R-PPA, and the obtained two fungi species could be potentially used for R-HPPA production.

Radish powder and chitosan were evaluated in fermented cooked sausages to replace sodium nitrite. Treatments of fermented cooked sausages were prepared and evaluated during the ripening process and storage time: control (150 ppm nitrite, CONT); and two levels of chitosan (0.25%, CHI1) and (0.5%, CHI2) with radish powder (0.5%) and without the addition of nitrite. During storage, pieces sliced or not were also evaluated regarding the physicochemical, microbiological and sensory. Pure chitosan was evaluated regarding determination of MIC and MBC to Enterobacter aerogenes, Listeria innocua and Lactobacillus rhamnosus, and antioxidant capacity. The addition of chitosan increased a w and decreased weight loss of fermented sausages along processing, but pH was not affected during ripening. Except for aroma, sensory attributes were affected by the addition of chitosan. The addition of 0.5% radish powder and 0.25% chitosan showed promising results to development of nitrite-free fermented cooked sausages, mainly regarding their physicochemical and microbiological stability., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest with the information shared in this article., (Copyright © 2020. Published by Elsevier Ltd.)

We evaluated the effects of a chemical additive on the microbial communities, fermentation profile, and aerobic stability of whole-plant corn silage with or without air stress during storage. Whole-plant corn was either untreated or treated with a chemical additive containing sodium benzoate, potassium sorbate, and sodium nitrite at 2 or 3 liters/t of fresh forage weight. Ten individually treated and replicated silos (7.5 liters) were made for each treatment. Half of the silos remained sealed throughout a 63-d storage period, and the other half was subjected to air stress for 2 h/wk. The composition of the bacterial and fungal communities of fresh forage and silages untreated or treated with 2 liters/t of fresh forage weight was analyzed by Illumina Miseq sequencing. Treated silage had greater (P < 0.05) aerobic stability than untreated, even when subjected to air stress during storage, but the numbers of yeasts culturable on selective agar were not affected. However, the additive reduced the relative abundance (RA) of the lactating-assimilating yeast Candida tropicalis (P < 0.01). In air-stressed silages, untreated silage had a greater (P < 0.05) RA of Pichia kudriavzevii (also a lactate assimilator) than treated silage, whereas treated silage was dominated by Candida humilis, which is usually unable to assimilate lactate or assimilates it slowly. The additive improved the aerobic stability by specifically preventing the dominance of yeast species that can consume lactate and initiate aerobic spoilage. To the best of our knowledge, this is the first work that identifies the specific action of this additive on shifting the microbial communities in corn silage., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The utility of plasma-treated winter mushroom powder (PWMP) as an alternative to synthetic nitrite and phosphate in ground ham was evaluated. Treatment of atmospheric non-thermal plasma generated nitrite in winter mushroom homogenate, and PWMP contained 4.87 g/kg of nitrite. Canned ground ham samples without nitrite and phosphate (NC), with sodium nitrite and sodium pyrophosphate (PC), and with PWMP (PWM) were manufactured, sterilized (F 0  > 4), and stored under accelerated conditions (40 °C) for 30 days. The nitrosyl-hemochrome content and color of PWM were similar to those of PC, without differences in the thiobarbituric acid reactive substances (TBARS) value. The proportion of jelly and melted fat exuded from ground hams in PWM exceeded that in PC but was lower than that in NC (P < .05). Therefore, PWMP can successfully replace synthetic nitrite, but it is an insufficient substitute for phosphate in ground ham., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nitrite is widely used in meat products as a multifunctional additive, combining flavour and colour properties with antioxidant and antimicrobial effects. However, nitrite may form reaction products (i.e., nitrosamine) that are potentially carcinogenic to humans. The meat industry, in response to consumers' demands for nitrite-free products, is seeking natural alternatives to nitrite, such as plant-based extracts. Three types of dry-fermented sausages were manufactured: NIT, containing 30 ppm of sodium nitrite; GSE, containing grape seed extract and olive pomace hydroxytyrosol; and CHE, containing chestnut extract and olive pomace hydroxytyrosol. Next-generation sequencing (NGS) was used to analyse microbial consortia, which were correlated with physical and chemical parameters. The prokaryotic community composition was similar among treatments, with a high relative abundance of Staphylococcus xylosus and Lactobacillus sakei, collectively accounting for 87% of the total community. However, significant differences were observed in both operational taxonomic unit (OTU) presence/absence and relative abundance. Ten genera varied in abundance between treatments. The increase in Lactobacillaceae in CHE may explain the reduced pH levels detected in these samples. In conclusion, NGS analysis showed that the prokaryotic community composition was similar in GSE and NIT, while CHE varied in both the composition and relative abundance of different taxa., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Organophosphate triesters (OPEs) have caused great concern as a class of emerging environmental contaminants due to their widespread use and their toxicity to organisms. However, the phototransformation behavior of OPE is still not fully understood, which is important for understanding their environmental fate. In the present study, the photodegradation of tricresyl phosphate (TCP), one of the most widely detected OPEs in aqueous environments, was investigated including the direct photolysis and in the presence of several natural water factors, NO 2 - , Fe 3+ and humic acid. The degradation process followed the pseudo-first-order kinetics, with rate constant increasing slightly with increasing initial TCP concentration. The presence of NO 2 - and Fe 3+ was observed to promote the photochemical loss of TCP, while humic acid played a negative role on TCP transformation. Electron spin resonance (EPR) analysis showed that carbon-centered radical was produced in the photolysis process of TCP, and hydroxyl radical contributed to the promotion of rate constant for Fe 3+ and NO 2 - . Four photolysis products were tentatively identified by HPLC-LTQ-Orbitrap MS analysis, and the possible degradation pathways of TCP were proposed. These findings provide a meaningful reference for the fate and transformation of OPEs in natural water., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Processed meats are classified by the International Agency for Research on Cancer (IARC) as carcinogenic to humans. However, information on the responsible agents and the influence of industrial processing on the increased risk of cancer is still lacking. This study aimed to use cultures of Lactobacillus delbrueckii subsp. bulgaricus LB-UFSC 01 to biodegrade harmful C-nitrous, N-nitro, and C-nitro compounds in processed meat matrix. Firstly, positive results for ethylnitrolic acid (ENA) (>5.00 μg kg -1 ) and 2-methyl-1,4-dinitro-pyrrole (DNMP) (>12.0 μg kg -1 ) were obtained in mortadellas produced under different experimental conditions employing preservatives and antioxidants. Mortadellas containing nitrite and sorbate in the ratio of 8:1 (w/w) yielded the highest concentrations of mutagens. However, the treatment with the LB-UFSC 01 culture was able to modulate the harmful compounds in the mortadella samples. Several analytical methods employing liquid chromatography coupled to mass spectrometry and statistical models were employed to identify the metabolites and reaction routes during microbial biotransformation. For the first time, relevant information regarding the formation and degradation of ENA and DNMP in a processed meat model simulating real conditions was presented., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The effect of coriander essential oil (CEO) at concentrations of 0.075-0.150 μL/g on pH, color, lipid oxidation (TBARS), residual nitrite concentration and microbial growth of cooked pork sausages produced with different levels of sodium nitrite (0, 50 and 100 mg/kg) was investigated. Artificial neural networks modeling and the multi-response optimization were used to determine the optimal combinations of process parameters and storage time. Reduced concentration of sodium nitrite (60 mg/kg) in combination with 0.12 μL/g of CEO resulted in satisfying redness (a* approx. 11.1) and improved oxidative (TBARS approx. 0.12 mg MDA/kg) and microbial stability (total plate count - TPC approx. 2.50 Log CFU/g) of cooked pork sausages during refrigerated storage. Therefore, the results of this paper revealed significant antioxidative and antimicrobial activity of CEO, and consequently its high potential of utilization in processing of cooked pork sausages with enhanced quality and shelf-life., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study was conducted to improve the quality characteristics of cured meat with natural nitrite. Control and treatment were conducted as follows: nitrite free, marinated with sodium nitrite and ascorbic acid, marinated with only fermented spinach (FS), and marinated with FS adding ascorbic acid, malic acid, citric acid, and tartaric acid. Treatments were pickled with regulated brine (8% salt and 0.08% nitrite). Cured meat with FS adding ascorbic acid, malic acid, and citric acid had higher redness values than sodium nitrite with ascorbic acid on cooked meat. There was a positive effect on lipid oxidation except for citric acid. Protein degradation appeared more in malic acid and tartaric acid treatment than others. Residual nitrite level was lower when adding organic acids. Among various organic acid, ascorbic acid had the highest efficient on quality properties of cured meat. Thus, ascorbic acid was a proper ingredient when curing meat product., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The objective of this study was to determine nitrite scavenging activity of fermented soy sauce in vitro and in a pork sausage model. In vitro nitrite scavenging activity (pH 6.0) of fermented soy sauce (10 mg/mL) was 13.2%. Within selected concentrations (10-100 mg/mL), in vitro nitrite scavenging activity and total phenol content of fermented soy sauce were highly dose-dependent (P < 0.001). In pork sausage model containing 120 mg/kg of NaNO 2 , fermented soy sauce resulted in significantly lower residual nitrite content (35.28 mg/kg) compared to only NaNO 2 addition (40.12 mg/kg) at initial storage. During 4 weeks of cold storage, however, fermented soy sauce showed little effect on the residual nitrite content. This study indicates that fermented soy sauce could initially contribute to reduce residual nitrite content, and the nitrite scavenging impact of fermented soy sauce was less effective in the pork sausage model than in vitro evaluation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

S-nitrosocaptopril (CapNO) possesses dual capacities of both Captopril and an NO donor with enhanced efficacy and reduced side effects. CapNO crystals are difficult to make due to its unstable S-NO bond. Here, we report a novel stable S-nitrosocaptopril monohydrate (CapNO·H 2 O) that is stabilized by intermolecular five-membered structure, where one H of H 2 O forms a hydrogen bond with O - of the stable resonance zwitterion Cap-S + =N-O - , and the O in H 2 O forms the dipole-dipole interaction with S + through two unpaired electrons. With the chelation and common ion effect, we synthesized and characterized CapNO·H 2 O that is stable at 4 °C for 180 days and thereafter without significant degradation. Compared to Captopril, CapNO showed direct vasorelaxation and beneficial effect on PAH rats, and could be self-assembled in rat stomach when Captopril and NaNO 2 were given separately. This novel CapNO·H 2 O with low entropy paves an avenue for its clinical trials and commercialization., (Copyright © 2018 Elsevier Inc. All rights reserved.)

This study investigated the effects of different curing agents on the physicochemical properties, microbiological properties and sensory evaluation of sausages formulated with and without nitrite over 4 weeks of cold storage. Seven batches were prepared: control, sodium nitrite 0.01%; celery powder 0.8% (T1); fruit extract powder 0.6% (T2); purple sweet potato powder 0.45% (T3); fruit and vegetable extract powders 0.5% (T4); gardenia red 0.04% (T5); paprika and blueberry powder 0.07% (T6). T1 produced significantly lighter, redder and yellower sausages compared to control and had a higher color intensity (C*) and hue (h). The residual nitrite ion concentration was the highest in the control and declined most rapidly in control, T1, and T2 during storage. The pH, thiobarbituric acid reactive substances (TBARS), volatile basic nitrogen (VBN) content, and total microbe counts were the same for T1 and the control. T1 received comparable sensory attributes as the control. These results suggest that celery powder effectively protects sausages from quality deterioration during storage., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Since cultivations of Arthrospira platensis have a high water demand, it is necessary to develop treatment methods for reusing the exhausted medium that may prevent environmental problems and obtaining useful biomass. The exhausted Schlösser medium obtained from A. platensis batch cultivation in bench-scale mini-tanks was treated by varying concentrations of different coagulants, ferric chloride (6, 10, and 14 mg L -1 ) or ferric sulfate (15, 25, and 35 mg L -1 ) and powdered activated carbon (PAC, 30 and 50 mg L -1 ). Such treated effluent was restored with NaNO 3 and reused in new cultivations of A. platensis performed in Erlenmeyer flasks. Reusing media through the cultivation of A. platensis showed satisfactory results, particularly in the medium treated with ferric chloride and PAC. The maximum cell concentration obtained in the flasks was 1093 mg L -1 , which corresponded to the medium treated with ferric chloride (6 mg L -1 ) and PAC (30 mg L -1 ). This cellular growth was higher than in the medium treated with ferric sulfate and PAC, in which values of maximum cell concentration did not exceed 796 mg L -1 . The cultures in the media after treatment did not modify the biomass composition. Thus, combined coagulation/adsorption processes, commonly used in water treatment processes, can be efficient and viable for treating exhausted medium of A. platensis, allowing the production of such biomass with the reduction of production cost and saving water.

We report a simple and promising synthetic method to oxidize peptide hydrazides containing N-terminal thiazolidine as a protected cysteine. This yields the corresponding thioester via a peptide azide without decomposition of the thiazolidine ring. The newly developed protocol was validated by the synthesis of the bioactive peptide LacZα.

5,10,15,20-Tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride (FeTPPS) is a water-soluble heme analog, which has been used as a scavenger of peroxynitrite in many studies. Similar to heme, it may also possess pseudo-peroxidase activity that could cause protein tyrosine nitration through the peroxidase-H 2 O 2 -NO 2 - pathway. In this paper, we used western blotting and spectrophotometry analysis to study the capability of FeTPPS in catalyzing protein tyrosine nitration. Furthermore, the capability of FeTPPS in catalyzing protein nitration in tissue homogenate and cultured cells was also investigated. Our results showed that FeTPPS induced bovine serum albumin (BSA) nitration in the presence of H 2 O 2 and NaNO 2 , and the reaction was dose-, time- and pH-dependent. In acidic condition, more protein was nitrated by FeTPPS than heme, which corresponded to their peroxidase activities. Meanwhile, our results also confirmed the catalytic effect of FeTPPS on protein tyrosine nitration in rat brain homogenate and human hepatocellular carcinoma (HepG2) cells. At the end of this study, we used liquid chromatography (LC)-tandem mass spectrometry (MS/MS) to investigate differences of site selectivity between heme and FeTPPS catalyzed protein tyrosine nitration. The result indicated that FeTPPS tended to catalyze tyrosine residues locating in more hydrophilic sites, whereas heme was more likely to induce nitration of tyrosine residues locating in relatively hydrophobic environment. Taken together, this is the first report that FeTPPS is an effective and convenient nitration catalyzer in vitro, and this study confirms that the hydrophilicity of the nitrating agents would play an important role in nitration site selection., (Copyright © 2018. Published by Elsevier Inc.)

The RNA-world hypothesis assumes that life on Earth started with small RNA molecules that catalyzed their own formation. Vital to this hypothesis is the need for prebiotic routes towards RNA. Contemporary RNA, however, is not only constructed from the four canonical nucleobases (A, C, G, and U), it also contains many chemically modified (noncanonical) bases. A still open question is whether these noncanonical bases were formed in parallel to the canonical bases (chemical origin) or later, when life demanded higher functional diversity (biological origin). Here we show that isocyanates in combination with sodium nitrite establish methylating and carbamoylating reactivity compatible with early Earth conditions. These reactions lead to the formation of methylated and amino acid modified nucleosides that are still extant. Our data provide a plausible scenario for the chemical origin of certain noncanonical bases, which suggests that they are fossils of an early Earth., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aim of this work is to introduce, model, and optimize a new non-acid-catalyzed system for a direct N[Formula: see text]N-C bond formation. By reacting naphthols or phenol with anilines in the presence of the sodium nitrite as nitrosonium ([Formula: see text] source and triethylammonium acetate (TEAA), a N[Formula: see text]N-C group can be formed in non-acid media. Modeling and optimization of the reaction conditions were investigated by response surface method. Sodium nitrite, TEAA, and water were chosen as variables, and reaction yield was also monitored. Analysis of variance indicates that a second-order polynomial model with F value of 35.7, a P value of 0.0001, and regression coefficient of 0.93 is able to predict the response. Based on the model, the optimum process conditions were introduced as 2.2 mmol sodium nitrite, 2.2 mL of TEAA, and 0.5 mL [Formula: see text] at room temperature. A quadratic (second-order) polynomial model, by analysis of variance, was able to predict the response for a direct N=N-C group formation. Predicted response values were in good agreement with the experimental values. Electrochemistry studies were done to introduce new Michael acceptor moieties. Broad scope, high yields, short reaction time, and mild conditions are some advantages of the presented method.

A spectrophotometric method based on diazotization of aniline with triclosan has been developed for the determination of triclosan in water samples. The diazotization process involves two steps: (1) reaction of aniline with sodium nitrite in an acidic medium to form diazonium ion and (2) reaction of diazonium ion with triclosan to form a yellowish-orange azo compound in an alkaline medium. The resulting yellowish-orange product has a maximum absorption at 352 nm which allows the determination of triclosan in aqueous solution in the linear concentration range of 0.1-3.0 μM with R 2 = 0.998. The concentration of hydrochloric acid, sodium nitrite, and aniline was optimized for diazotization reaction to achieve good spectrophotometric determination of triclosan. The optimization of experimental conditions for spectrophotometric determination of triclosan in terms of concentration of sodium nitrite, hydrogen chloride and aniline was also carried out by using Box-Behnken design of response surface methodology and results obtained were in agreement with the experimentally optimized values. The proposed method was then successfully applied for analyses of triclosan content in water samples.

Accelerated stability studies of pharmaceutical products are commonly conducted at various combinations of temperature and relative humidity (RH). The RH of the sample environment can be controlled to set points using humidity-controlled stability chambers or via storage of the sample in a closed container in the presence of a saturated aqueous salt solution. Herein we report an unexpected N-nitrosation reaction that occurs upon storage of carvedilol- or propranolol-excipient blends in a stability chamber in the presence of saturated sodium nitrite (NaNO 2 ) solution to control relative humidity (∼60% RH). In both cases, the major products were identified as the corresponding N-nitroso derivatives of the secondary amine drugs based on mass spectrometry, UV-vis and retention time. These degradation products were not observed upon storage of the samples at the same temperature and humidity but in the presence of saturated potassium iodide (KI) solution (∼60% RH) for humidity control. The levels of the N-nitrosamine derivatives varied with the pH of various NaNO 2 batches. The presence of volatile NOx species in the headspace of a container containing saturated NaNO 2 solution was confirmed via the Griess assay. The process for formation of the N-nitrosamine derivatives is proposed to involve volatilization of nitric oxide (NO) from aqueous nitrite solution into the headspace of the container followed by diffusion into the solid drug-excipient blend and subsequent reaction of NOx with the secondary amine., (Copyright © 2017 Elsevier B.V. All rights reserved.)

N-nitrosodiethanolamine (NDELA), a type of nitrosamine, is a possible human carcinogen that may form in cosmetic products. The aim of this study was to examine the formation and inhibition of NDELA through chemical reactions of secondary amines including mono-ethanolamine, di-ethanolamine (DEA), and tri-ethanolamine (TEA), and sodium nitrite (SN) under varying conditions such as pH, temperature, and fluorescent, ultraviolet (UV), and visual light (VIS) using liquid chromatography-mass spectroscopy. In a mixture of TEA and SN under acidic conditions pH 2, residual NDELA concentrations rose significantly under various storage conditions in the following order: 50°C > 40°C > UV (2 W/m 2 ) > VIS (4000 lux) > fluorescent light > 25°C > 10°C. In a mixture of DEA and SN under the same acidic pH 2 conditions, NDELA formation was significantly elevated in the following order: UV (2 W/m 2 ) > VIS (4000 lux) > 50°C > 40°C > fluorescent light > 25°C > 10°C. Inhibition of NDELA formation by d-mannitol, vitamin C (Vit C), or vitamin E (Vit E) was determined under varying conditions of pH, temperature, and fluorescent, UV, and VIS. At high concentrations of 100 or 1000 µg/ml, Vit E significantly decreased residual NDELA compared with control levels under acidic pH 2, but not under basic pH 6. Among various antioxidants, Vit E reacted more effectively with many nitrosating agents such as nitrate and nitrite found in cosmetic products. Therefore, to reduce NDELA, it is recommended that cosmetics be stored under cool/amber conditions and that Vit E or Vit C inhibitors of nitrosation be optimally added to cosmetic formulations at concentrations between 100 and 1000 µg/ml.

Dimethyl trisulfide (DMTS) is a promising antidotal candidate for cyanide intoxication. DMTS acts as a sulfur donor in the conversion of cyanide to the less-toxic thiocyanate. The alternate reaction pathways of DMTS in the blood are not well understood. We report changes in the hemoglobin absorption spectrum upon reaction with DMTS. These changes closely match those induced by the known methemoglobin former, sodium nitrite. The kinetics of methemoglobin formation with DMTS is slower than with sodium nitrite. These results support the hypothesis that a potentially significant side-reaction of the therapeutically administered DMTS is the oxidization of hemoglobin to methemoglobin.

Increasing concern about chemical additives in processed meat has led to an increased market of uncured and alternatively cured meat products. However, the use of vegetable extracts or the exclusion of curing salt may increase the risk of greater bacterial growth and alteration of several physicochemical parameters. Therefore, in this study mortadella-type sausages, manufactured with 1.07 (V3), 2.14 (V4) and 4.29 (V5) g parsley extract powder/kg sausage meat were produced. These sausage variants were compared to an uncured (V2) and a traditionally nitrite-cured control (V1). A significantly lower Listeria monocytogenes growth was observed for V5 compared to all other variants during the storage time of 28days (P<0.05). Compared to V1, V5 presented a residual nitrite content reduced by 40% and similar a* values until day 21. Concerning texture parameters, L* and a w values, no differences between the variants were detected. Sensory analysis showed that overall acceptance of V4 and V5 was comparable with V1., (Copyright © 2017. Published by Elsevier Ltd.)

Ferrous oxyhemoglobin (HbO 2 ) in red blood cells (RBCs) invariably and slowly autoxidizes to form ferric methemoglobin (metHb). However, the level of metHb is always maintained below 0.5% by intracellular metHb reduction of enzymatic systems with coenzymes, such as reduced nicotinamide adenine dinucleotide (NADH), and by superoxide dismutase (SOD) and catalase (CAT), which eliminate reactive oxygen species. Unquestionably, NADH cannot reduce metHb without the corresponding enzymatic system. Our study, however, demonstrated that a high concentration of NADH (100-fold of normal level, equimolar to HbO 2 ) retarded autoxidation of HbO 2 in a highly purified Hb solution with no enzymatic system. Furthermore, an inhibitory effect of NADH on metHb formation was observed with additions of oxidants such as H 2 O 2 , NO, and NaNO 2 . Our mechanism assessment elucidated extremely high pseudo-CAT and pseudo-SOD activities of NADH with coexistence of HbO 2 , and reactivity of NADH with NO. We prepared a model of RBCs (Hb-vesicles, Hb-V) encapsulating purified HbO 2 solution and NADH, but no enzymatic system within liposome. We confirmed the inhibitory effect of NADH on both autoxidation and oxidant-induced metHb formation. In addition, an intravenous administration of these Hb-Vs to rats caused significant retardation of metHb formation by approximately 50% compared to the case without NADH coencapsulation. Based on these results, we elucidated a new role of NADH, that is, antioxidative effect via interaction with Hb, in addition to its classical role as a coenzyme.

Solution plasma (SP) treatment in combination with oxidizing agents, i.e., hydrogen peroxide (H 2 O 2 ), potassium persulfate (K 2 S 2 O 8 ) and sodium nitrite (NaNO 2 ) were adopted to chitosan degradation in order to achieve fast degradation rate, low chemicals used and high yield of low-molecular-weight chitosan and chitooligosaccharide (COS). Among the studied oxidizing agents, H 2 O 2 was found to be the best choice in terms of appreciable molecular weight reduction without major change in chemical structure of the degraded products of chitosan. By the combination with SP treatment, dilute solution of H 2 O 2 (4-60mM) was required for effective degradation of chitosan. The combination of SP treatment and dilute solution of H 2 O 2 (60mM) resulted in the great reduction of molecular weight of chitosan and water-soluble chitosan was obtained as a major product. The resulting water-soluble chitosan was precipitated to obtain COS. An inhibitory effect against cervical cancer cell line (HeLa cells) of COS was also examined., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Peroxynitrite and heme peroxidases (or heme)-H 2 O 2 -NaNO 2 system are the two common ways to cause protein tyrosine nitration in vitro, but the effects of antioxidants on reducing these two pathways-induced protein nitration and oxidation are controversial. Both nitrating systems can dose-dependently induce triosephosphate isomerase (TIM) nitration, however, heme-H 2 O 2 -NaNO 2 was less destructive to protein secondary structures and led to more nitrated tyrosine residue than 3-morpholinosydnonimine hydrochloride (SIN-1, a peroxynitrite donor). Both of desferrioxamine and catechin could inhibit TIM nitration induced by heme-H 2 O 2 -NaNO 2 and SIN-1 and protein oxidation induced by SIN-1, but promoted heme-H 2 O 2 -NaNO 2 -induced protein oxidation. Moreover, the antagonism of natural phenolic compounds on SIN-1-induced tyrosine nitration was consistent with their radical scavenging ability, but no similar consensus was found in heme-H 2 O 2 -NaNO 2 -induced nitration. Our results indicated that peroxynitrite and heme-H 2 O 2 -NaNO 2 -induced protein nitration was different, and the later one could be a better model for anti-nitration compounds screening., (© 2017 Wiley Periodicals, Inc.)

Staphylococcus aureus is frequently isolated in the setting of infections of indwelling medical devices, which are mediated by the microbe's ability to form biofilms on a variety of surfaces. Biofilm-embedded bacteria are more resistant to antimicrobial agents than their planktonic counterparts and often cause chronic infections and sepsis, particularly in patients with prolonged hospitalizations. In this study, we demonstrate that sustained nitric oxide-releasing nanoparticles (NO-np) interfere with S. aureus adhesion and prevent biofilm formation on a rat central venous catheter (CVC) model of infection. Confocal and scanning electron microscopy showed that NO-np-treated staphylococcal biofilms displayed considerably reduced thicknesses and bacterial numbers compared to those of control biofilms in vitro and in vivo, respectively. Although both phenotypes, planktonic and biofilm-associated staphylococci, of multiple clinical strains were susceptible to NO-np, bacteria within biofilms were more resistant to killing than their planktonic counterparts. Furthermore, chitosan, a biopolymer found in the exoskeleton of crustaceans and structurally integrated into the nanoparticles, seems to add considerable antimicrobial activity to the technology. Our findings suggest promising development and translational potential of NO-np for use as a prophylactic or therapeutic against bacterial biofilms on CVCs and other medical devices., (Copyright © 2016 American Society for Microbiology.)

The effects of sodium nitrite and ascorbate on lipid and protein oxidation were studied during the ripening process of dry fermented sausages. Samples were taken at day 0, 2, 8, 14, 21 and 28 of ripening to assess lipid (malondialdehyde) and protein (carbonyls and sulfhydryl groups) oxidation. Sodium ascorbate and nitrite were separately able to reduce the formation of malondialdehyde. Their combined addition resulted in higher amounts of carbonyl compounds compared to their separate addition or the treatment without any of both compounds. Moreover, sodium nitrite limited the formation of γ-glutamic semialdehyde whereas sodium ascorbate showed a pro-oxidant effect. A loss of thiol groups was observed during ripening, which was not affected by the use of sodium ascorbate nor sodium nitrite. In conclusion, sodium nitrite and ascorbate affected protein and lipid oxidation in different manners. The possible pro-oxidant effect of their combined addition on carbonyl formation might influence the technological and sensory properties of these products., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Biomimetic coatings to increase endothelialization of blood-contacting materials in biomedical devices are promising to improve the biocompatibility of these devices. Although a stable extracellular matrix protein coating on a biomaterial's surface is a prerequisite for endothelial cell attachment, it also stimulates platelet adhesion. Therefore, antithrombotic additives, such as nitric oxide donors, to a stable protein coating might lead to successful endothelialization of a material's surface. We aimed to test whether immobilized bioactive nitrite and acidified nitrite-generating sodium nitrite-collagen conjugate on silicone tubes enhances endothelialization by increasing the number of endothelial cells as well as growth hormone production and by decreasing platelet adhesion. Stable collagen immobilization on acrylic acid-grafted silicone tubes decreased the water contact angle from 102° to 56°. Initial 25 µM sodium nitrite in conjugate resulted in maximal growth hormone production (2.5-fold increase) and endothelial cell number (1.8-fold increase) after 2 days. A 95% confluent endothelial cell monolayer on sodium nitrite-collagen conjugate coating was obtained after 6 days. Maximum (2.7-fold) inhibition of platelet adhesion was reached with initial 500 µM sodium nitrite in conjugate. Our data showing that sodium nitrite-collagen conjugate coating with 25-50 µM sodium nitrite on silicone tubes increases the number of endothelial cells attached and inhibits platelet adhesion suggest that this coating is highly promising for use in blood-contacting parts of biomedical devices. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1311-1321, 2016., (© 2015 Wiley Periodicals, Inc.)

A method for aerobic oxidation of aldehydes to carboxylic acids has been developed using organic nitroxyl and NOx cocatalysts. KetoABNO (9-azabicyclo[3.3.1]nonan-3-one N-oxyl) and NaNO2 were identified as the optimal nitroxyl and NOx sources, respectively. The mildness of the reaction conditions enables sequential asymmetric hydroformylation of alkenes/aerobic aldehyde oxidation to access α-chiral carboxylic acids without racemization. The scope, utility, and limitations of the oxidation method are further evaluated with a series of achiral aldehydes bearing diverse functional groups.

Biocompatibility of artificial lungs can be improved by endothelialization of hollow fibers. Bioavailability of growth-inducing and anti-thrombotic agents on the hollow fiber-blood interface inhibits thrombosis. We investigated if nanoliposomal growth-inducing growth hormone (nGH) and anti-thrombotic sodium nitrite (nNitrite) incorporation into collagen-coating on silicone hollow fibers improves blood biocompatibility by increasing endothelial cell growth and nitrite bioavailability under flow. Nitrite production rate was assessed under varying flow conditions. Finite element (FE) modeling was used to simulate nitrite transport within the parallel-plate flow chamber, and nitrite bioavailability on the fiber-blood interface at 1-30 dyn/cm(2) shear stress. Endothelial cell number on fibers coated with nNitrite-nGH-collagen conjugate was 1.5-fold higher than on collagen-coated fibers. For collagen-coated fibers, nitrite production reached a maximum at 18 dyn/cm(2) shear stress. When fibers were coated with nNitrite-nGH-collagen conjugate, nitrite production increased continuously by increasing shear stress. FE modeling revealed that nitrite concentrations at the fiber-blood interface were affected by shear stress-induced nitrite production, and diffusion/convection-induced nitrite removal. Highest nitrite concentrations and lowest thrombus deposition were observed on fibers coated with nNitrite-nGH-collagen conjugate exposed to 6-12 dyn/cm(2) shear stress. In conclusion, our results suggest that nNitrite-nGH-Col conjugate coatings promote endothelialization of silicone hollow fibers in biohybrid artificial lungs.

The influence of NaNO2 content on protein oxidation and nitrosation was investigated in cooked sausages at different concentrations (0, 50, 100, 200 and 400 mg NaNO2/kg). Dependent on concentration, NaNO2 had both anti- and pro-oxidant effects on protein oxidation. The antioxidant effects of NaNO2 on the protein oxidation were evidenced by significantly lower carbonyl contents, higher free amines and lower surface hydrophobicities. The pro-oxidant effects of NaNO2 on protein oxidation resulted in a decrease of sulfhydryls and an increase of disulfide bonds. NaNO2 also improved the protein nitrosation inducing the formation of 3-nitrotyrosine (3-NT). Moreover, 3-NT had significant correlations with parameters of protein oxidation, indicating that 3-NT may be a possible marker for protein oxidation. Results of this study contribute to an understanding of the impact of NaNO2 on food quality and help to identify optimal formulations of cured meat products., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Nitrite reductase (NiR) activity was examined in a series of dicopper P.a. azurin variants in which a surface binding copper site was added through site-directed mutagenesis. Four variants were synthesized with copper binding motifs inspired by the catalytic type 2 copper binding sites found in the native noncoupled dinuclear copper enzymes nitrite reductase and peptidylglycine α-hydroxylating monooxygenase. The four azurin variants, denoted Az-NiR, Az-NiR3His, Az-PHM, and Az-PHM3His, maintained the azurin electron transfer copper center, with the second designed copper site located over 13 Å away and consisting of mutations Asn10His,Gln14Asp,Asn16His-azurin, Asn10His,Gln14His,Asn16His-azurin, Gln8Met,Gln14His,Asn16His-azurin, and Gln8His,Gln14His,Asn16His-azurin, respectively. UV-visible absorption spectroscopy, EPR spectroscopy, and electrochemistry of the sites demonstrate copper binding as well as interaction with small exogenous ligands. The nitrite reduction activity of the variants was determined, including the catalytic Michaelis-Menten parameters. The variants showed activity (0.34-0.59 min(-1)) that was slower than that of native NiRs but comparable to that of other model systems. There were small variations in activity of the four variants that correlated with the number of histidines in the added copper site. Catalysis was found to be reversible, with nitrite produced from NO. Reactions starting with reduced azurin variants demonstrated that electrons from both copper centers were used to reduce nitrite, although steady-state catalysis required the T2 copper center and did not require the T1 center. Finally, experiments separating rates of enzyme reduction from rates of reoxidation by nitrite demonstrated that the reaction with nitrite was rate limiting during catalysis.

Streptococcus pneumoniaeis one of the key pathogens responsible for otitis media (OM), the most common infection in children and the largest cause of childhood antibiotic prescription. Novel therapeutic strategies that reduce the overall antibiotic consumption due to OM are required because, although widespread pneumococcal conjugate immunization has controlled invasive pneumococcal disease, overall OM incidence has not decreased. Biofilm formation represents an important phenotype contributing to the antibiotic tolerance and persistence ofS. pneumoniaein chronic or recurrent OM. We investigated the treatment of pneumococcal biofilms with nitric oxide (NO), an endogenous signaling molecule and therapeutic agent that has been demonstrated to trigger biofilm dispersal in other bacterial species. We hypothesized that addition of low concentrations of NO to pneumococcal biofilms would improve antibiotic efficacy and that higher concentrations exert direct antibacterial effects. Unlike in many other bacterial species, low concentrations of NO did not result inS. pneumoniaebiofilm dispersal. Instead, treatment of bothin vitrobiofilms andex vivoadenoid tissue samples (a reservoir forS. pneumoniaebiofilms) with low concentrations of NO enhanced pneumococcal killing when combined with amoxicillin-clavulanic acid, an antibiotic commonly used to treat chronic OM. Quantitative proteomic analysis using iTRAQ (isobaric tag for relative and absolute quantitation) identified 13 proteins that were differentially expressed following low-concentration NO treatment, 85% of which function in metabolism or translation. Treatment with low-concentration NO, therefore, appears to modulate pneumococcal metabolism and may represent a novel therapeutic approach to reduce antibiotic tolerance in pneumococcal biofilms., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Candida albicansis a leading nosocomial pathogen. Today, candidal biofilms are a significant cause of catheter infections, and such infections are becoming increasingly responsible for the failure of medical-implanted devices.C. albicansforms biofilms in which fungal cells are encased in an autoproduced extracellular polysaccharide matrix. Consequently, the enclosed fungi are protected from antimicrobial agents and host cells, providing a unique niche conducive to robust microbial growth and a harbor for recurring infections. Here we demonstrate that a recently developed platform comprised of nanoparticles that release therapeutic levels of nitric oxide (NO-np) inhibits candidal biofilm formation, destroys the extracellular polysaccharide matrices of mature fungal biofilms, and hinders biofilm development on surface biomaterials such as the lumen of catheters. We found NO-np to decrease both the metabolic activity of biofilms and the cell viability ofC. albicansin vitroandin vivo Furthermore, flow cytometric analysis found NO-np to induce apoptosis in biofilm yeast cellsin vitro Moreover, NO-np behave synergistically when used in combination with established antifungal drug therapies. Here we propose NO-np as a novel treatment modality, especially in combination with standard antifungals, for the prevention and/or remediation of fungal biofilms on central venous catheters and other medical devices., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Falcipain-2 (FP2) is an important hemoglobinase from the malaria parasite Plasmodium falciparum and a suitable target for the development of an antimalarial chemotherapy. Many reports have indicated that radical nitrogen species (RNS) including nitric oxide (NO) are inhibitors of P. falciparum growth and promoters of recovery from malaria symptoms. In this scenario, FP2 emerges as a potential target of RNS, since its inhibition partially hinders the parasite growth. We report that in vitro FP2 did not undergo S-nitrosylation when exposed to the NO-donor GSNO. However, it was modified by a combined mechanism of methionine oxidation and tyrosine nitration in response to SIN-1, and NaNO2- H2O2 treatment. The treatments with the nitrating agents caused a pronounced decrease in protease activity most likely induced by a disruption on the secondary and tertiary structure of FP2. Our data also demonstrate that at least four tyrosine residues were nitrated and found on the surface of the enzyme, partially or completely exposed to the solvent. Although performed in vitro, these results suggest that falcipain-2 may be a target of RNS activity and its inhibition could explain the hindering of the parasite growth when exposed to these radicals. The understanding of the molecular mechanisms involving free radicals and its inhibition activity towards FP2 may be effective in the development of antimalarial therapies.

The stability of cured meat products is increased by the protection of its proteins from oxidation by sodium nitrite (NaNO2) during processing. This study investigated the effects of NaNO2 (0, 50, 100, 200, and 400 mg/kg) on the physiochemical and structural characteristics of myofibrillar protein (MP) in raw and cooked ham. The NaNO2 showed a dose-dependent antioxidant effect, by inhibiting carbonyl formation, dityrosine formation, and denaturation of MP, and a nitrosative effect, through the formation of 3-Nitrotyrosine (3-NT). The 3-NT content within MP of raw ham had distinct negative correlations with sulfhydryl content and surface hydrophobicity. The 3-NT content within MP of cooked ham had significantly negative correlations with carbonyl, sulfhydryl content and turbidity and had significantly positive correlations with disulfide content. These results indicated that 3-NT may be a potential marker for protein oxidation in raw and cooked cured meat products.

Leishmania spp are the causative agents of a spectrum of diseases termed leishmaniasis that affect mammals, including humans and dogs. Although reactive nitrogen species are employed in the control of parasitism by the immune system, it is known that Leishmania can withstand this oxidative stress. As the mechanism by which these species are resistant to nitric oxide (NO) is poorly understood, the main objective of this study was to evaluate the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in Leishmania amazonensis and Leishmania chagasi promastigotes showing natural resistance to NO. GAPDH transcript levels were quantified by real-time polymerase chain reaction amplification, and GAPDH activity (assessed by levels of NADH oxidation) was measured by spectrophotometry. The level of nitration in total protein was assessed by immunoblotting. The results demonstrated an increase in GAPDH expression in resistant isolates of both species compared to susceptible isolates. The increase in GAPDH expression led to an increase in the activity of GAPDH in L. amazonensis human isolates resistant to NO. The pattern of protein nitration did not differ between sensitive and resistant isolates. Our results suggest that changes in expression of GAPDH may be responsible, at least in part, to natural resistance to NO found in human and canine Leishmania spp.

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.