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The objective of this study was to develop and validate a novel microfluidic paper-based analytical device (µPADpH) for determining the pH levels in foods. Anthocyanins from red cabbage aqueous extract (RCAE) were used as its analytical sensor. Whatman No. 1 filter paper was the most suitable for the device due to its porosity and fiber organization, which allows for maximum color intensity and minimal color heterogeneity of the RCAE in the detection zone of the µPADpH. To ensure the color stability of the RCAE for commercial use of the µPADpH, gum arabic was added. The geometric design of the µPADpH, including the channel length and separation zone diameter, was systematically optimized using colored food. The validation showed that the µPADpH did not differ from the pH meter when analyzing natural foods. However, certain additives in processed foods were found to increase the pH values.
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Antocianinas , Brassica , Goma Arábica , Antocianinas/química , Antocianinas/análise , Brassica/química , Concentração de Íons de Hidrogênio , Goma Arábica/química , Papel , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
AIM: To analyze the accuracy and costs of bedside methods for confirming the position of a nasoenteral feeding tube newly inserted blindly by nurses. DESIGN: Diagnostic accuracy study of three clinical methods (ultrasound, epigastric auscultation, and pH measurement) compared to radiography. The direct costs of each method used to confirm the positioning of the nasoenteral tube were also measured. METHODS: Seventy-six adult patients underwent a total of 87 nasoenteral tube insertion procedures in hospital units located within the Northeast region of the State of São Paulo, Brazil. The clinical methods were conducted on all study participants in the specified sequence: ultrasound (as index test), followed by epigastric auscultation and pH measurement (also index tests). RESULTS: The outcomes regarding the confirmation of the accurate positioning of the nasoenteral tube are as follows: ultrasonography demonstrated sensitivity and specific of 79.0% and 66.7%, respectively. Epigastric auscultation exhibited a sensitivity of 81.3% and specificity of 83.3%. The pH measurement method displayed sensitivity and specificity of 89.3% and 100% respectively. Additionally, in terms of estimated direct costs, the pH measurement method incurred a higher cost (USD $8.31) compared to the other methods, with a difference of USD $6.68. CONCLUSIONS: Based on these results, X-ray examination remains the primary method for confirming the placement of nasoenteral tubes recently inserted blindly at the bedside. However, when considering the costs of the evaluated methods, it is advisable to consider the variations in expenses between non-radiological methods and X-ray examinations.
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This study evaluated the efficacy of semisolid systems (gels) and films containing a combination of metronidazole (MTZ) and metronidazole benzoate after scaling and root-planing (SRP) for periodontitis. In total, 45 patients with stage I or II periodontitis were enrolled and divided into 3 groups: 1-SRP-control; 2-SRP + Film with MTZ; 3-SRP + Gel with MTZ. The pH of gingival crevicular fluid (GCF) before/after treatments, MTZ concentrations, and drug release using high-performance liquid chromatography were investigated. The effects were evaluated by longitudinal monitoring of clinical parameters (probing depth-PD, clinical attachment level-CAL, and bleeding on probing-BP). MTZ and MTZ-benzoate concentrations in the periodontal pocket and pH showed no statistical difference after application. SRP + Gel presented the lowest CAL values. For SRP + Film and SRP + Gel, higher PD values were observed at T0 compared to all groups. A relevant reduction in BP was observed in SRP + Film and SRP + Gel groups at all times compared to T0. Both therapies improved periodontal health compared to SRP alone, reducing PD and BP, and increasing CAL for the gel group, suggesting they are promising for periodontal disease treatment.
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In this study, poly(HEMA-PEGxMEM-IA) hydrogels were prepared by radical copolymerization of poly(ethylene glycol) methyl ether methacrylate (PEGxMEM), 2-hydroxyethyl methacrylate (HEMA), and itaconic acid (IA). The reaction was carried out in ethanolic solution using N,N'-methylenebisacrylamide (MBA) as a crosslinking agent and 1-hydroxycyclohexyl phenyl ketone (HCPK) as a photo-initiator. The poly(HEMA-PEGxMEM-IA) hydrogels (HGx) were evaluated as a delivery system for ursolic acid (UA), a phytochemical extracted from the plant Clinopodium revolutum, "flor de arena". The hydrogels were characterized by Fourier-transform infrared spectroscopy (FTIR-ATR), Raman spectroscopy, X-Ray diffraction (XRD), thermogravimetric analysis (TGA), and scanning electron microscopy (SEM). The swelling behavior was studied in buffer solutions from pH 2 to 10, specifically at pH 2.2 (gastric environment) and 7.4 (intestinal environment). It was found that the hydrogels studied showed sensitivity to pH. At pH 2.2, the degree of swelling for HG5 and HG9 hydrogels was 0.45 and 0.93 (g water/g hydrogel), respectively. At pH 7.4, the degree of swelling for HG5 and HG9 hydrogels was 1.97 and 2.64 (g water/g hydrogel), respectively. The SEM images show the variation in pore size as a function of pH, and the UA crystals in the pores of the hydrogels can also be observed. The in vitro UA release data best fit the Korsmeyer-Peppas kinetic model and the diffusion exponent indicates that the release mechanism is governed by Fickian diffusion.
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The use of pyrethroids in aquaculture has been an important component of achieving a thriving salmon farming industry in Chile. While the residual presence of such substances is known to depend on environmental conditions, most ecotoxicological studies to date have not considered environmental context. Here, we conducted oceanographic monitoring combined with experiments aiming to estimate the effects of two pyrethroids on the feeding rates of larvae of farmed mussels, Mytilus chilensis. In additional experiments, mussel spats were exposed to both pyrethroids, but under contrasting temperature/pH so as to mimic winter and summer conditions. Experiments mimicking spring conditions revealed that both pyrethroid substances affected the feeding of mussel larvae as a function of concentration. Conversely, significant impact of pyrethroids on adults were not observed with regard to temperature and pH, but a significant impact of low temperature/low pH condition on ingestion rates was confirmed. Given the current status of increasing ocean acidification, the results of this study are expected to provide useful information with regard to achieving sustainable mussel aquaculture, especially considering both activities occur in similar geographic areas, and the expansion of salmon farming areas is ongoing in Chile.
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Platelet concentrates undergo progressive changes during storage, such as a decrease in pH. Additionally, pH and lactate production showed the strongest correlation with platelet survival in posttransfusion viability studies. pH measurement is a straightforward method for evaluating the quality control of blood components in blood bank practice. Our aim was to compare three pH assessment methods for canine platelet concentrates. The pH values of the canine platelet concentrates were assessed on the first day of storage using a calibrated pH meter, a portable gas analyzer and pH-indicator strips. The results from the pH meter and portable gas analyzer measurements were similar. The pH indicator strips presented higher average values compared to the other more reliable methods evaluated, which could result in the use of inadequate blood components. In conclusion, it is recommended to implement pH measurements using a pH meter for quality control in veterinary blood banks.
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The aim of the present study was to evaluate the characteristics of carcasses, monitor their pH evolution during the first 24 h post-mortem, and determine the time required for the establishment and resolution of rigor mortis in different categories of Botucatu rabbits. Live weight at slaughter, carcass weight, and yield were higher in 12-month-old animals compared to 3-month-old rabbits, regardless of sex. There was an effect of muscle type, age, and sex on the kinetics of muscle acidification, with the Biceps femoris showing a significantly higher pH than the Longissimus lumborum from 4 h post-mortem onward. The establishment of rigor mortis occurred at 5 h post-mortem in young rabbits and bucks and at 6 h post-mortem in does, along with pH stabilization, while the resolution of rigor occurred at 18 h post-mortem for all types of carcasses evaluated. In conclusion, Botucatu rabbit carcasses should be chilled continuously at 4 °C for a minimum of 18 h to ensure efficient rigor mortis progression and muscle-to-meat transformation throughout the carcass.
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The use of wild species as a source of genetic variability is a valued tool in the framework of crop breeding. Hordeum chilense Roem. et Schult is a wild barley species that can be a useful genetic donor for sustainable wheat breeding which carries genes conferring resistance to some diseases or increasing grain quality, among others. Septoria tritici blotch (STB), caused by the Zymoseptoria tritici fungus, is one of the most important wheat diseases worldwide, affecting both bread and durum wheat and having a high economic impact. Resistance to STB has been previously described in H. chilense chromosome 4Hch. In this study, we have developed introgression lines for H. chilense chromosome 4Hch in durum wheat using interspecific crosses, advanced backcrosses, and consecutive selfing strategies. Alien H. chilense chromosome segments have been reduced in size by genetic crosses between H. chilense disomic substitution lines in durum wheat and durum wheat lines carrying the Ph1 deletion. Hordeum chilense genetic introgressions were identified in the wheat background through several plant generations by fluorescence in situ hybridisation (FISH) and simple sequence repeat (SSR) markers. An STB infection analysis has also been developed to assess STB resistance to a specific H. chilense chromosome region. The development of these H. chilense introgression lines with moderate to high resistance to STB represents an important advance in the framework of durum breeding and can be a valuable tool for plant breeders.
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Background: Carbon dioxide (CO2), traditionally viewed as a mere byproduct of cellular respiration, plays a multifaceted role in human physiology beyond simple elimination through respiration. CO2 may regulate the tumor microenvironment by significantly affecting the release of oxygen (O2) to tissues through the Bohr effect and by modulating blood pH and vasodilation. Previous studies suggest hypercapnia (elevated CO2 levels) might trigger optimized cellular mechanisms with potential therapeutic benefits. The role of CO2 in cellular stress conditions within tumor environments and its impact on O2 utilization offers a new investigative area in oncology. Objectives: This study aims to explore CO2's role in the tumor environment, particularly how its physiological properties and adaptive responses can influence therapeutic strategies. Methods: By applying a structured translational approach using the Work Breakdown Structure method, the study divided the analysis into six interconnected work packages to comprehensively analyze the interactions between carbon dioxide and the tumor microenvironment. Methods included systematic literature reviews, data analyses, data integration for identifying critical success factors and exploring extracellular environment modulation. The research used SMART criteria for assessing innovation and the applicability of results. Results: The research revealed that the human body's adaptability to hypercapnic conditions could potentially inform innovative strategies for manipulating the tumor microenvironment. This could enhance O2 utilization efficiency and manage adaptive responses to cellular stress. The study proposed that carbon dioxide's hormetic potential could induce beneficial responses in the tumor microenvironment, prompting clinical protocols for experimental validation. The research underscored the importance of pH regulation, emphasizing CO2 and carbonic acid's role in modulating metabolic and signaling pathways related to cancer. Conclusion: The study underscores CO2 as vital to our physiology and suggests potential therapeutic uses within the tumor microenvironment. pH modulation and cellular oxygenation optimization via CO2 manipulation could offer innovative strategies to enhance existing cancer therapies. These findings encourage further exploration of CO2's therapeutic potential. Future research should focus on experimental validation and exploration of clinical applications, emphasizing the need for interdisciplinary and collaborative approaches to tackle current challenges in cancer treatment.
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Monitoring equine parturition effectively is essential for preemptive intervention in periparturient issues and ensuring the overall well-being of both mares and foals. However, its implementation in breeding farms is challenging due to variable gestational lengths and nocturnal births. Predictive techniques have the potential to streamline the monitoring process, reduce labor intensity, and minimize costs. Research on foaling prediction in mares carrying mule or equine clone fetuses is scarce. Therefore, this study aimed to comparatively analyze foaling prediction parameters in mares pregnant with mule, equine, or equine clone fetus. The study included vulvar relaxation, sacroiliac ligament tension, pH, BRIX index, and concentrations of calcium, phosphorus, magnesium, sodium, and potassium in prepartum mammary secretions. Sixty pregnant mares were used for this study and grouped as follows: 25 mares with mule fetuses (MF), 20 with equine clone fetuses (CF), and 15 with equine control fetuses (EF). Results showed significant differences in vulvar relaxation and sacroiliac ligament tension only in MF group (p < 0.05) on the day of parturition compared to the other days evaluated, different from the other groups. Levels of pH notably decreased on parturition day (mean 5.7 ± 0.04, p < 0.0001), with lower values in MF (6.05 ± 0.02) and CF (6.08 ± 0.04) compared to EF (6.26 ± 0.04) (p < 0.03). The BRIX index showed variation across mares and was not a good parameter for foaling prediction. Electrolytes correlated positively with impending parturition, showing no significant differences among groups. The MF and CF groups exhibited a substantial increase (102.13 % and 110.66 %, respectively) in mean calcium concentrations on the day before foaling, unlike EF (38.29 %). In conclusion, the pH values were different in mammary secretions between mares carrying mule and clone fetuses, in contrast to equine control fetuses. Nevertheless, there was a trend of decreasing pH values closer to parturition in all groups. Conversely, the BRIX index serves as a valuable indicator of colostrum quality yet does not offer insights into the proximity of parturition. While electrolyte concentrations did not reveal significant differences among groups, it is worth noting that the evaluation of phosphorus emerges as a new parameter to explore in mares nearing parturition, since it obtained a pattern similar to calcium.
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Parto , Prenhez , Animais , Cavalos/fisiologia , Feminino , Gravidez , Parto/fisiologia , Prenhez/fisiologia , Clonagem de Organismos/veterináriaRESUMO
This work covers the formation of a passive state for two different alloys used in the aeronautical industry. The aim of this study is to investigate the effectiveness of passivation treatments on 17-7PH and 410 SS (stainless steel) samples, specifically when performed with citric and nitric acid solutions at 49 °C using an immersion time of 90 min and subsequent exposure in 3.5 wt.% NaCl solution. Employing the cyclic potentiodynamic polarization (CPP) technique, the corrosion properties of the passivated material were evaluated according to the ASTM G65-11 standard. A microstructural analysis was performed using scanning electron microscopy (SEM). The passivated layer was characterized via X-ray photoelectron spectroscopy. In the results, the CPP curves showed positive hysteresis, indicating pitting localized corrosion, and 17-7PH steel passivated at 49 °C for 90 min in citric acid exhibited lower corrosion rate values equivalent to ×10-3 mm/year.
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This study investigated the impact of ocean acidification on the photodegradation of three microplastics (MPs): polypropylene (PP), expanded polystyrene (EPS), and ethylene-vinyl acetate (EVA), under accelerated UV radiation at three pH levels (i.e., 8.1, 7.8, and 7.5), simulating marine conditions. The acidification system simulated current and projected future environmental conditions. As expected, an increase in partial pressure of CO2, total inorganic carbon, bicarbonate ion, and CO2 resulted in more acidic pH levels, with the reverse being true for the carbonate ion. Structural changes of MPs were evaluated, revealing that all weathered samples underwent higher degradation rate compared to the virgin samples. The oxidation state and crystallinity of PP and EVA MPs were higher in samples exposed to the lowest pH, whereas no significant increase in the degradation rate of EPS samples was observed. Saltwater acidification in this study contributed to enhance the photo-oxidation of MPs depending on their polymeric composition.
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Microplásticos , Fotólise , Água do Mar , Poluentes Químicos da Água , Microplásticos/análise , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/química , Concentração de Íons de Hidrogênio , Água do Mar/química , Polipropilenos/química , Poliestirenos/química , Oceanos e Mares , Acidificação dos OceanosRESUMO
This study aimed to evaluate the effect of chemical gasification and HEPES as alternative systems to pH control during in vitro maturation on bovine oocytes competence. Groups of 20 bovine cumulus oocytes complexes (COCs) were randomly distributed and cultured for 24 h in one of the following experimental groups: (i) chemical reaction (ChRG) system: CO2 generated from sodium bicarbonate and citric acid reaction (ii) culture media TCM-HEPES (HEPES-G); and (iii) control group (CNTG) in conventional incubator. After in vitro maturation (IVM), the COCs were in vitro fertilized (IVF), and in vitro cultivated (IVC) in a conventional incubator. We evaluated oocyte nuclear maturation, cleavage and blastocyst rates, in addition to the relative mRNA expression of BAX, BMP-15, AREG and EREG genes in oocytes and cumulus cells. The proportion of oocytes in metaphase II was higher in CNTG and ChRG (77.57% and 77.06%) than in the HEPES-G (65.32%; p = .0408 and .0492, respectively). The blastocyst production was similar between CNTG and ChRG (26.20% and 28.47%; p = .4232) and lower (p = .001) in the HEPES-G (18.71%). The relative mRNA expression of BAX gene in cumulus cells was significantly higher (p = .0190) in the HEPES-G compared to the CNTG. Additionally, the relative mRNA expression of BMP-15 gene was lower (p = .03) in oocytes from HEPES-G compared to the CNTG. In conclusion, inadequate atmosphere control has a detrimental effect on oocyte maturation. Yet, the use of chemical gasification can be an efficient alternative to bovine COCs cultivation.
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Fertilização in vitro , Técnicas de Maturação in Vitro de Oócitos , Oócitos , Animais , Bovinos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/efeitos dos fármacos , Fertilização in vitro/veterinária , Feminino , Meios de Cultura , Blastocisto/efeitos dos fármacos , Células do Cúmulo/efeitos dos fármacos , Dióxido de Carbono/farmacologia , Bicarbonato de Sódio/farmacologia , Ácido Cítrico/farmacologia , Técnicas de Cultura Embrionária/veterináriaRESUMO
Currently, coffee fermentation is visually operated, which results in incomplete or excessive processes and coffees with undesirable characteristics. In front of it, pH and total soluble solids (TSS) have been shown to be good fermentation indicators, although this requires rapid, accurate, and chemical-free measurement techniques such as NIR spectroscopy. However, the complexity of the NIR spectra requires optimization steps in which variable selection techniques simplify profiles and subsequent models. This work tests a new covering array feature selection (CAFS) approach on NIR spectra to optimize prediction models in coffee samples during fermentation. Spectral profiles in the range 1100-2100 nm were extracted from coffee beans (Typica, Caturra, and Catimor varieties) raw and during fermentation (4, 8, 12, 16, 20, and 24 h). Partial least-squares regressions (PLSR) were performed using full spectra using a five-fold cross-validation strategy for training and validation. The relevant wavelengths were then selected using the ß coefficients, the important projection of variables (VIP), and the CAFS method. Finally, optimized models were performed using the relevant wavelengths and compared among these using their statistical metrics. The models performed using the selected variables (22-47) of CAFS showed the best performance in predicting pH (R 2 = 0.825-0.903, RMSE = 0.096-0.158, RPD = 6.33-10.38) and TSS (R 2 = 0.865-0.922, RMSE = 0.688-1.059, RPD = 0.94-1.45) compared to the other methods. These findings suggest that simple and efficient models could be performed and implemented in routine analysis due to the maximum coverage and minimum cardinality of CAFS.
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This paper presents an innovative application of chitosan material to be used as pH-responsive valves for the precise control of lateral flow in microfluidic paper-based analytical devices (µPADs). The fabrication of µPADs involved wax printing, while pH-responsive valves were created using a solution of chitosan in acetic acid. The valve-forming solution was applied, and ready when dry; by exposure to acidic solutions, the valve opens. Remarkably, the valves exhibited excellent compatibility with alkaline, neutral, and acidic solutions with a pH higher than 4. The valve opening process had no impact on the flow rate and colorimetric analysis. The potential of chitosan valves used for flow control was demonstrated for µPADs employed for nitrate determination. Valves were used to increase the conversion time of nitrate to nitrite, which was further analyzed using the Griess reaction. The µPAD showed a linear response in the concentration range of 10-100 µmol L-1, with a detection limit of 5.4 µmol L-1. As a proof of concept, the assay was successfully applied to detect nitrate levels in water samples from artificial lakes of recreational parks. For analyses that require controlled kinetics and involve multiple sequential steps, the use of chitosan pH-responsive valves in µPADs is extremely valuable. This breakthrough holds great potential for the development of simple and high-impact microfluidic platforms that can cater to a wide range of analytical chemistry applications.
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INTRODUCTION: Esophageal pH-impedance monitoring is a tool for diagnosing gastroesophageal reflux in children. The position of the pH catheter is essential for a reliable reading and the current formulas for calculating catheter insertion length are not completely accurate. The aim of the present study was to develop a new formula for adequate insertion of the pH catheter. MATERIAL AND METHODS: A cross-sectional study was conducted on children that underwent pH-impedance monitoring and later radiographic control, to calculate the correct catheter insertion length. The documented variables were age, sex, weight, height, naris to tragus distance, tragus to sternal notch distance, sternal notch to xiphoid process distance, and initial insertion length determined by the Strobel and height interval formulas. A multivariate regression analysis was carried out to predict the final insertion length. Regression ANOVA and Pearson's adjusted R-squared tests were performed. RESULTS: Forty-five pH-impedance studies were carried out, 53% of which were in males. The age and weight variables were not normally distributed. In the initial regression model, the variables that did not significantly correlate with the final insertion length were: sex (P 0.124), length determined by the Strobel or height interval formulas (P 0.078), naris to tragus distance (P 0.905), and tragus to sternal notch distance (P 0.404). The final equation: 5.6 + (height in cm * 0.12) + (sternal notch to xiphoid process distance * 0.57) produced an R2 of 0.93 (P 0.000). CONCLUSIONS: This formula can be considered a valid option for placement of the pH-impedance monitoring catheter in pediatrics.
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Impedância Elétrica , Monitoramento do pH Esofágico , Humanos , Masculino , Feminino , Estudos Transversais , Criança , Pré-Escolar , Lactente , Adolescente , Refluxo GastroesofágicoRESUMO
Oral drug administration, especially when composed of mucoadhesive delivery systems, has been a research trend due to increased residence time and contact with the mucosa, potentially increasing drug bioavailability and stability. In this context, this study aimed to develop self-assembly mucoadhesive beads composed of blends of κ-carrageenan and sericin (κ-Car/Ser) loaded with the anti-inflammatory drug indomethacin (IND). We investigated the swelling, adhesion behaviour, and mechanical/physical properties of the beads, assessing their effects on cell viability, safety and permeation characteristics in both 2D and triple-culture model. The swelling ratio of the beads indicated pH-responsiveness, with maximum water absorption at pH 6.8, and strong mucoadhesion, increasing primarily with higher polymer concentrations. The beads exhibited thermal stability and no chemical interaction with IND, showing improved mechanical properties. Furthermore, the beads remained stable during accelerated and long-term storage studies. The beads were found to be biocompatible, and IND encapsulation improved cell viability (>70 % in both models, 79 % in VN) and modified IND permeation through the models (6.3 % for F5 formulation (κ-Car 0.90 % w/v | Ser 1.2 % w/v| IND 3.0 g); 10.9 % for free IND, p < 0.05). Accordingly, κ-Car/Ser/IND beads were demonstrated to be a promising IND drug carrier to improve oral administration while mitigating the side effects of non-steroidal anti-inflammatories.
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Carragenina , Preparações de Ação Retardada , Indometacina , Sericinas , Indometacina/química , Indometacina/administração & dosagem , Indometacina/farmacocinética , Carragenina/química , Administração Oral , Humanos , Sericinas/química , Preparações de Ação Retardada/química , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Sobrevivência Celular/efeitos dos fármacos , Microesferas , Animais , Células CACO-2 , Concentração de Íons de HidrogênioRESUMO
Bacterial nanocellulose (BNC) is a sustainable, renewable, and eco-friendly nanomaterial, which has gained great attentions in both academic and industrial fields. Two bacterial nanocellulose-producing strains (CVV and CVN) were isolated from apple vinegar sources, presenting high 16S rRNA gene sequence similarities (96%-98%) with Komagataeibacter species. The biofilm was characterized by scanning electron microscopy (SEM), revealing the presence of rod-shaped bacteria intricately embedded in the polymeric matrix composed of nanofibers of bacterial nanocellulose. FTIR spectrum and XRD pattern additionally confirmed the characteristic chemical structure associated with this material. The yields and productivities achieved during 10 days of fermentation were compared with Komagataeibacter xylinus ATCC 53524, resulting in low levels of BNC production. However, a remarkable increase in the BNC yield was achieved for CVV (690% increase) and CVN (750% increase) strains at day 6 of the fermentation upon adding 22 mM citrate buffer into the medium. This effect is mainly attributed to the buffering capacity of the modified Yakamana medium, which allowed to maintain pH close to 4.0 until day 6, though in combination with additional factors including stimulation of the gluconeogenesis pathway and citrate assimilation as a carbon source. In addition, the productivities determined for both isolated strains (0.850 and 0.917 g L-1 d-1) compare favorably to previous works, supporting current efforts to improve fermentation performance in static cultures and the feasibility of scaling-up BNC production in these systems.
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Firefly luciferases emit yellow-green light and are pH-sensitive, changing the bioluminescence color to red in the presence of heavy metals, acidic pH and high temperatures. These pH and metal-sensitivities have been recently harnessed for intracellular pH indication and toxic metal biosensing. However, whereas the structure of the pH sensor and the metal binding site, which consists mainly of two salt bridges that close the active site (E311/R337 and H310/E354), has been identified, the specific role of residue H310 in pH and metal sensing is still under debate. The Amydetes vivianii firefly luciferase has one of the lowest pH sensitivities among the group of pH-sensitive firefly luciferases, displaying high bioluminescent activity and special spectral selectivity for cadmium and mercury, which makes it a promising analytical reagent. Using site-directed mutagenesis, we have investigated in detail the role of residue H310 on pH and metal sensitivity in this luciferase. Negatively charged residues at position 310 increase the pH sensitivity and metal sensitivity; H310G considerably increases the size of the cavity, severely impacting the activity, H310R closes the cavity, and H310F considerably decreases both pH and metal sensitivities. However, no substitution completely abolished pH and metal sensitivities. The results indicate that the presence of negatively charged and basic side chains at position 310 is important for pH sensitivity and metals coordination, but not essential, indicating that the remaining side chains of E311 and E354 may still coordinate some metals in this site. Furthermore, a metal binding site search predicted that H310 mutations decrease the affinity mainly for Zn, Ni and Hg but less for Cd, and revealed the possible existence of additional binding sites for Zn, Ni and Hg.
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Vaga-Lumes , Histidina , Luciferases de Vaga-Lume , Mutagênese Sítio-Dirigida , Concentração de Íons de Hidrogênio , Animais , Luciferases de Vaga-Lume/metabolismo , Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/genética , Vaga-Lumes/enzimologia , Histidina/química , Histidina/metabolismo , Cor , Metais Pesados/química , Metais Pesados/metabolismo , Mercúrio/química , Mercúrio/metabolismo , Cádmio/química , Cádmio/metabolismoRESUMO
The change in the corrosion activities of SS304 and the carbon steel A36 were studied during their exposure for 30 days to hybrid pumice-Portland cement extract (CE), to simulate the concrete-pore environment. The ionic composition and the initial pH (12.99) of the CE were influenced by the reduction of Portland cement (PC) content, volcanic pumice oxides and alkaline activators. Because of the air CO2 dissolution, the pH decreased and maintained a constant value ≈ 9.10 (established dynamic ionic equilibrium). The CE promoted the passivation of both steels and their free corrosion potential (OCP) reached positive values. On the surfaces, Fe and Cr oxides were formed, according to the nature of the steel. Over the time of exposure, the presence of chloride ions in the pumice caused a localized pitting attack, and for carbon steel, this fact may indicate an intermediate risk of corrosion. The chloride effect was retarded by the accumulation of SO42- ions at the steel surfaces. Based on electrochemical impedance (EIS), the polarization resistance (Rp) and the thickness of the passive layers were calculated. Their values were compared with those previously reported for the steels exposed to CEs of Portland and supersulfated cements, and the hybrid cement was considered as a PC "green" alternative.