Effects of Hydrofluoric Acid Concentrations, Commercial Brands, and Adhesive Application on the Bond Strength of a Resin Luting Agent to Lithium Disilicate Glass Ceramic.

OBJECTIVES: To evaluate the surface topography/roughness and bond strength of a resin luting agent to a lithium disilicate glass ceramic after etching with different concentrations of hydrofluoric acid (HF) and commercial brands. METHODS: For bond strength evaluation, 260 lithium disilicate glass ceramic (EMX) discs were randomly distributed into 13 groups based on concentrations of HF and commercial brands (n=20): 5% and 10%, Lysanda (LY5 and LY10); 5% and 10%, Maquira (MA5 and MA10); 5% and 10%, FGM (FG5 and FG10); 4.8%, Ivoclar Vivadent (IV5); 5% and 10%, PHS do Brasil (PH5 and PH10); 5% and 10%, BM4 (BM5 and BM10); 9%, Ultradent Inc (UL10); and Dentsply (DE10). A further random distribution (n=10) was made based on the application (+) or absence (-) of an adhesive layer. Resin luting agent cylinders (1 mm in diameter) were added on EMX surfaces, light-cured, and stored for 24 hours in deionized water at 37°C. On a universal testing machine (DL 500, EMIC), specimens were submitted to a microshear bond strength test at a crosshead speed of 1 mm/min until failure. A representative etched EMX disc from each group underwent surface topography analysis using field-emission scanning electron microscopy (n=1), and five (n=5) etched EMX discs from each group were tested for surface roughness. Data were statistically analyzed using analysis of variance and Tukey test (α=0.05). RESULTS: A less conditioned and smoother surface was observed for 5% HF compared to 10%. Additionally, commercial brands of HF were shown to affect bond strength. When the adhesive layer was not used (-), a 10% concentration promoted higher bond strengths to EMX. However, when adhesive was applied (+), the concentrations of HF and commercial brands had no effect on bond strength results. CONCLUSIONS: A 10% concentration of HF results in higher bond strength than a 5% concentration. If an adhesive layer is applied, neither this distinction nor the influence of commercial brands is observed.

Simultaneous speciation analysis of Hg and Se in fish by high-performance liquid chromatography and inductively coupled plasma-mass spectrometry following microwave-assisted enzymatic hydrolysis.

This study reports the development and validation of a new analytical method for simultaneous speciation analysis of Se and Hg in fish muscle. For this purpose, four Se species (selenite/Se(IV), selenate/Se(VI), selenomethionine/SeMet, and selenocysteine/SeCys) and two Hg species (inorganic mercury/iHg and methylmercury/MeHg) were extracted simultaneously by microwave-assisted enzymatic hydrolysis and then separated by HPLC in less than 15 min by using a column with both anion and cation exchange mechanisms and a mobile phase consisting of a mixture of methanol 5% (v/v), 45 mM HNO3, 0.015% 2-mercaptoethanol, and 1.5 mM sodium 3-mercapto-1-propanesulfonate. The separated species of Hg and Se were detected online by inductively coupled plasma-mass spectrometry (ICP-MS). The speciation analysis method was validated by means of the accuracy profile approach by carrying out three series of measurements in duplicate on three different days over a time-span of 3 weeks. The limits of quantification (LOQ) are in the range of 0.010-0.013 mg/kg wet weight (ww) for all selenium species, except for Se(IV) (0.15 mg/kg ww), while the coefficient of variation in terms of intermediate reproducibility (CVR) was < 7%. The LOQ for MeHg was 0.006 mg/kg ww, while the CVR was 3%. The method was successfully applied to the analysis of muscle samples from four different fish species: rainbow trout, tuna, swordfish, and dogfish.

The geographical distribution and socioeconomic risk factors of COVID-19, tuberculosis and leprosy in Fortaleza, Brazil.

BACKGROUND: Fortaleza (Brazil) is high endemic for coronavirus disease 2019 (COVID-19), tuberculosis (TB) and leprosy. These three diseases share respiratory droplets through coughing or sneezing as the main mode of transmission but differ in incubation time, with COVID-19 having a short and leprosy a long incubation time. Consequently, contacts of a patient are at higher risk of infection and developing these diseases. There might be scope for combined preventive measures, but a better understanding of the geographical distribution and relevant socioeconomic risk factors of the three diseases is needed first. This study aims to describe the geographic distribution of COVID-19, TB and leprosy incidence and to identify common socioeconomic risk factors. METHODS: The total number of new cases of COVID-19, TB and leprosy, as well as socioeconomic and demographic variables, were retrieved from official registers. The geographical distribution of COVID-19, TB and leprosy rates per neighbourhood was visualised in Quantum GIS, and spatial autocorrelation was measured with Moran's I in GeoDa. A spatial regression model was applied to understand the association between COVID-19, TB, leprosy rates, and socioeconomic factors. RESULTS: COVID-19 and TB showed a more homogenous distribution, whereas leprosy is located more in the south and west of Fortaleza. One neighbourhood (Pedras) in the southeast was identified as high endemic for all three diseases. Literacy was a socioeconomic risk factor for all three diseases: a high literacy rate increases the risk of COVID-19, and a low literacy rate (i.e., illiteracy) increases the risk of TB and leprosy. In addition, high income was associated with COVID-19, while low income with TB. CONCLUSIONS: Despite the similar mode of transmission, COVID-19, TB and leprosy show a different distribution of cases in Fortaleza. In addition, associated risk factors are related to wealth in COVID-19 and to poverty in TB and leprosy. These findings may support policymakers in developing (partially combined) primary and secondary prevention considering the efficient use of resources.

Previous fracture and subsequent fracture risk: a meta-analysis to update FRAX.

A large international meta-analysis using primary data from 64 cohorts has quantified the increased risk of fracture associated with a previous history of fracture for future use in FRAX. INTRODUCTION: The aim of this study was to quantify the fracture risk associated with a prior fracture on an international basis and to explore the relationship of this risk with age, sex, time since baseline and bone mineral density (BMD). METHODS: We studied 665,971 men and 1,438,535 women from 64 cohorts in 32 countries followed for a total of 19.5 million person-years. The effect of a prior history of fracture on the risk of any clinical fracture, any osteoporotic fracture, major osteoporotic fracture, and hip fracture alone was examined using an extended Poisson model in each cohort. Covariates examined were age, sex, BMD, and duration of follow-up. The results of the different studies were merged by using the weighted ß-coefficients. RESULTS: A previous fracture history, compared with individuals without a prior fracture, was associated with a significantly increased risk of any clinical fracture (hazard ratio, HR = 1.88; 95% CI = 1.72-2.07). The risk ratio was similar for the outcome of osteoporotic fracture (HR = 1.87; 95% CI = 1.69-2.07), major osteoporotic fracture (HR = 1.83; 95% CI = 1.63-2.06), or for hip fracture (HR = 1.82; 95% CI = 1.62-2.06). There was no significant difference in risk ratio between men and women. Subsequent fracture risk was marginally downward adjusted when account was taken of BMD. Low BMD explained a minority of the risk for any clinical fracture (14%), osteoporotic fracture (17%), and for hip fracture (33%). The risk ratio for all fracture outcomes related to prior fracture decreased significantly with adjustment for age and time since baseline examination. CONCLUSION: A previous history of fracture confers an increased risk of fracture of substantial importance beyond that explained by BMD. The effect is similar in men and women. Its quantitation on an international basis permits the more accurate use of this risk factor in case finding strategies.

Comparing Prevalence of Sarcopenia Using Twelve Sarcopenia Definitions in a Large Multinational European Population of Community-Dwelling Older Adults.

OBJECTIVES: Multinational prevalence data on sarcopenia among generally healthy older adults is limited. The aim of the study was to assess prevalence of sarcopenia in the DO-HEALTH European trial based on twelve current sarcopenia definitions. SETTING AND PARTICIPANTS: This is an analysis of the DO-HEALTH study including 1495 of 2157 community-dwelling participants age 70+ years from Germany, France, Portugal, and Switzerland with complete measurements of the sarcopenia toolbox including muscle mass by DXA, grip strength, and gait speed. MEASUREMENTS: The twelve sarcopenia definitions applied were Asian Working Group on Sarcopenia (AWGS1), AWGS2, Baumgartner, Delmonico, European Working Group on Sarcopenia in Older People (EWGSOP1), EWGSOP2, EWGSOP2-lower extremities, Foundation for the National Institutes of Health (FNIH1), FNIH2, International Working Group on Sarcopenia in Older People (IWGS), Morley, and Sarcopenia Definitions and Outcomes Consortium (SDOC). RESULTS: Mean age was 74.9 years (SD 4.4); 63.3% were women. Sarcopenia prevalence ranged between 0.7% using the EWGSOP2 or AWGS2 definition, up to 16.8% using the Delmonico definition. Overall, most sarcopenia definitions, including Delmonico (16.8%), Baumgartner (12.8%), FNIH1(10.5%), IWGS (3.6%), EWGSOP1 (3.4%), SDOC (2.0%), Morley (1.3%), and AWGS1 (1.1%) tended to be higher than the prevalence based on EWGSOP2 (0.7%). In contrast, the definitions AWGS2 (0.7%), EWGSOP2-LE (1.1%), FNIH2 (1.0%) - all based on muscle mass and muscle strength - showed similar lower prevalence as EWGSOP2 (0.7%). Moreover, most sarcopenia definitions did not overlap on identifying sarcopenia on an individual participant-level. CONCLUSION: In this multinational European trial of community-dwelling older adults we found major discordances of sarcopenia prevalence both on a population- and on a participant- level between various sarcopenia definitions. Our findings suggest that the concept of sarcopenia may need to be rethought to reliably and validly identify people with impaired muscle health.

Update of the fracture risk prediction tool FRAX: a systematic review of potential cohorts and analysis plan.

We describe the collection of cohorts together with the analysis plan for an update of the fracture risk prediction tool FRAX with respect to current and novel risk factors. The resource comprises 2,138,428 participants with a follow-up of approximately 20 million person-years and 116,117 documented incident major osteoporotic fractures. INTRODUCTION: The availability of the fracture risk assessment tool FRAX® has substantially enhanced the targeting of treatment to those at high risk of fracture with FRAX now incorporated into more than 100 clinical osteoporosis guidelines worldwide. The aim of this study is to determine whether the current algorithms can be further optimised with respect to current and novel risk factors. METHODS: A computerised literature search was performed in PubMed from inception until May 17, 2019, to identify eligible cohorts for updating the FRAX coefficients. Additionally, we searched the abstracts of conference proceedings of the American Society for Bone and Mineral Research, European Calcified Tissue Society and World Congress of Osteoporosis. Prospective cohort studies with data on baseline clinical risk factors and incident fractures were eligible. RESULTS: Of the 836 records retrieved, 53 were selected for full-text assessment after screening on title and abstract. Twelve cohorts were deemed eligible and of these, 4 novel cohorts were identified. These cohorts, together with 60 previously identified cohorts, will provide the resource for constructing an updated version of FRAX comprising 2,138,428 participants with a follow-up of approximately 20 million person-years and 116,117 documented incident major osteoporotic fractures. For each known and candidate risk factor, multivariate hazard functions for hip fracture, major osteoporotic fracture and death will be tested using extended Poisson regression. Sex- and/or ethnicity-specific differences in the weights of the risk factors will be investigated. After meta-analyses of the cohort-specific beta coefficients for each risk factor, models comprising 10-year probability of hip and major osteoporotic fracture, with or without femoral neck bone mineral density, will be computed. CONCLUSIONS: These assembled cohorts and described models will provide the framework for an updated FRAX tool enabling enhanced assessment of fracture risk (PROSPERO (CRD42021227266)).

Assessment of the mercury-selenium antagonism in rainbow trout fish.

This study aims at the assessment of mercury (Hg)-selenium (Se) antagonism in fish. For this purpose, rainbow trout fish (Oncorhynchus mykiss) were exposed to methylmercury (MeHg) under controlled conditions, in the presence or absence of selenomethionine (SeMet) using an in-house prepared diet (enriched with MeHg and SeMet at 0.2 µg/g and 5.0 µg/g, respectively). The total duration of the exposure study was 3 months. Fish was sampled and analysed for total Se (SeT) and total mercury (HgT) content after 1, 2 and 3 months of exposure. Six feeding protocols were compared, depending on the exposure type: (i) no MeHg nor SeMet exposure (control group); (ii) exposure to SeMet solely; (iii) exposure to MeHg solely; (iv) exposure to both MeHg and SeMet; (v) exposure first to MeHg during 1 month and then to SeMet during 2 months and (vi), exposure to SeMet during 1 month and then to MeHg for 2 months. The levels of SeT and HgT in the fish (control and supplemented with MeHg/SeMet) were measured by inductively coupled plasma-mass spectrometry (ICPMS). Steadily (linear) bioaccumulation of MeHg in the fish muscle occurred when the fish were exposed individually to this species during the period. The bioaccumulation of MeHg is diminished when the fish are firstly exposed to SeMet and then to MeHg, hence indicating the MeHg detoxification due to SeMet supplementation.

Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766) / Obtenção e caracterização de proteases aspárticas digestórias do peixe Caranx hippos (Linnaeus, 1766)

This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

Este trabalho objetivou obter proteases aspárticas de interesse industrial e biotecnológico a partir do estômago do peixe xaréu (Caranx hippos). Para isso, foi obtido um extrato bruto do estômago, o qual foi submetido a uma purificação parcial por salting-out onde se obteve o extrato enzimático (EE). As proteases do EE foram caracterizadas físico-quimicamente e através de zimograma. Além disso, o efeito de agentes químicos sobre sua atividade também foi avaliado. Através de salting-out foi possível obter uma purificação de 1,6 vezes com rendimento de 49,4%. Foram observadas duas proteases ácidas presentes no EE através de zimograma. A temperatura ótima e a estabilidade térmica para as proteases ácidas do EE foram de 55 ºC e 45 °C, respectivamente. O pH ótimo e a estabilidade ao pH encontrados para estas enzimas foram o pH 1,5 e 7,0, respectivamente. Observou-se a inibição total da atividade proteolítica ácida do EE na presença de pepstatina A. O ditiotreitol (DTT) e o Ca2+ não promoveram efeito significativo na atividade enzimática. Na presença de metais pesados, como Al3+, Cd2+ e Hg2+, o EE manteve mais de 70% de atividade enzimática do EE. Os resultados mostram que é possível obter, a partir do estômago de C. hippos, proteases aspárticas com alta atividade proteolítica e características que demonstram potencial para aplicações industriais e biotecnológicas.

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications / Estômago de Carangoides bartholomaei (Cuvier, 1833): uma fonte de proteases aspárticas para aplicações industriais e biotecnológicas

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U·mg-¹. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

As vísceras e outros resíduos do processamento de peixes são geralmente descartados pela indústria pesqueira. Esses resíduos podem ser uma fonte de enzimas digestivas com potencial industrial e biotecnológico. Neste estudo, objetivamos a extração, caracterização e aplicação de proteases aspárticas do estômago de Carangoides bartholomaei (Cuvier, 1833). Um extrato bruto do estômago foi obtido e submetido a um processo de purificação parcial, que obteve um Extrato Purificado (EP) com uma atividade proteolítica específica de 54,0 U·mg-¹. Foi obtida uma purificação de 1,9 vezes e um rendimento de 41%. O EP apresenta duas isoformas de proteases ácidas e atividade proteolítica máxima a 45 °C e pH 2,0. A atividade proteolítica do EP foi estável na faixa de pH de 1,5 a 7,0 e temperatura de 25 °C a 50 °C. O EP manteve 35% de sua atividade proteolítica na presença de NaCl a 15% (m/v), mas foi totalmente inibida pela pepstatina A. As proteases ácidas do EP apresentaram alta atividade na presença de metais pesados como o Cd2+, Hg2+, Pb2+, Al3+ e Cu2+. A utilização de EP como aditivo enzimático na extração de colágeno a partir de escamas de tilápia do Nilo dobrou o rendimento do processo. Os resultados indicam um potencial dessas proteases para aplicações industriais e biotecnológicas.

Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766)

Abstract This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

Resumo Este trabalho objetivou obter proteases aspárticas de interesse industrial e biotecnológico a partir do estômago do peixe xaréu (Caranx hippos). Para isso, foi obtido um extrato bruto do estômago, o qual foi submetido a uma purificação parcial por salting-out onde se obteve o extrato enzimático (EE). As proteases do EE foram caracterizadas físico-quimicamente e através de zimograma. Além disso, o efeito de agentes químicos sobre sua atividade também foi avaliado. Através de salting-out foi possível obter uma purificação de 1,6 vezes com rendimento de 49,4%. Foram observadas duas proteases ácidas presentes no EE através de zimograma. A temperatura ótima e a estabilidade térmica para as proteases ácidas do EE foram de 55 ºC e 45 °C, respectivamente. O pH ótimo e a estabilidade ao pH encontrados para estas enzimas foram o pH 1,5 e 7,0, respectivamente. Observou-se a inibição total da atividade proteolítica ácida do EE na presença de pepstatina A. O ditiotreitol (DTT) e o Ca2+ não promoveram efeito significativo na atividade enzimática. Na presença de metais pesados, como Al3+, Cd2+ e Hg2+, o EE manteve mais de 70% de atividade enzimática do EE. Os resultados mostram que é possível obter, a partir do estômago de C. hippos, proteases aspárticas com alta atividade proteolítica e características que demonstram potencial para aplicações industriais e biotecnológicas.

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications

Abstract The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by-products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 Umg-1. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

Resumo As vísceras e outros resíduos do processamento de peixes são geralmente descartados pela indústria pesqueira. Esses resíduos podem ser uma fonte de enzimas digestivas com potencial industrial e biotecnológico. Neste estudo, objetivamos a extração, caracterização e aplicação de proteases aspárticas do estômago de Carangoides bartholomaei (Cuvier, 1833). Um extrato bruto do estômago foi obtido e submetido a um processo de purificação parcial, que obteve um Extrato Purificado (EP) com uma atividade proteolítica específica de 54,0 Umg-1. Foi obtida uma purificação de 1,9 vezes e um rendimento de 41%. O EP apresenta duas isoformas de proteases ácidas e atividade proteolítica máxima a 45 °C e pH 2,0. A atividade proteolítica do EP foi estável na faixa de pH de 1,5 a 7,0 e temperatura de 25 °C a 50 °C. O EP manteve 35% de sua atividade proteolítica na presença de NaCl a 15% (m/v), mas foi totalmente inibida pela pepstatina A. As proteases ácidas do EP apresentaram alta atividade na presença de metais pesados como o Cd2+, Hg2+, Pb2+, Al3+ e Cu2+. A utilização de EP como aditivo enzimático na extração de colágeno a partir de escamas de tilápia do Nilo dobrou o rendimento do processo. Os resultados indicam um potencial dessas proteases para aplicações industriais e biotecnológicas

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications / Estômago de Carangoides bartholomaei (Cuvier, 1833): uma fonte de proteases aspárticas para aplicações industriais e biotecnológicas

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These byproducts can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U·mg-1. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

As vísceras e outros resíduos do processamento de peixes são geralmente descartados pela indústria pesqueira. Esses resíduos podem ser uma fonte de enzimas digestivas com potencial industrial e biotecnológico. Neste estudo, objetivamos a extração, caracterização e aplicação de proteases aspárticas do estômago de Carangoides bartholomaei (Cuvier, 1833). Um extrato bruto do estômago foi obtido e submetido a um processo de purificação parcial, que obteve um Extrato Purificado (EP) com uma atividade proteolítica específica de 54,0 U·mg-1. Foi obtida uma purificação de 1,9 vezes e um rendimento de 41%. O EP apresenta duas isoformas de proteases ácidas e atividade proteolítica máxima a 45 °C e pH 2,0. A atividade proteolítica do EP foi estável na faixa de pH de 1,5 a 7,0 e temperatura de 25 °C a 50 °C. O EP manteve 35% de sua atividade proteolítica na presença de NaCl a 15% (m/v), mas foi totalmente inibida pela pepstatina A. As proteases ácidas do EP apresentaram alta atividade na presença de metais pesados como o Cd2+, Hg2+, Pb2+, Al3+ e Cu2+. A utilização de EP como aditivo enzimático na extração de colágeno a partir de escamas de tilápia do Nilo dobrou o rendimento do processo. Os resultados indicam um potencial dessas proteases para aplicações industriais e biotecnológicas.

Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766) / Obtenção e caracterização de proteases aspárticas digestórias do peixe Caranx hippos (Linnaeus, 1766)

This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

Este trabalho objetivou obter proteases aspárticas de interesse industrial e biotecnológico a partir do estômago do peixe xaréu (Caranx hippos). Para isso, foi obtido um extrato bruto do estômago, o qual foi submetido a uma purificação parcial por salting-out onde se obteve o extrato enzimático (EE). As proteases do EE foram caracterizadas físico-quimicamente e através de zimograma. Além disso, o efeito de agentes químicos sobre sua atividade também foi avaliado. Através de salting-out foi possível obter uma purificação de 1,6 vezes com rendimento de 49,4%. Foram observadas duas proteases ácidas presentes no EE através de zimograma. A temperatura ótima e a estabilidade térmica para as proteases ácidas do EE foram de 55 ºC e 45 °C, respectivamente. O pH ótimo e a estabilidade ao pH encontrados para estas enzimas foram o pH 1,5 e 7,0, respectivamente. Observou-se a inibição total da atividade proteolítica ácida do EE na presença de pepstatina A. O ditiotreitol (DTT) e o Ca2+ não promoveram efeito significativo na atividade enzimática. Na presença de metais pesados, como Al3+, Cd2+ e Hg2+, o EE manteve mais de 70% de atividade enzimática do EE. Os resultados mostram que é possível obter, a partir do estômago de C. hippos, proteases aspárticas com alta atividade proteolítica e características que demonstram potencial para aplicações industriais e biotecnológicas.

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications.

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These by-products can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U⋅mg-1. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

Red-cell alloimmunization profile in multi transfused patients: Findings and insights of a blood transfusion service.

OBJECTIVES: Blood transfusion is a key intervention for decreasing morbidity and mortality in many cases and, besides its importance, potentially fatal consequences of incompatible transfusion are a great risk to patients. This study evaluated the incidence and specificity of erythrocyte alloantibodies in multi-transfused patients enrolled at an important Regional Blood Center. MATERIALS/METHODS: This was a single-center retrospective cohort study that eveluated patients enrolled at a Regional Blood Center in a period of four years. A total of 29,128 patient samples were screened, out of which 79 (0.27%) were multiple-transfused patients with alloantibodies identified. RESULTS: The most common alloantibody found was anti-E (22.55%) followed by anti-D (14.71%), anti-C (5.88%), anti-c (5.88%), anti-e (1.96%) and anti-Cw (0.98%). We also identified combinations of alloantibodies (25.32%), 5.88% of which showed an IgG autoantibody isolated or combined with alloantibodies. The most frequent reason for the need of blood transfusion included cases of surgery, emergency and urgency (36.71%). CONCLUSIONS: A low rate of development of alloantibodies in multi-transfused patients was found, which could be a consequence of the implementation of red blood cell phenotyping for patients who may receive frequent transfusions, as in the case of some hematological neoplasms and hemoglobinopathies. However, the most common alloantibodies identified were against the Rh and/or Kell systems, with high clinical significance since both can cause delayed hemolytic transfusion reactions. Thus, the implementation of reliable antibody screening tests and the transfusion of phenotyped units for selected patients in all transfusion services represent important measures to increase transfusion safety.

Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766).

This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

Patients' and rheumatologists' perspectives on the efficacy and safety of low-dose glucocorticoids in rheumatoid arthritis-an international survey within the GLORIA study.

OBJECTIVE: To evaluate the current perspectives of patients and health professionals regarding the efficacy and safety of low-dose glucocorticoids (GCs) in RA. METHODS: Two online surveys were disseminated to patients and health professionals, in their native language, through national patient organizations and national rheumatology medical societies, respectively. SurveyMonkey®, MediGuard.org and the Glucocorticoid Low-dose Outcome in RA Study (GLORIA) website were used to offer and deliver these surveys. RESULTS: A total of 1221 RA patients with exposure to GCs, and 414 rheumatologists completed the surveys. Patients and rheumatologists reported high levels of agreement regarding the efficacy of low-dose GCs: at least 70% considered that they are very rapid and effective in the control of signs and symptoms of RA. However, half of the patients also reported having suffered serious adverse events with GCs, and 83% described concerns about safety. The majority of rheumatologists estimated that endocrine, ophthalmologic and cutaneous adverse events affect >4% of all patients treated with low-dose GCs for 2 years, based on a heat map. CONCLUSIONS: RA patients with self-reported exposure to GCs express high levels of satisfaction with low-dose GCs efficacy, as do rheumatologists. However, both expressed excessive concerns regarding the safety of GCs (greatly exceeding the published evidence data), which may compromise the optimal use of this medication. This study indicates that there is an unmet need for appropriately designed prospective trials that shed light on the real risk associated with low-dose GCs, as well as a need for renovated educational programs on the real benefits and harms of low-dose GCs, for both patients and physicians.

Biomechanical and biochemical investigation of erythrocytes in late stage human leptospirosis.

Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira, which can cause lipid changes in the erythrocyte membrane. Optical tweezers were used to characterize rheological changes in erythrocytes from patients with leptospirosis in the late stage. Biochemical methods were also used for quantification of plasma lipid, erythrocyte membrane lipid, and evaluation of liver function. Our data showed that the mean elastic constant of erythrocytes from patients with leptospirosis was around 67% higher than the control (healthy individuals), indicating that patient's erythrocytes were less elastic. In individuals with leptospirosis, several alterations in relation to control were observed in the plasma lipids, however, in the erythrocyte membrane, only phosphatidylcholine showed a significant difference compared to control, increasing around 41%. With respect to the evaluation of liver function of individuals with leptospirosis, there was a significant increase in levels of alanine transaminase (154%) and aspartate transaminase (150%), whereas albumin was 43.8% lower than control (P<0.01). The lecithin-cholesterol acyltransferase fractional activity was 3.6 times lower in individuals with leptospirosis than in the healthy individuals (P<0.01). The decrease of the erythrocyte elasticity may be related to the changes of erythrocyte membrane phospholipids composition caused by disturbances that occur during human leptospirosis, with phosphatidylcholine being a strong candidate in the erythrocyte rheological changes.