Development and characterization of a multimeric recombinant protein using the spike protein receptor binding domain as an antigen to induce SARS-CoV-2 neutralization.

BACKGROUND: SARS-CoV2 virus, responsible for the COVID-19 pandemic, has four structural proteins and 16 nonstructural proteins. S-protein is one of the structural proteins exposed on the virus surface and is the main target for producing neutralizing antibodies and vaccines. The S-protein forms a trimer that can bind the angiotensin-converting enzyme 2 (ACE2) through its receptor binding domain (RBD) for cell entry. AIMS: The goal of this study was to express in HEK293 cells a new RBD recombinant protein in a constitutive and stable manner in order to use it as an alternative immunogen and diagnostic tool for COVID-19. MATERIALS & METHODS: The protein was designed to contain an immunoglobulin signal sequence, an explanded C-terminal section of the RBD, a region responsible for the bacteriophage T4 trimerization inducer, and six histidines in the pCDNA-3.1 plasmid. Following transformation, the cells were selected with geneticin-G418 and purified from serum-fre culture supernatants using Ni2+-agarand size exclusion chromatography. The protein was structurally identified by cross-linking and circular dichroism experiments, and utilized to immunize mice in conjuction with AS03 or alum adjuvants. The mice sera were examined for antibody recognition, receptor-binding inhibition, and virus neutralization, while spleens were evaluated for γ-interferon production in the presence of RBD. RESULTS: The protein released in the culture supernatant of cells, and exhibited a molecular mass of 135 kDa with a secondary structure like the monomeric and trimeric RBD. After purification, it formed a multimeric structure comprising trimers and hexamers, which were able to bind the ACE2 receptor. It generated high antibody titers in mice when combined with AS03 adjuvant (up to 1:50,000). The sera were capable of inhibiting binding of biotin-labeled ACE2 to the virus S1 subunit and could neutralize the entry of the Wuhan virus strain into cells at dilutions up to 1:2000. It produced specific IFN-γ producing cells in immunized mouse splenocytes. DISCUSSION: Our data describe a new RBD containing protein, forming trimers and hexamers, which are able to induce a protective humoral and cellular response against SARS-CoV2. CONCLUSION: These results add a new arsenal to combat COVID-19, as an alternative immunogen or antigen for diagnosis.

Blood DNA methylation marks discriminate Chagas cardiomyopathy disease clinical forms.

Chagas disease is a parasitic disease from South America, affecting around 7 million people worldwide. Decades after the infection, 30% of people develop chronic forms, including Chronic Chagas Cardiomyopathy (CCC), for which no treatment exists. Two stages characterized this form: the moderate form, characterized by a heart ejection fraction (EF) ≥ 0.4, and the severe form, associated to an EF < 0.4. We propose two sets of DNA methylation biomarkers which can predict in blood CCC occurrence, and CCC stage. This analysis, based on machine learning algorithms, makes predictions with more than 95% accuracy in a test cohort. Beyond their predictive capacity, these CpGs are located near genes involved in the immune response, the nervous system, ion transport or ATP synthesis, pathways known to be deregulated in CCCs. Among these genes, some are also differentially expressed in heart tissues. Interestingly, the CpGs of interest are tagged to genes mainly involved in nervous and ionic processes. Given the close link between methylation and gene expression, these lists of CpGs promise to be not only good biomarkers, but also good indicators of key elements in the development of this pathology.

Blood DNA methylation marks discriminate Chagas cardiomyopathy disease clinical forms

Chagas disease is a parasitic disease from South America, affecting around 7 million people worldwide. Decades after the infection, 30% of people develop chronic forms, including Chronic Chagas Cardiomyopathy (CCC), for which no treatment exists. Two stages characterized this form: the moderate form, characterized by a heart ejection fraction (EF) ≥ 0.4, and the severe form, associated to an EF < 0.4. We propose two sets of DNA methylation biomarkers which can predict in blood CCC occurrence, and CCC stage. This analysis, based on machine learning algorithms, makes predictions with more than 95% accuracy in a test cohort. Beyond their predictive capacity, these CpGs are located near genes involved in the immune response, the nervous system, ion transport or ATP synthesis, pathways known to be deregulated in CCCs. Among these genes, some are also differentially expressed in heart tissues. Interestingly, the CpGs of interest are tagged to genes mainly involved in nervous and ionic processes. Given the close link between methylation and gene expression, these lists of CpGs promise to be not only good biomarkers, but also good indicators of key elements in the development of this pathology.

Prospecting of potentially probiotic lactic acid bacteria from bovine mammary ecosystem: imminent partners from bacteriotherapy against bovine mastitis / Prospección de bacterias ácido lácticas potencialmente probióticas del ecosistema mamario bovino: socios inminentes de la bacterioterapia contra la mastitis bovina

Mastitis is one of the most important causes of loss of cattle production, burdening producers due to the increased cost of milk production and decreased herd productivity. The development of alternative methods for the treatment and prevention of mastitis other than traditional chemical antibiotic therapy needs to be implemented to meet international pressures to reduce the use of these drugs and promote the elimination of multiresistant microbial strains from the environment. Treatment with probiotic bacteria or yeast strains offers a possible strategy for the control of mastitis. The objective of this work was to isolate, identify, and characterize lactic bacteria from milk and the intramammary duct of Gyr, Guzerat, Girolando 1/2, and Holstein cattle breeds from Brazil. Samples of 115 cows were taken, a total of 192 bacteria isolates belonging to 30 species were obtained, and 81 were selected to evaluate their probiotic potential in in vitro characterization tests. In general, bacteria isolated from the mammary gland have low autoaggregation, cell surface hydrophobicity, and co-aggregation with mastitis etiological bacteria Staphylococcus aureus and Escherichia coli. Also, they have biofilm assembly capacity, inability to produce exopolysaccharides, high production of H2O2, and strong antagonism against mastitis pathogens. Ten lactic bacteria isolates were used in co-culture with human MDA-MB-231 breast epithelial cells to assess their adhesion capacity and impairment of the S. aureus invasion. Our results, therefore, contribute to the future production of new prevention and treatment tools for bovine mastitis.(AU)

Prospecting of potentially probiotic lactic acid bacteria from bovine mammary ecosystem: imminent partners from bacteriotherapy against bovine mastitis.

Mastitis is one of the most important causes of loss of cattle production, burdening producers due to the increased cost of milk production and decreased herd productivity. The development of alternative methods for the treatment and prevention of mastitis other than traditional chemical antibiotic therapy needs to be implemented to meet international pressures to reduce the use of these drugs and promote the elimination of multiresistant microbial strains from the environment. Treatment with probiotic bacteria or yeast strains offers a possible strategy for the control of mastitis. The objective of this work was to isolate, identify, and characterize lactic bacteria from milk and the intramammary duct of Gyr, Guzerat, Girolando 1/2, and Holstein cattle breeds from Brazil. Samples of 115 cows were taken, a total of 192 bacteria isolates belonging to 30 species were obtained, and 81 were selected to evaluate their probiotic potential in in vitro characterization tests. In general, bacteria isolated from the mammary gland have low autoaggregation, cell surface hydrophobicity, and co-aggregation with mastitis etiological bacteria Staphylococcus aureus and Escherichia coli. Also, they have biofilm assembly capacity, inability to produce exopolysaccharides, high production of H2O2, and strong antagonism against mastitis pathogens. Ten lactic bacteria isolates were used in co-culture with human MDA-MB-231 breast epithelial cells to assess their adhesion capacity and impairment of the S. aureus invasion. Our results, therefore, contribute to the future production of new prevention and treatment tools for bovine mastitis.

SPHINCTEROTOMY ALONE VERSUS SPHINCTEROTOMY AND BILIARY STENT PLACEMENT IN THE TREATMENT OF BILE LEAKS: 10 YEAR EXPERIENCE AT A QUATERNARY HOSPITAL.

BACKGROUND: Hepatobiliary surgery and hepatic trauma are frequent causes of bile leaks and this feared complication can be safely managed by endoscopic retrograde cholangiopancreatography (ERCP). The approach consists of sphincterotomy alone, biliary stenting or a combination of the two but the optimal form remains unclear. OBJECTIVE: The aim of this study is to compare sphincterotomy alone versus sphincterotomy plus biliary stent placement in the treatment of post-surgical and traumatic bile leaks. METHODS: We retrospectively analyzed 31 patients with the final ERCP diagnosis of "bile leak". Data collected included patient demographics, etiology of the leak and the procedure details. The treatment techniques were divided into two groups: sphincterotomy alone vs. sphincterotomy plus biliary stenting. We evaluated the volume of the abdominal surgical drain before and after each procedure and the number of days needed until cessation of drainage post ERCP. RESULTS: A total of 31 patients (18 men and 3 women; mean age, 51 years) with bile leaks were evaluated. Laparoscopic cholecystectomy was the etiology of the leak in 14 (45%) cases, followed by conventional cholecystectomy in 9 (29%) patients, hepatic trauma in 5 (16%) patients, and hepatectomy secondary to neoplasia in 3 (9.7%) patients. The most frequent location of the leaks was the cystic duct stump with 12 (38.6%) cases, followed by hepatic common duct in 10 (32%) cases, common bile duct in 7 (22%) cases and the liver bed in 2 (6.5%) cases. 71% of the patients were treated with sphincterotomy plus biliary stenting, and 29% with sphincterotomy alone. There was significant difference between the volume drained before and after both procedures (P<0.05). However, when comparing sphincterotomy alone and sphincterotomy plus biliary stenting, regarding the volume drained and the days needed to cessation of drainage, there was no statistical difference in both cases (P>0.005). CONCLUSION: ERCP remains the first line treatment for bile leaks with no difference between sphincterotomy alone vs sphincterotomy plus stent placement.

Esfincterotomia isolada versus esfincterotomia associada a passagem de prótese biliar no tratamento de fístulas biliares: 10 anos de experiência de um hospital quaternário / Sphincterotomy alone versus sphincterotomy and biliary stent placement in the treatment of bile leaks: 10 year experience at a quaternary hospital

ABSTRACT BACKGROUND: Hepatobiliary surgery and hepatic trauma are frequent causes of bile leaks and this feared complication can be safely managed by endoscopic retrograde cholangiopancreatography (ERCP). The approach consists of sphincterotomy alone, biliary stenting or a combination of the two but the optimal form remains unclear. OBJECTIVE: The aim of this study is to compare sphincterotomy alone versus sphincterotomy plus biliary stent placement in the treatment of post-surgical and traumatic bile leaks. METHODS: We retrospectively analyzed 31 patients with the final ERCP diagnosis of "bile leak". Data collected included patient demographics, etiology of the leak and the procedure details. The treatment techniques were divided into two groups: sphincterotomy alone vs. sphincterotomy plus biliary stenting. We evaluated the volume of the abdominal surgical drain before and after each procedure and the number of days needed until cessation of drainage post ERCP. RESULTS: A total of 31 patients (18 men and 3 women; mean age, 51 years) with bile leaks were evaluated. Laparoscopic cholecystectomy was the etiology of the leak in 14 (45%) cases, followed by conventional cholecystectomy in 9 (29%) patients, hepatic trauma in 5 (16%) patients, and hepatectomy secondary to neoplasia in 3 (9.7%) patients. The most frequent location of the leaks was the cystic duct stump with 12 (38.6%) cases, followed by hepatic common duct in 10 (32%) cases, common bile duct in 7 (22%) cases and the liver bed in 2 (6.5%) cases. 71% of the patients were treated with sphincterotomy plus biliary stenting, and 29% with sphincterotomy alone. There was significant difference between the volume drained before and after both procedures (P<0.05). However, when comparing sphincterotomy alone and sphincterotomy plus biliary stenting, regarding the volume drained and the days needed to cessation of drainage, there was no statistical difference in both cases (P>0.005). CONCLUSION: ERCP remains the first line treatment for bile leaks with no difference between sphincterotomy alone vs sphincterotomy plus stent placement.

RESUMO CONTEXTO: Cirurgia hepatobiliar e trauma hepático são causas frequentes de fístulas biliares, e esta temida complicação pode ser manejada de forma segura através da colangiopancreatografia retrógrada endoscópica (CPRE). O procedimento consiste em esfincterotomia isolada, passagem de prótese biliar ou combinação das duas técnicas, porém a forma ideal permanece incerta. OBJETIVO: O objetivo desse estudo é comparar a realização de esfincterotomia isolada versus locação de prótese biliar no tratamento de fístulas pós-cirúrgicas e traumáticas. MÉTODOS: Foram analisados de forma retrospectiva 31 CPREs com diagnóstico final de "fístula biliar". A informação colhida incluía dados demográficos dos pacientes, etiologia das fístulas e detalhes dos procedimentos. As técnicas de tratamentos foram divididas em dois grupos: esfincterotomia isolada vs esfincterotomia associada a locação de prótese biliar. Foram analisados os volumes dos drenos abdominais cirúrgicos antes e depois de cada procedimento e o número de dias necessários para que ocorresse cessação da drenagem pelo dreno abdominal cirúrgico após a CPRE. RESULTADOS: Um total de 31 pacientes (18 homens e 3 mulheres; idade média de 51 anos) com fístulas biliares foram avaliados. Colecistectomia laparoscópica foi a etiologia da fístula em 14 (45%) casos, seguida de colecistectomia convenvional em 9 (29%) pacientes, trauma hepático em 5 (16%) pacientes, e hepatectomia secundária a neoplasia em 3 (9,7%) pacientes. As localizações mais frequentes das fístulas foram: coto do ducto císticos com 12 (38,6%) casos, seguido de ducto hepático comum em 10 (32%) casos, ducto colédoco em 7 (22%) cases e leito hepático em 2 (6,5%) casos. 71% dos pacientes foram tratados com esfincterotomia associada a passagem de prótese biliar e 29% com esfincterotomia isolada. Houve diferença estatística em relação ao volume drenado antes e depois de ambos os procedimentos (P<0,05). Entretanto, quando comparada esfincterotomia isolada e esfincterotomia associada a passagem de prótese biliar, em relação ao volume drenado e ao número de dias necessários para cessação da drenagem, não houve diferença estatística em ambos os casos (P>0,005). CONCLUSÃO: A CPRE permanece como tratamento de primeira linha no tratamento de fístulas biliares, sem diferença entra a esfincterotomia isolada versus esfincterotomia associada a passagem de prótese biliar.