Inactivated and Immunogenic SARS-CoV-2 for Safe Use in Immunoassays and as an Immunization Control for Non-Clinical Trials.

Successful SARS-CoV-2 inactivation allows its safe use in Biosafety Level 2 facilities, and the use of the whole viral particle helps in the development of analytical methods and a more reliable immune response, contributing to the development and improvement of in vitro and in vivo assays. In order to obtain a functional product, we evaluated several inactivation protocols and observed that 0.03% beta-propiolactone for 24 h was the best condition tested, as it promoted SARS-CoV-2 inactivation above 99.99% and no cytopathic effect was visualized after five serial passages. Moreover, RT-qPCR and transmission electron microscopy revealed that RNA quantification and viral structure integrity were preserved. The antigenicity of inactivated SARS-CoV-2 was confirmed by ELISA using different Spike-neutralizing monoclonal antibodies. K18-hACE2 mice immunized with inactivated SARS-CoV-2, formulated in AddaS03TM, presented high neutralizing antibody titers, no significant weight loss, and longer survival than controls from a lethal challenge, despite RNA detection in the oropharyngeal swab, lung, and brain. This work emphasizes the importance of using different techniques to confirm viral inactivation and avoid potentially disastrous contamination. We believe that an efficiently inactivated product can be used in several applications, including the development and improvement of molecular diagnostic kits, as an antigen for antibody production as well as a control for non-clinical trials.

Process development and characterization of recombinant nucleocapsid protein for its application on COVID-19 diagnosis.

COVID-19 pandemic was caused by the severe acute respiratory syndrome coronavirus 2 (Sars-CoV-2). The nucleocapsid (N) protein from Sars-CoV-2 is a highly immunogenic antigen and responsible for genome packing. Serological assays are important tools to detect previous exposure to SARS-CoV-2, complement epidemiological studies, vaccine evaluation and also in COVID-19 surveillance. SARS-CoV-2 N (r2N) protein was produced in Escherichia coli, characterized, and the immunological performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and beads-based array immunoassay. r2N protein oligomers were evidenced when it is associated to nucleic acid. Benzonase treatment reduced host nucleic acid associated to r2N protein, but crosslinking assay still demonstrates the presence of higher-order oligomers. Nevertheless, after RNase treatment the higher-order oligomers reduced, and dimer form increased, suggesting RNA contributes to the oligomer formation. Structural analysis revealed nucleic acid did not interfere with the thermal stability of the recombinant protein. Interestingly, nucleic acid was able to prevent r2N protein aggregation even with increasing temperature while the protein benzonase treated begin aggregation process above 55 °C. In immunological characterization, ELISA performed with 233 serum samples presented a sensitivity of 97.44% (95% Confidence Interval, CI, 91.04%, 99.69%) and a specificity of 98.71% (95% CI, 95.42%, 99.84%) while beads-based array immunoassay carried out with 217 samples showed 100% sensitivity and 98.6% specificity. The results exhibited an excellent immunological performance of r2N protein in serologic assays showing that, even in presence of nucleic acid, it can be used as a component of an immunoassay for the sensitive and specific detection of SARS-CoV-2 antibodies.

A tríade COVID-19, cascata inflamatória e diabetes: implicações para o cuidado de enfermagem / The COVID-19 triad, inflammatory cascade and diabetes: implications for nursing care / La triada COVID-19, cascada inflamatoria y diabetes: implicaciones para la atención de enfermería

Objetivo: refletir sobre a relação entre diabetes, cascata inflamatória e COVID-19 e estratégias de cuidados de enfermagem aos pacientes diabéticos para a redução dos riscos de COVID-19 e suas complicações. Método: estudo teórico e reflexivo desenvolvido segundo as etapas do Método do Arco entre maio e julho de 2020 por participantes de grupo de apoio às pessoas diabéticas instituição de ensino do sul do Brasil. Resultados: a cascata metabólica envolvida na hiperglicemia e a resistência à insulina estão diretamente relacionadas à cascata inflamatória e à maior propensão a infecções e desfechos desfavoráveis diante da COVID-19 em diabéticos e o cuidado de enfermagem e de saúde exigem o uso de novas tecnologias para manutenção da devida atenção à saúde, destaca-se as tecnologias virtuais. Conclusão: Reafirma-se a importância do controle do diabetes, que no cenário do isolamento social encontra apoio na atenção dos profissionais da saúde e uso da tecnologia. (AU).

Objective: to reflect on the relationship between diabetes, inflammatory cascade and COVID- 19 and nursing care strategies for diabetic patients to reduce the risk of COVID-19 and its complications. Method: theoretical and reflective study developed according to the steps of the Arch Method between May and July 2020 by participants of a support group for diabetic people, an educational institution in southern Brazil. Results: the metabolic cascade involved in hyperglycemia and insulin resistance are directly related to the inflammatory cascade and greater propensity to infections and unfavorable outcomes in the face of COVID-19 in diabetics and nursing and health care require the use of new technologies to maintenance of proper health care, virtual technologies stand out. Conclusion: It reaffirms the importance of controlling diabetes, which in the scenario of social isolation finds support in the care of health professionals and the use of technology. (AU).

Objetivo: reflexionar sobre la relación entre diabetes, cascada inflamatoria y COVID-19 y las estrategias de atención de enfermería al diabético para reducir el riesgo de COVID-19 y sus complicaciones. Método: estudio teórico y reflexivo desarrollado según los pasos del Método Arco entre mayo y julio de 2020 por participantes de un grupo de apoyo a personas diabéticas, en una institución educativa de Brasil. Resultados: la cascada metabólica involucrada en la hiperglucemia y la resistencia a la insulina está relacionada con la cascada inflamatoria y una mayor propensión a infecciones y desenlaces desfavorables frente al COVID-19 y la enfermería y la atención de la salud requieren el uso de nuevas tecnologías para el mantenimiento de el cuidado. Conclusión: Se reafirma la importancia del control de la diabetes, que en el escenario de aislamiento social encuentra apoyo en la atención de los profesionales de la salud y el uso de la tecnología. (AU).

BRAF and NRAS mutated melanoma: Different Ca2+ responses, Na+/Ca2+ exchanger expression, and sensitivity to inhibitors.

Calcium is a ubiquitous intracellular second messenger, playing central roles in the regulation of several biological processes. Alterations in Ca2+ homeostasis and signaling are an important feature of tumor cells to acquire proliferative and survival advantages, which include structural and functional changes in storage capacity, channels, and pumps. Here, we investigated the differences in Ca2+ homeostasis in vemurafenib-responsive and non-responsive melanoma cells. Also, the expression of the Na+/Ca2+ exchanger (NCX) and the impact of its inhibition were studied. For this, it was used B-RAFV600E and NRASQ61R-mutated human melanoma cells. The intracellular Ca2+ chelator BAPTA-AM decreased the viability of SK-MEL-147 but not of SK-MEL-19 and EGTA sensitized NRASQ61R-mutated cells to vemurafenib. These cells also presented a smaller response to thapsargin and ionomycin regarding the cytosolic Ca2+ levels in relation to SK-MEL-19, which was associated to an increased expression of NCX1, NO basal levels, and sensitivity to NCX inhibitors. These data highlight the differences between B-RAFV600E and NRASQ61R-mutated melanoma cells in response to Ca2+ stimuli and point to the potential combination of clinically used chemotherapeutic drugs, including vemurafenib, with NCX inhibitors as a new therapeutic strategy to the treatment of melanoma.

Cytotoxicity of 4-substituted quinoline derivatives: Anticancer and antileishmanial potential.

Chemical modifications of quinoline moiety have been recognized as a useful strategy to development of new drugs. Here, the cytotoxicity of a set of twenty-four 4-substituted quinolines (named HTI) was screened for their antitumor and antileishmanial potential in vitro, and the underlying mechanisms investigated. HTI 21 and HTI 22 exhibited the highest cytotoxicity, being selected to the subsequent studies. Both derivatives induced caspase-dependent apoptosis associated to the dissipation of the mitochondrial transmembrane potential (ΔΨ) and ROS generation. HTI-induced cell death was calcium dependent, associated to thiol oxidation and cysteine proteases activation. In isolated mitochondria, HTI derivatives promoted mitochondrial permeabilization by different mechanisms. The inhibition of BCL-2 by venetoclax enhanced the HTI-induced cytotoxicity. Regarding the inhibition of cysteine proteases type B of Leishmania mexicana, HTI 15 exhibited the most potent inhibitory activity through a linear non-competitive mechanism. These data highlight the therapeutic potential of 4-substituted quinolines as antitumor and antileishmanial drugs.

In vitro and in vivo biological performance of porous Ti alloys prepared by powder metallurgy.

Titanium (Ti) and Ti-6 Aluminium-4 Vanadium alloys are the most common materials in implants composition but ß type alloys are promising biomaterials because they present better mechanical properties. Besides the composition of biomaterial, many factors influence the performance of the biomaterial. For example, porous surface may modify the functional cellular response and accelerate osseointegration. This paper presents in vitro and in vivo evaluations of powder metallurgy-processed porous samples composed by different titanium alloys and pure Ti, aiming to show their potential for biomedical applications. The porous surfaces samples were produced with different designs to in vitro and in vivo tests. Samples were characterized with scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and elastic modulus analyses. Osteogenic cells from newborn rat calvaria were plated on discs of different materials: G1-commercially pure Ti group (CpTi); G2-Ti-6Al-4V alloy; G3-Ti-13 Niobium-13 Zirconium alloy; G4-Ti-35 Niobium alloy; G5-Ti-35 Niobium-7 Zirconium-5 Tantalum alloy. Cell adhesion and viability, total protein content, alkaline phosphatase activity, mineralization nodules and gene expression (alkaline phosphatase, Runx-2, osteocalcin and osteopontin) were assessed. After 2 and 4 weeks of implantation in rabbit tibia, bone ingrowth was analyzed using micro-computed tomography (µCT). EDS analysis confirmed the material production of each group. Metallographic and SEM analysis revealed interconnected pores, with mean pore size of 99,5µm and mean porosity of 42%, without significant difference among the groups (p>0.05). The elastic modulus values did not exhibit difference among the groups (p>0.05). Experimental alloys demonstrated better results than CpTi and Ti-6Al-4V, in gene expression and cytokines analysis, especially in early experimental periods. In conclusion, our data suggests that the experimental alloys can be used for biomedical application since they contributed to excellent cellular behavior and osseointegration besides presenting lower elastic modulus.

Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor.

BACKGROUND: Leptospirosis is a zoonose that is increasingly endemic in built-up areas, especially where there are communities living in precarious housing with poor or non-existent sanitation infrastructure. Leptospirosis can kill, for its symptoms are easily confused with those of other diseases. As such, a rapid diagnosis is required so it can be treated effectively. A test for leptospirosis diagnosis using Leptospira Immunoglobulin-like (Lig) proteins is currently at final validation at Fiocruz. RESULTS: In this work, the process for expression of LigB (131-645aa) in E. coli BL21 (DE3)Star™/pAE was evaluated. No significant difference was found for the experiments at two different pre-induction temperatures (28 °C and 37 °C). Then, the strain was cultivated at 37 °C until IPTG addition, followed by induction at 28°C, thereby reducing the overall process time. Under this condition, expression was assessed using central composite design for two variables: cell growth at which LigB (131-645aa) was induced (absorbance at 600 nm between 0.75 and 2.0) and inducer concentration (0.1 mM to 1 mM IPTG). Both variables influenced cell growth and protein expression. Induction at the final exponential growth phase in shaking flasks with Abs(ind) = 2.0 yielded higher cell concentrations and LigB (131-645aa) productivities. IPTG concentration had a negative effect and could be ten-fold lower than the concentration commonly used in molecular biology (1 mM), while keeping expression at similar levels and inducing less damage to cell growth. The expression of LigB (131-645aa) was associated with cell growth. The induction at the end of the exponential phase using 0.1 mM IPTG at 28 °C for 4 h was also performed in microbioreactors, reaching higher cell densities and 970 mg/L protein. LigB (131-645aa) was purified by nickel affinity chromatography with 91% homogeneity. CONCLUSIONS: It was possible to assess the effects and interactions of the induction variables on the expression of soluble LigB (131-645aa) using experimental design, with a view to improving process productivity and reducing the production costs of a rapid test for leptospirosis diagnosis.

Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage.

The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.

The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis.

Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar Copenhageni. Immunization of Golden Syrian hamsters with Lig fragments in Freund's adjuvant induced robust antibody responses against recombinant protein and native protein, as detected by ELISA and immunoblot, respectively. A single fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of the LigA molecule, conferred immunoprotection against mortality (67-100%, P<0.05) in hamsters which received a lethal inoculum of L. interrogans serovar Copenhageni. However, immunization with this fragment did not confer sterilizing immunity. These findings indicate that the carboxy-terminal portion of LigA is an immunoprotective domain and may serve as a vaccine candidate for human and veterinary leptospirosis.

Premature ovarian failure and FRAXA premutation: Positive correlation in a Brazilian survey.

Fragile X syndrome (FRAXA) is the most common form of inherited mental retardation (MR). The mutational mechanism leading to the disease involves an expansion of a trinucleotide repeat located at the 5' UTR region of the gene FMR-1. Four types of alleles can be identified in the population, based on the number of repeats: normal (6-40), gray-zone (41-60), premutated (61-200), and fully mutated (>200). Despite only full mutations being associated with the development of the disorder, some authors propose a correlation between FRAXA premutation and the occurrence of premature ovarian failure (POF). We have undertaken a study in 58 women from 24 fragile X syndrome families ascertained for FRAXA testing. Using Southern blotting for direct DNA analysis we have identified 19 normal, 33 premutation carriers, and 6 fully mutated individuals (including 4 somatic mosaics showing premutated and fully mutated alleles). Among the premutated women, 11 experienced menopause before the age of 40 (POF), including one somatic mosaic, which was different from the ones with normal pattern who did not experience POF. Our data corroborate the notion that females carrying alleles in the premutation range are at high risk of experiencing POF.