Recombinant multiepitope proteins expressed in Escherichia coli cells and their potential for immunodiagnosis.

Recombinant multiepitope proteins (RMPs) are a promising alternative for application in diagnostic tests and, given their wide application in the most diverse diseases, this review article aims to survey the use of these antigens for diagnosis, as well as discuss the main points surrounding these antigens. RMPs usually consisting of linear, immunodominant, and phylogenetically conserved epitopes, has been applied in the experimental diagnosis of various human and animal diseases, such as leishmaniasis, brucellosis, cysticercosis, Chagas disease, hepatitis, leptospirosis, leprosy, filariasis, schistosomiasis, dengue, and COVID-19. The synthetic genes for these epitopes are joined to code a single RMP, either with spacers or fused, with different biochemical properties. The epitopes' high density within the RMPs contributes to a high degree of sensitivity and specificity. The RMPs can also sidestep the need for multiple peptide synthesis or multiple recombinant proteins, reducing costs and enhancing the standardization conditions for immunoassays. Methods such as bioinformatics and circular dichroism have been widely applied in the development of new RMPs, helping to guide their construction and better understand their structure. Several RMPs have been expressed, mainly using the Escherichia coli expression system, highlighting the importance of these cells in the biotechnological field. In fact, technological advances in this area, offering a wide range of different strains to be used, make these cells the most widely used expression platform. RMPs have been experimentally used to diagnose a broad range of illnesses in the laboratory, suggesting they could also be useful for accurate diagnoses commercially. On this point, the RMP method offers a tempting substitute for the production of promising antigens used to assemble commercial diagnostic kits.

Pharmacophoric characteristics of dengue virus NS2B/NS3pro inhibitors: a systematic review of the most promising compounds.

Dengue virus (DENV) infection can lead to a wide range of clinical manifestations, including fatal hemorrhagic complications. There is a need to find effective pharmacotherapies to treat this disease due to the lack of specific immunotherapies and antiviral drugs. That said, the DENV NS2B/NS3pro protease complex is essential in both the viral multiplication cycle and in disease pathogenesis, and is considered a promising target for new antiviral therapies. Here, we performed a systematic review to evaluate the pharmacophoric characteristics of promising compounds against NS2B/NS3pro reported in the past 10 years. Online searches in the PUBMED/MEDLINE and SCOPUS databases resulted in 165 articles. Eight studies, which evaluated 3,384,268 molecules exhibiting protease inhibition activity, were included in this review. These studies evaluated anti-dengue activity in vitro and the IC50 and EC50 values were provided. Most compounds exhibited non-competitive inhibition. Cytotoxicity was evaluated in BHK-21, Vero, and LLC-MK2 cells, and the CC50 values obtained ranged from < 1.0 to 780.5 µM. Several groups were associated with biological activity against dengue, including nitro, catechol, halogen and ammonium quaternaries. Thus, these groups seem to be potential pharmacophores that can be further investigated to treat dengue infections.

Dengue outbreaks in Divinopolis, south-eastern Brazil and the geographic and climatic distribution of Aedes albopictus and Aedes aegypti in 2011-2012.

OBJECTIVE: To entomologically monitor Aedes spp. and correlate the presence of these vectors with the recent epidemic of dengue in Divinopolis, Minas Gerais State, Brazil. METHODS: Ovitraps were installed at 44 points in the city, covering six urban areas, from May 2011 to May 2012. After collection, the eggs were incubated until hatching. In the 4th stage of development, the larvae were classified as Ae. aegypti or Ae. albopictus. RESULTS: In total, 25 633 Aedes spp. eggs were collected. February was the month with the highest incidence, with 5635 eggs collected and a hatching rate of 46.7%. Ae. aegypti eggs had the highest hatching rate, at 72.3%, whereas Ae. albopictus eggs had 27.7%. Climate and population density influenced the number of eggs found. Indicators of vector presence were positively correlated with the occurrence of dengue cases. CONCLUSION: These data reinforce the need for entomological studies, highlight the relevance of Ae. albopictus as a possible disease vector and demonstrate its adaptation. Ae. albopictus, most commonly found in forested areas, comprised a substantial proportion of the urban mosquito population.

A Biotechnological Review on Patents Applied to Rubella Diagnosis.

BACKGROUND: Rubella, caused by the Rubella virus (RV), is considered a mild self-limited illness. However, RV has teratogenic potential. Laboratory investigation plays an important role in both diagnosis and surveillance of the disease. The main methods for diagnosing Rubella are serological assays for the detection of specific IgM and molecular assays for detecting viral RNA. However, some laboratories perform IgG avidity testing, virus isolation and analysis of genetic sequence as tools to help Rubella eradication. The importance of the diagnosis of Rubella involves the appropriate treatment of the disease, because the Rubella clinical symptoms may be similar to those of other diseases, and the population monitoring to avoid new emergent cases. This study addresses different methods of diagnosing Rubella and contributes as a source of knowledge to assist health systems in controlling the disease. OBJECTIVE: The main objective of this study was to review the available patents regarding Rubella diagnosis published in intellectual property databases, and provides an overview of the technologies available for the diagnosis of Rubella. METHOD: The search strategy was based on the keywords searched separately or together using a Boolean operator either in the patent title or abstract the time interval was restricted to patents filed or granted from January 2009 until February 2022. The database used was Google Patents. RESULTS: This study analyzed 24 patent documents regarding strategies for the diagnosis of Rubella. Of these, 15 patents disclose strategies for detecting Rubella antibodies, 7 patents the detection of Rubella virus nucleic acid, and 2 patents the production of antibodies applied in Rubella diagnosis. CONCLUSION: Rubella is still a public health problem in some countries, mainly those in development, especially due to congenital Rubella syndrome, which can cause malformation or fetal death. However, its diagnosis is challenging, due to similarity of symptoms with other diseases, and for this reason, laboratory diagnosis is essential. Studies like this encourage researchers and governments to invest in research to continue the development of new products, using different areas of biotechnology, to solve society's problems, especially diseases that have an impact on global health, such as Rubella.

A Mini-review on Potentials Proteins/Peptides Applied for Serodiagnosis of Human Monkeypox Infection and Future Trends.

Monkeypox is a zoonosis that re-emerged in 2022, generating cases in non-endemic countries for the disease and creating a public health issue. The rapid increase in the number of cases kindles a need for quick, inexpensive diagnostic tests for the epidemiological control of the disease. The high cost of molecular tests can make this control more difficult to access in poorer regions, with immunological tests being a more viable option. In this mini-review, a search was conducted in the main databases for peptide and protein options that could be used in the development of serological diagnostic tests. Nine viable registres were found, and seven were selected (two patents and five studies). The main studies used the B21R peptide sequence as it is a high immunogenic epitope. In addition, studies on the improvement of these sequences were also found to avoid cross-reactions against other viruses of the same family, proposing a rational approach using multiepitope recombinant proteins. These approaches demonstrated high sensitivity and specificity values and are seen as viable options for developing new tests. New effective serological testing options, when combined with awareness, disease surveillance, early diagnosis, and rapid communication, form a set of key strategies used by health systems to control the spread of the monkeypox virus.

A mini-review on ELISA-Based Diagnosis of Schistosomiasis.

BACKGROUND: schistosomiasis is a neglected tropical parasitic disease caused by trematode worms of the genus schistosoma, which affects approximately 240 million people worldwide. the diagnosis of the disease can be performed by parasitological, molecular, and/or immunological methods, however, the development of new diagnostic methods still essential to guide policy decisions, monitor disease trends and assess the effectiveness of interventions. OBJECTIVE: in this sense, the current work summarizes the findings of a systematic review regarding antigens applied in the enzyme-linked immunosorbent assay test, which were patented and published over the last ten years. METHODS: the literature search strategy used medical subject heading (mesh) terms to define as descriptors. "schistosoma mansoni" was used in arrangement with the descriptors "immunoassay", "enzyme-linked immunosorbent assay", "elisa", and "antigens", using the "and" connector. the patent search was done using keywords, including diagnosis and schistosoma or schistosomiasis or schistosome. several databases were employed for the patent search, such as intellectual property national institute; european patent office; the united states patent and trademark office; patent scope, and google patents. RESULTS: forty-one articles were retrieved, of which only five met the eligibility criteria. seventeen patents were taken from the databases, and a brief description of the most relevant inventions is given here. CONCLUSION: schistosomiasis is considered the most important helminthic disease in worldwide. therefore, it is important to of searching for and develops diagnostic methods based on serology to reduce morbidity and mortality caused by the disease.

Proof of Concept of a Novel Multiepitope Recombinant Protein for the Serodiagnosis of Patients with Chagas Disease.

Chagas disease remains a neglected disease that is considered to be a public health problem. The early diagnosis of cases is important to improve the prognosis of infected patients and prevent transmission. Serological tests are the method of choice for diagnosis. However, two serological tests are currently recommended to confirm positive cases. In this sense, more sensitive and specific serological tests need to be developed to overcome these current diagnosis problems. This study aimed to develop a new recombinant multiepitope protein for the diagnosis of Chagas disease, hereafter named rTC. The rTC was constructed based on amino acid sequences from different combinations of Trypanosoma cruzi antigens in the same polypeptide and tested using an enzyme-linked immunosorbent assay (ELISA) to detect different types of Chagas disease. rTC was able to discriminate between indeterminate (IND) and cardiac (CARD) cases and cross-reactive diseases, as well as healthy samples, with 98.28% sensitivity and 96.67% specificity, respectively. These data suggest that rTC has the potential to be tested in future studies against a larger serological panel for the diagnosis of Chagas disease.

Patents Related to Pathogenic Human Coronaviruses.

BACKGROUND: Coronaviruses have caused outbreaks of respiratory disease since the beginning of the 21st century, representing a significant threat to public health. Together, the severe acute respiratory syndrome coronavirus (SARS-CoV), the respiratory syndrome coronavirus (MERS-CoV), and, more recently, the novel coronavirus (SARS-CoV-2) have caused a large number of deaths around the world. Thus, investments in research and the development of strategies aimed at diagnosing, treating, and preventing these infections are urgently needed. OBJECTIVE: The objective of this study was to analyze the patents that address pathogenic coronaviruses in Google Patents databases in the last year (2019-2020). METHODS: The search strategy was carried out in April 2020, based on the keywords "SARS", "SARS-CoV", "MERS", "MERS-CoV", "SARS-CoV-2" and "COVID-19. Out of the patents examined, 25 were selected for a short description in this study. RESULTS: A total of 191 patents were analyzed, 149 of which were related to SARS-CoV, and 29 and 12 were related to MERS-CoV and SARS- CoV2, respectively. The patents addressed the issues of diagnosis, therapeutic agents, prevention and control, along with other applications. CONCLUSION: Several promising strategies have been documented in intellectual property databases favoring the need for further studies on the pathogenesis and optimization of the diagnosis and therapeutic treatment for these emerging infections.

Bauhinia holophylla (Bong.) Steud. leaves-derived extracts as potent anti-dengue serotype 2.

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen and made the disease a major health concern worldwide. However, specific antiviral drugs against this arbovirose or vaccines are not yet available for treatment or prevention. Thus, here we aimed to study the antiviral activity of hydroethanolic extract, fraction ethyl acetate and subfractions of the leaves of Bauhinia holophylla (Fabaceae:Cercideae), a native plant of the Brazilian Cerrado, against DENV-2 by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method in mammalian cells culture. As results, the hydroethanolic extract showed the most potent effect, with an inhibitory concentration (IC50) of 3.2 µg mL-1 and selectivity index (SI) of 27.6, approximately 16-times higher anti-DENV-2 activity than of the ribavirin (IC50 52.8 µg mL-1). Our results showed in this study appointed that B. holophylla has a promising anti-dengue activity, which was associated mainly with the presence of flavonoids.

Heated distilled water with or without continuous ultrasonic irrigation improves final irrigation efficacy and reduces dentine erosion.

OBJECTIVES: The present study evaluated the effects of using heated distilled water (HDW), with or without continuous ultrasonic irrigation (CUI), on smear layer removal and deterioration of root dentine microstructure. MATERIALS AND METHODS: After chemomechanical preparation, 60 human teeth were longitudinally cleaved into two halves for smear layer quantification at the cervical-third, middle-third and apical root-third of the canal space. After reassembly, the root canals were irrigated with 17 % EDTA, followed by one of the six final irrigation protocols (n = 10) - G1: conventional irrigation (CI)+NaOCl at 25 °C; G2: NaOCl at 25 °C + CUI; G3: CI with DW at 25 °C; G4: DW at 25 °C + CUI; G5: CI + HDW at 65 °C; G6: HDW at 65 °C + CUI. Tooth-halves were processed and examined by scanning electron microscopy. The percentage of opened dentinal tubules in the irrigated areas of the canal space was expressed as a percentage of the total surface area. Dentine erosion was classified by numeric scores. Smear layer removal was analysed with ANOVA and Student-Newman-Keuls tests; dentine erosion was analysed with Cohran-Mantel-Haenstel statistic (α = 0.05). RESULTS: The cervical-third had a higher percentage of open dentinal tubules for all groups (p < 0.05). In G2, the middle-third had a lower percentage of open tubules, which was significantly different from the other groups (p < 0.05). Groups that utilised NaOCl as the final irrigant had more extensive erosion when CUI was used. CONCLUSION: Heated distilled water, with or without CUI, was as efficient as 1 % NaOCl in the final cleaning of the instrumented canal space. The use of HDW produces less extensive dentine erosion. CLINICAL SIGNIFICANCE: Heated distilled water removes smear layers with less deleterious effects on dentine microstructure and may be considered a final irrigant after mechanical preparation and EDTA chelation.

Effect of different water-to-powder ratios on the dimensional stability and compressive strength of mineral aggregate-based cements.

PURPOSE: The aim of this study was to evaluate the effect of different water-to-powder ratios on the dimensional stability and compressive strength of Portland cement and Mineral Trioxide Aggregate (MTA). MATERIALS AND METHODS: Five different volumes of distilled water (0.26; 0.28; 0.30; 0.33 and 0.35 mL) were used for every 1 g of the cements. Twelve samples (12 mm long x 6 mm in diameter) were prepared in Teflon molds. After measuring the initial length, the specimens were stored in distilled water for 24 hours or 30 days. At the end of these time intervals, the specimens were measured again, and the dimensional change was calculated. The same samples used in the previous test were submitted to compression in a universal test machine (1 mm/min-1). RESULTS: Analysis of the dimensional stability results showed no statistical difference between the cements, proportions and time intervals tested, or between their interactions. After 24 hours, MTA was more resistant than Portland cement (p<0.05). At 30 day-period, both cements had similar, and significantly higher resistance than they did at 24 hours (p<0.05). CONCLUSION: The powder/water ratio had no influence on the dimensional stability of cements. Compressive strength of Portland cement was affected at the proportions of 0.30 and 0.35 mL/g.