Diagnosis of Canine Visceral Leishmaniasis by Flow Cytometry Serology using the rMELEISH Multiepitope Antigen Coupled in a Functional Bead.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonotic disease, with dogs being the main reservoir of the Leishmania infantum parasite. OBJECTIVE: To develop a new flow cytometry test to diagnosis canine VL (CVL) diagnosis. METHODS: The current study addresses a new flow cytometry test using beads coupled to the multiepitope antigen rMELEISH. RESULTS: In the study set of samples a sensitivity (87.1%) and specificity (89.9%) was observed. Considering the dogs' clinical status, 20/20 (100.0%) of the symptomatic sera tested positive, while 19/22 (86.4%) of the oligosymptomatic and 16/20 (80.0%) of asymptomatic were positive. In the non-infected control, all samples (0/30) tested as negative. In the cross-reaction control, the test was more efficient in dogs infected with L. braziliensis (2/10) and Trypanosoma cruzi (0/10), than those with Babesia canis (4/10) and Ehrlichia canis (4/10). Dogs immunized with different vaccines (Leishmune, Leish-Tec®, or LBSap) did not present serological reactivity. CONCLUSION: The flow cytometry serology through coupling the antigen rMELEISH in functional beads showed high accuracy in diagnosing CVL.

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.

Recent Patents on the Industrial Application of Alpha-amylases.

BACKGROUND: Alpha-amylases are enzymes capable of degrading polysaccharides, such as starch and glycogen. Found in various organisms, such as fungi and bacteria, these enzymes have great biotechnological potential due to their insertion in several industrial sectors ranging from food to biofuels. OBJECTIVE: The aim of this study was to analyze patents deposited in intellectual property databases on alpha-amylases in the fields of food, beverages, detergents, animal feeds, biofuels, pharmaceuticals and textiles, with the search period being 5 years. METHODS: This study targeted the use of alpha-amylases in various industrial sectors, so searches were carried out on the intellectual property database Espacenet website (European Patent Office - EPO) which contains more than 90 million patents deposited in its database. RESULTS: During the search for patents filed in the last 5 years, 186 were found related to the use of alpha-amylases. These were disturbed as follows: 84 (biofuel), 41 (drinks), 16 (pharmaceuticals), 15 (detergents), 11 (food), 10 (animal feed), 9 (textiles). From the total number of patents found, we selected 6 from each area, except pharmaceutical products, to discuss and provide information on the application of this enzyme. CONCLUSION: This study demonstrated that the sectors of beverages and animal feed have preferences for thermostable alpha-amylases while sectors such as food, biofuels and textiles only regarded the importance of enzymatic efficiency. The detergent sector presented the greatest use of alpha-amylases which had distinct biochemical characteristics as solvent resistance and thermostability. The pharmaceutical sector was the one that presented less patents related to the application of alpha amylases. In addition, this work showed that China is the country with the highest patent registration for the use of alpha-amylases in the analyzed period.

Diagnostic markers selected by immunoproteomics and phage display applied for the serodiagnosis of canine leishmaniosis.

Canine leishmaniosis (CanL) is one of the most important parasitic diseases found in several countries worldwide. Dogs are considered important domestic reservoirs of the parasites, being relevant in the maintenance of transmission cycle of the disease between sandflies and humans. However, the prevalence of asymptomatic infection is considerably higher than that of apparent clinical illness in the infected animals; thus making promptly necessary to diagnose the infection in these animals, which could help to allow to the adoption of more efficient control measures against disease. Parasitological tests, which are considered as gold standard to demonstrate the infection and diagnose the disease, present problems related with their sensitivity. Also, the sample´s collect is considered invasive. As consequence, serological tests could be applied as an additional tool to detect the asymptomatic and symptomatic CanL. For this purpose, distinct recombinant antigens have been studied; however, problems in their sensitivity and/or specificity have been still registered. The present review focus in advances in the identification of new diagnostic targets applied for the CanL diagnose, represented here by recombinant single, combined or chimeric proteins, as well as by peptides that mimic epitopes (mimotopes); which were selected by means of immunoproteomics and phage display.

A Custom-Designed Recombinant Multiepitope Protein for Human Cytomegalovirus Diagnosis.

BACKGROUND: The Human Cytomegalovirus (HCMV) has infected more than 90% of the world population and its prevalence can be related to the individuals geographical and socialeconomic status. Serological tests based on ELISA are pivotal for HCMV diagnosis. Due to the lack of standardization in the production/purification of antigens from viral preparations, ELISA tests are based on several recombinant proteins or peptides. As an alternative, multiepitope proteins may be employed. OBJECTIVE: In this work, we developed a recombinant multiepitope protein (rMEHCMV) for HCMV diagnosis based on conserved and immunodominant epitopes derived from tegument (pp150, pp65 and pp28), glycoprotein gB (pp38) and DNA polymerase subunit (pp52) of HCMV. METHODS: The rMEHCMV gene was synthesized de novo and overexpressed in Escherichia coli cells. The recombinant protein was purified to homogeneity using a Ni-NTA column. Biophysical analysis of recombinant protein was performed by circular dichroism. A preliminary biological activity test was performed using 12 positive human sera samples by using an in-house IgG ELISA. The following patents database were consulted: Espacenet, Google Patents and the National Institute of Intellectual Property (INPI, Brazil). RESULTS: The recombinant multiepitope protein was successfully expressed in E. coli. The structural data obtained by circular dichroism spectroscopy showed that rMEHCMV is structurally disordered. An in-house IgG ELISA test with rMEHCMV was successfully used to recognized IgG from human serum samples. CONCLUSION: Together, our results show that rMEHCMV should be considered as a potential antigenic target for HCMV diagnosis.

Recent Patents Applications in Red Biotechnology: A Mini-Review.

BACKGROUND: The different fields of biotechnology can be classified by colors, as a "rainbow" methodology. In this sense, the red biotechnology, focused on the preservation of health, has been outstanding in helping to solve this challenge through the provision of technologies, including diagnostic kits, molecular diagnostics, vaccines, innovations in cancer research, therapeutic antibodies and stem cells. OBJECTIVE: The main goal of this work is to highlight the different areas within the red Biotechnology. In this sense, we revised some patents regarding red biotechnology as examples to cover this subject. METHODS: A literature search of patents was performed from the followings Patents Database: INPI, USPTO, Esp@cenet, WIPO and Google Patents. RESULTS: Our analysis showed the following numbers from patents found: cancer research (8), diagnosis kit (9), vaccines (8), stem cells (9) and therapeutic antibodies (5), where the United States is the leader for most filled patents in Red Biotechnology. CONCLUSION: This mini-review has provided an update of some patents on Recent Patents in Red Biotechnology. As far as we know, this is the first mini-review report on Red Biotechnology based on patents.

Expression and Biochemical Characterization of a Yersinia intermedia Phytase Expressed in Escherichia coli.

BACKGROUND: Phytases are enzymes capable of degrading phytic acid and used in animal feed supplementation in order to improve digestibility through the release of minerals such as phosphorus. OBJECTIVE: The main goal of this study was to express and characterize a Yersinia intermedia phytase expressed in Escherichia coli cells. METHODS: The Y. intermedia phytase gene was synthesized and overexpressed in Escherichia coli cells. The phytase recombinante (rPHY) was purified to homogeneity using a Ni-NTA column. The biochemical and biophysical properties of the rPHY were measured in order to fully characterize the recombinant enzyme. The following patents database were consulted: Espacenet, USPTO, LATIPAT, Patent Scope, WIPO and Google Patents. RESULTS: The results showed that the rPHY is active at 37-40ºC and presented an optimal pH and temperature of 8.0 and 40°C, respectively. The phytase rPHY was activated by Cu2+ ion and showed resistance to trypsin and pepsin, retaining 55% of the activity at the ratio of 0.02. Furthermore, the dissociation constant (Kd = 1.1150 ± 0.0087 mM), as estimated by a fluorescence binding assay, suggests a medium affinity of the enzyme with the substrate. CONCLUSION: The results of this article can be considered as innovative and for this reason, they were protected by Intellectual Property Law in Brazil. Take together, the biochemical properties of the rPHY could be useful in future for its industrial application of this enzyme as an additive in the monogastric feed.