Synthesis of new non-natural L-glycosidic flavonoid derivatives and their evaluation as inhibitors of Trypanosoma cruzi ecto-nucleoside triphosphate diphosphohydrolase 1 (TcNTPDase1).

Trypanosoma cruzi is the pathogen of Chagas disease, a neglected tropical disease that affects more than 6 million people worldwide. There are no vaccines to prevent infection, and the therapeutic arsenal is very minimal and toxic. The unique E-NTPDase of T. cruzi (TcNTPDase1) plays essential roles in adhesion and infection and is a virulence factor. Quercetin is a flavonoid with antimicrobial, antiviral, and antitumor activities. Its potential as a partial inhibitor of NTPDases has also been demonstrated. In this work, we synthesized the non-natural L-glycoside derivatives of quercetin and evaluated them as inhibitors of recombinant TcNTPDase1 (rTcNTPDase1). These compounds, and quercetin and miquelianin, a natural quercetin derivative, were also tested. Compound 16 showed the most significant inhibitory effect (94%). Quercetin, miquelianin, and compound 14 showed inhibition close to 50%. We thoroughly investigated the inhibitory effect of 16. Our data suggested a competitive inhibition with a Ki of 8.39 µM (± 0.90). To better understand the interaction of compound 16 and rTcNTPDase1, we performed molecular dynamics simulations of the enzyme and docking analyses with the compounds. Our predictions show that compound 16 binds to the enzyme's catalytic site and interacts with important residues for NTPDase activity. As an inhibitor of a critical T. cruzi enzyme, (16) could be helpful as a starting point in the developing of a future treatment for Chagas disease. Furthermore, the discovery of (16) as an inhibitor of TcNTPDase1 may open new avenues in the study and development of new inhibitors of E-NTPDases.

Heterologous expression and biochemical characterization of the recombinant nucleoside triphosphate diphosphohydrolase 2 (LbNTPDase2) from Leishmania (Viannia) braziliensis.

Leishmania braziliensis is a pathogenic protozoan parasite that causes American Tegumentary Leishmaniasis (ATL), an important tropical neglected disease. ENTPDases are nucleotidases that hydrolyze intracellular and/or extracellular nucleotides. ENTPDases are known as regulators of purinergic signalling induced by extracellular nucleotides. Leishmania species have two isoforms of ENTPDase, and, particularly, ENTPDase2 seems to be involved in infectivity and virulence. In this study, we conducted the heterologous expression and biochemical characterization of the recombinant ENTPDase2 of L. braziliensis (rLbNTPDase2). Our results show that this enzyme is a canonical ENTPDase with apyrase activity, capable of hydrolysing triphosphate and diphosphate nucleotides, and it is dependent on divalent cations (calcium or magnesium). Substrate specificity was characterized as UDP>GDP>ADP>GTP>ATP=UTP. The enzyme showed optimal activity at a neutral to basic pH and was partially inhibited by suramin and DIDS. Furthermore, the low apparent Km for ADP suggests that the enzyme may play a role in adenosine-mediated signalling. The biochemical characterization of this enzyme can open new avenues for using LbNTPDase2 as a drug target.

Urea, salts, and Tween 20 influence on adsorption of IgG and Leishmania rNTPDase2 to nitrocellulose.

Lateral flow immunochromatography is a widely used technique for immunological assays. Construction of test and control lines is mostly done by antigen adsorption to nitrocellulose membranes, a process not fully understood. This study aimed to evaluate the influence of urea, salts, and Tween 20, on adsorption. The performance of canine IgG in water and in buffer containing urea and salts (pH 8.3) were compared to observe if the interferents would lead to protein stripping when challenged with increasing concentrations of Tween 20 in the lateral flow buffer. Immobilization of the rLiNTPDase2, an antigen for Canine Leishmaniasis diagnosis, was evaluated and compared to the rLbNTPDase2 by the same method. There were no differences between adsorption coefficients of IgG in water and in buffer, but high salt and urea concentrations seems to stabilize and enhance IgG immobilization. Adsorption performance between canine IgG and rNTPDases had different patterns, but was highly similar between rNTPDases, indicating that protein identity may have an important role. Also, low concentrations of Tween 20 in the flow solution may aid the maintenance of rNTPDase2 on the strips. Our results bring insights about protein adsorption and perspectives about the influence of urea, salts and Tween 20 on this process.

Genetic diversity of porcine circovirus 3 strains and the first detection of two different PCV3 strains coinfecting the same host in Minas Gerais, Brazil.

Porcine circovirus 3 (PCV3) is a recently emerged circovirus discovered in 2016 that has drawn the attention of the swine industry worldwide. In this study, we evaluated the genetic diversity of PCV3 strains on pig farms. A total of 261 samples from sows, weaning pigs, growing pigs, and stillborn/mummified fetuses were analyzed by quantitative real-time PCR. The results revealed that at least two main lineages of PCV3 are circulating in Brazil. For the first time, it was possible to detect the presence of two different PCV3 strains in the same host.

Effect of the topical administration of N-(2-(4-bromophenylamino)-5-(trifluoromethyl)phenyl)nicotinamide compound in a murine subcutaneous melanoma model.

Conventional treatments for metastatic melanomas are still ineffective and generate numerous side effects, justifying the search for new therapies. The antimetastatic effect of the named N-(2-(4-bromophenylamino)-5-(trifluoromethyl)phenyl)nicotinamide (SRVIC30) compound has been previously demonstrated in murine melanoma. Herein, we aimed to evaluate its effect when topically administrated in a murine subcutaneous melanoma model. For that, mice C57BL/6 were injected subcutaneously with 2 × 10 B16-F10 cells. Topical treatment began when tumors became visible on animal's back. Therefore, tumor volume was measured three times a week until it reaches 12 mm approximately. At this point, 40 mg oil-in-water cream (Lanette) without (control mice; n = 10) or with SRVIC30 compound (SRVIC30 group; n = 10 animals) were spread daily over the tumor external surface using a small brush for 14 days. The treatments increased the percentage of peroxidase antioxidant enzyme and dead cells via caspase-3 activation, with a consequent deposit of collagen fibers in the tumors. In addition, the skin of treated animals showed the presence of inflammatory infiltrate. Finally, SRVIC30 did not show signs of toxicity. Thus, we concluded that the topic administration of SRVIC30 was able to influence crucial anticancer processes such as tumor cells apoptosis and surrounding microenvironment.

Correction to: Genetic variation of Mycoplasma hyopneumoniae from Brazilian field samples.

Following publication of the original article [1], we have been notified that two of the author names were incomplete or incorrect.

Genetic variation of Mycoplasma hyopneumoniae from Brazilian field samples.

BACKGROUND: Porcine enzootic pneumonia is a worldwide problem in swine production. The infected host demonstrates a respiratory disease whose etiologic agent is Mycoplasma hyopneumoniae (Mhp). A total of 266 lung samples with Mycoplasma-like lesions were collected from two slaughterhouses. We analyzed the genetic profile of Mhp field samples using 16 genes that encode proteins involved in the mechanisms of bacterial pathogenesis and/or the immune responses of the host. Bioinformatic analyses were performed to classify the Mhp field samples based on their similarity according to the presence of the studied genes. RESULTS: Our results showed variations in the frequency of the 16 studied genes among different Mhp field samples. It was also noted that samples from the same farm were genetically different from each other and samples from different regions could be genetically similar, which is evidence of the presence of different genetic profiles among the Mhp field strains that circulate in Brazilian swine herds. CONCLUSION: This work demonstrated the genetic diversity of several Mhp field strains based on 16 selected genes related to virulence and/or immune response in Brazil. Our findings demonstrate the difference between Mhp field strains could influence the virulence, and we hypothesize that the most frequent genes in Mhp field strains could possibly be used as vaccine candidates. Based on our results, we suspect that Mhp genetic variability may be associated with the frequency of genes among the field strains and we have demonstrated that some Mhp field samples could not have many important genes described in the literature.

Antimetastatic effect of the pharmacological inhibition of serine/arginine-rich protein kinases (SRPK) in murine melanoma.

The Serine/arginine-rich protein kinases (SRPK) are involved in pre-mRNA splicing control through the phosphorylation of the SR protein family of splicing factors. Over the last years, several studies have shown the relevance of SRPK for human cancers and their potential as promising drug targets. In this context, we have previously selected three trifluoromethyl arylamides (named here as SRVIC24, SRVIC30 and SRVIC36) with improved in vitro antileukemia effect and ability of impairing the cellular activity of SRPK. Given the increasing amount of reports on the implication of these kinases in metastatic cancers, in this study, we have evaluated the antimetastatic effect of these compounds and the known SRPK inhibitor (SRPIN340) on a murine model of metastatic melanoma. The compounds were able to impact the melanoma cell metastatic behavior by decreasing migration, invasion, adhesion, and colony formation in in vitro assays. Also, they presented antimetastatic in vivo activity, without apparent signs of systemic toxicity after treatments, as revealed by the histology of organs and analysis of key serum biochemical markers. Moreover, the effect of the treatments on SRPK1 nuclear translocation and SR protein phosphorylation was observed. Finally, molecular docking studies were carried out to gain structural information on the SRPK-compound complexes. Together, these data suggest that SRPK pharmacological inhibition should be considered as an interesting therapeutic strategy against metastatic cancers.

Utilization of phage display to identify antigenic regions in the PCV2 capsid protein for the evaluation of serological responses in mice and pigs.

Porcine circovirus 2 (PCV2) is associated with a series of swine diseases. There is a great interest in improving our understanding of the immunology of PCV2, especially the properties of the viral capsid protein Cap-PCV2 and how they relate to the immunogenicity of the virus and the subsequent development of vaccines. Phage display screening has been widely used to study binding affinities for target proteins. The aim of this study was to use phage display screening to identify antigenic peptides in the PCV2 capsid protein. After the selection of peptides, five of them presented similarity to sequences found in cap-PCV2, and four peptides were synthesized and used for immunization in mice: 51-CTFGYTIKRTVT-62 (PS14), 127-CDNFVTKATALTY-138 (PS34), 164-CKPVLDSTIDY-173 (PC12), and 79-CFLPPGGGSNT-88 (PF1). Inoculation with the PC12 peptide led to the highest production of antibodies. Furthermore, we used the PC12 peptide as an antigen to examine the humoral response of swine serum by ELISA. The sensitivity and specificity of this assay was 88.9% and 92.85%, respectively. Altogether, characterization of immunogenic epitopes in the capsid protein of PCV2 may contribute to the improvement of vaccines and diagnostics.

Evolutionary analysis of Porcine circovirus 3 (PCV3) indicates an ancient origin for its current strains and a worldwide dispersion.

Porcine circovirus 3 (PCV3) is an emerging virus that was identified in the United States in 2016. Since its first identification, PCV3 has been identified in Brazil, China, United States, Poland, and Republic of Korea. In this study, we used molecular phylogenetic analysis of available sequences to address questions surrounding the emergence of PCV3 in porcine world industry. Our data indicate that PCV3 did not emerge through recombination events among currently known circoviruses and that its speciation is not a recent evolutionary event. The most common recent ancestor analysis suggests that PCV3 lineages have emerged over the past 50 years. PCV3 is not genetically closely related with other Porcine circovirus and it has been evolving undetected for some time in swine and probably in bovine population. We also found groups of genetically related isolates of PCV3 originated from different countries that may be associated with dispersal routes, suggesting that PCV3 has already been circulating in pig-producing countries for some time before its first detection.

Evaluation of the genetic variability found in Brazilian commercial vaccines for infectious bronchitis virus.

Infectious bronchitis virus (IBV) is currently one of the most important pathogens in the poultry industry. The H120 and Ma5 are the only viral strains approved by the Brazilian government as the constituent of vaccines. Despite the systematic vaccination in Brazil, IBV has not yet been controlled and diseases associated with this virus have been reported in vaccinated chickens. Here, we investigated the genetic variability of H120 and Ma5 strains present in the IBV vaccines from different Brazilian manufacturers. We performed DNA sequencing analyses of the S1 spike glycoprotein gene to investigate its genetic variability and the presence of viral subpopulations among vaccines, between batches, and also in each vaccine after a single passage was performed in chicken embryonated eggs. Our results revealed up to 13 amino acid substitutions among vaccines and some of them were localized in regions of the S1 glycoprotein that play a role in virus-host interaction. Secondary nucleotide peaks identified in the chromatogram for the S1 gene sequence revealed that all original vaccines (H120 and Ma5) were composed by different subpopulations of IBV. Moreover, new viral subpopulations were also found in vaccines after a single passage in chicken embryonated eggs. These findings indicate that H120 and Ma5 viral strains used in vaccines market in Brazil can still mutate very rapidly during replication, leading to amino acid substitutions in proteins involved in the stimulation of the immune response, such as the S1 glycoprotein. Therefore, our data suggest that the genetic variability of these viral strains should be taken into consideration to ensure an effective immune response against IBV.

6-methylmercaptopurine riboside, a thiopurine nucleoside with antiviral activity against canine distemper virus in vitro.

BACKGROUND: Canine distemper (CD) is a widespread infectious disease that can severely impact a variety of species in the order Carnivora, as well as non-carnivore species such as non-human primates. Despite large-scale vaccination campaigns, several fatal outbreaks have been reported in wild and domestic carnivore populations. This, in association with expansion of the disease host range and the development of vaccine-escape strains, has contributed to an increased demand for therapeutic strategies synergizing with vaccine programs for effectively controlling canine distemper. 6-methylmercaptopurine riboside (6MMPr) is a modified thiopurine nucleoside with known antiviral properties against certain RNA viruses. METHODS: We tested the inhibitory effects of 6MMPr against a wild-type CDV strain infection in cell culture. We measured infectious particle production and viral RNA levels in treated and untreated CDV-infected cells. Ribavirin (RIB) was used as a positive control. RESULTS: Here, we report for the first time the antiviral effects of 6MMPr against canine distemper virus (CDV) in vitro. 6MMPr was able to reduce viral RNA levels and to inhibit the production of infectious CDV particles. The therapeutic selectivity of 6MMPr was approximately six times higher than that of ribavirin. CONCLUSION: Our results indicate that 6MMPr has high anti-CDV potential and warrants further testing against other paramyxoviruses, as well as clinical testing of the compound against CDV.

Achievement of constitutive fluorescent pLEXSY-egfp Leishmania braziliensis and its application as an alternative method for drug screening in vitro.

BACKGROUND: Gene reporter-fluorescent cells have emerged as alternative method for drug screening. OBJECTIVE: Achievement of constitutive expression of fluorescent protein GFP by Leishmania braziliensis as alternative method for drug screening. METHODS: L. braziliensis-GFP was generated using Leishmania tarentolae pLEXSY-egfp for constitutive expression of GFP. Fluorescent cells were selected and subjected to standardisation tests of anti-promastigote and anti-intracellular amastigote assays. FINDINGS: Our results showed that L. braziliensis-GFP method is faster and more sensitive than Allamar Blue-resazurin. MAIN CONCLUSION: Transfected parasites maintained stable fluorescence after successive in vitro passages and pLEXSY system can be used to achieve non-L. tarentolae fluorescent cells.

Distribution of infectious bronchitis virus strains in different organs and evidence of vertical transmission in natural infection.

On the basis of partial sequencing of the infectious bronchitis virus (IBV) S1 gene, this study investigated the molecular diversity of the virus in two life periods of a batch of breeding hens at the field level. The chicks were vaccinated against IBV on the second day of life with the vaccine Ma5, but at the age of 18 days, they exhibited clinical signs and macroscopic lesions compatible with avian infectious bronchitis (IB). In the clinical disease stage, the Ma5 vaccine strain was detected in the trachea, lungs, and small intestine of the chicks, while IBV variants were detected in the bursa of Fabricius and kidneys. Subsequently, new samples were collected from the same batch at the end of the production cycle. In this phase, the Ma5 vaccine strain was detected in the kidneys, small intestine, and oviduct of the hens. However, a previously unidentified IBV variant was found in the cecal tonsils. Additionally, a fragment of viral RNA with that was completely identical to the corresponding region of the Ma5 vaccine was detected in the allantoic fluid of viable embryos from the hens under study after 18 days of incubation. These findings suggest that, in addition to the Ma5 vaccine, other strains of IBV variants can coexist, seeming to establish a chronic infection in the chickens, and that they can potentially be transmitted vertically. These results may assist in immunoprophylaxis control programs against IBV.

The genome of Anopheles darlingi, the main neotropical malaria vector.

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector-human and vector-parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.

Leishmania infantum NTPDase1 and NTPDase2 play an important role in infection and nitric oxide production in macrophages.

Leishmania infantum, the causative agent of American Visceral Leishmaniasis (VL), is known for its ability to modulate the host immune response to its own favor. Ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) represents a family of enzymes that hydrolyze nucleotides and are involved in nucleotide-dependent biological processes. L. infantum has two ENTPDases, namely LiNTPDase1 and LiNTPDase2. Here, we used genetic tools to overexpress or abolish the expression of LiNTPDase1 and -2 to assess their role in parasite growth in culture and macrophage infection. While LiNTPDase1 or 2-overexpressing clones showed no morphological or growth changes in promastigotes, LiNTPDase2 overexpression increased macrophage adhesion and infection by 50% and 30%, respectively. The individual LiNTPDase1 and 2 knockout mutants showed lag in growth profile, which was reversed by the addition of adenine and guanine to the culture media. Moreover, the morphology of the knockout mutants even in supplemented media was changed to an amastigote-like form. The double knockout of both genes was lethal and a mechanism of compensation of deletion of one isoform was detected in these mutants. Correspondingly, the absence of LiNTPDase1 or LiNTPDase2 led to a dramatic reduction in in vitro infection (∼90%). Interestingly, nitric oxide production was decreased in both knockout mutants during infection, which suggests that both LiNTPDases can inhibit macrophage responses against the parasite. Overall, our results show important roles of LiNTPDase1 and -2 concerning in vitro macrophage infection and reinforce their use as potential targets to control Leishmania infections.

First evidence of a serine arginine protein kinase (SRPK) in leishmania braziliensis and its potential as therapeutic target.

Leishmaniasis is a parasitic disease found in tropical and subtropical regions around the world, caused by parasites of the genus Leishmania. The disease is a public health concern and presents clinical manifestations that can cause death, disability, and mutilation. The parasite has promastigote (vector) and amastigote (vertebrate host) forms and kinase enzymes are involved in this differentiation process. In the present investigation, we show, for the first time, evidence of a serine/arginine protein kinase in Leshmania braziliensis (LbSRPK). Our results show that amastigotes express more LbSRPK than promastigotes.  Analogues of SRPIN340 (a known inhibitor of SRPK) were evaluated for their leishmanicidal activity and two of them, namely SRVIC22 and SRVIC32 showed important leishmanicidal activity in vitro. SRVIC22 and SRVIC32 were able to reduce the infection rate in macrophages and the number of intracellular amastigotes by 55 and 60%, respectively. Bioinformatics analysis revealed the existence of two different amino acid residues in the active site of LbSRPK compared to their human homologue (Tyr/Leu-and Ser/Tyr), which could explain the absence of leishmanicidal activity of SRPIN340 on infected macrophages. In order to enhance leishmanicidal activity of the analogues, optimizations were proposed in the structures of the ligands, suggesting strong interactions with the catalytic site of LbSRPK. Although the evidence on the action of inhibitors upon LbSRPK is only indirect, our studies not only reveal, for the first time, evidence of a SRPK in Leishmania, but also shed light on a new therapeutic target for drug development.

Immunomodulatory activity of trifluoromethyl arylamides derived from the SRPK inhibitor SRPIN340 and their potential use as vaccine adjuvant.

The serine/arginine-rich protein kinases (SRPK) specifically phosphorylate their substrates at RS-rich dipeptides, which are abundantly found in SR splicing factors. SRPK are classically known for their ability to affect the splicing and expression of gene isoforms commonly implicated in cancer and diseases associated with infectious processes. Non-splicing functions have also been attributed to SRPK, which highlight their functional plasticity and relevance as therapeutic targets for pharmacological intervention. In this sense, different SRPK inhibitors have been developed, such as the well-known SRPIN340 and its derivatives, with anticancer and antiviral activities. Here we evaluated the potential immunomodulatory activity of SRPIN340 and three trifluoromethyl arylamide derivatives. In in vitro analysis with RAW 264.7 macrophages and primary splenocytes, all the compounds modulated the expression of immune response mediators and antigen-presentation molecules related to a tendency for M2 macrophage polarization. Immunization experiments were carried out in mice to evaluate their potential as vaccine immunostimulants. When administrated alone, the compounds altered the expression of immune factors at the injection site and did not produce macroscopic or microscopic local reactions. In addition, when prepared as an adjuvant with inactivated EHV-1 antigens, all the compounds increased the anti-EHV-1 neutralizing antibody titers, a change that is consistent with an increased Th2 response. These findings demonstrate that SRPIN340 and its derivatives exhibit a noticeable capacity to modulate innate and adaptative immune cells, disclosing their potential to be used as vaccine adjuvants or in immunotherapies.

The SRPK inhibitor N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl) isonicotinamide (SRPIN340) increases the immune response against metastatic melanoma in mice.

Cancers have a strong relationship with immune cells in their microenvironment, which significantly influences tumor proliferation and progression. Thus, pharmacological strategies that stimulate the immune system to combat tumor cells are promising for better therapeutic efficacy. Deregulated expression of the splicing regulatory serine arginine protein kinases (mostly SRPK1 and SRPK2) has been found in different cancer types, leading to the expression of isoforms related to tumor growth and metastasis. The microenvironment of melanoma exhibits a strong presence of immune cells, which significantly influences tumor progression, and around 50% of cutaneous melanoma patients benefit from targeted immunotherapy. Here, we analyzed human malignant melanoma single-cell gene expression data and observed that SRPK1/2 overexpression correlates with immune system pathway alterations. In further analysis, we observed an increased presence of immune cells in biopsies from mice bearing metastatic melanoma treated with SRPIN340, a well-known SRPK1/2 pharmacological inhibitor. Local treatments increased the expression of proinflammatory cytokines at the tumor lesions and the activity of the spleen, accompanied by reduced pulmonary metastasis foci, edema formation, and alveolar congestion. In in vitro assays, SRPIN340 also potentiated immunological susceptibility, by increasing the expression of the antigen presenting MHCI and MHCII molecules and by increasing the ability of B16F10 cells to attract splenic cells in transwell assays. Taken together, these results reveal that the antimetastatic effect of SRPIN340 can also involve an increased immune response, which suggests additional functional clues for SRPKs in tumor biology.

Retrospective study on Porcine circovirus-2 by nested pcr and real time pcr in archived tissues from 1978 in brazil.

Porcine circovirus-2 (PCV-2) infection is currently considered an important disease of swine. The pathogenic agent was first described in Brazil in 2000. This study detected the PCV-2 DNA in four Brazilian pig tissues collected between 1978 and 1979. This observation is the oldest description of this virus in Brazil.