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Melanoma is one of the most aggressive tumors, and its lethality is associated with the ability of malignant cells to migrate and invade surrounding tissues to colonize distant organs and to generate widespread metastasis. The serine/arginine protein kinases 1 and 2 (SRPK1 and SRPK2) are classically related to the control of pre-mRNA splicing through SR protein phosphorylation and have been found overexpressed in many types of cancer, including melanoma. Previously, we have demonstrated that the pharmacological inhibition of SRPKs impairs pulmonary colonization of metastatic melanoma in mice. As the used compounds could target at least both SRPK1 and SRPK2, here we sought to obtain additional clues regarding the involvement of these paralogs in melanoma progression. We analyzed single-cell RNA sequencing data of melanoma patient cohorts and found that SRPK2 expression in melanoma cells is associated with poor prognosis. Consistently, CRISPR-Cas9 genome targeting of SRPK2, but not SRPK1, impaired actin polymerization dynamics as well as the proliferative and invasive capacity of B16F10 cells in vitro. In further in vivo experiments, genetic targeting of SRPK2, but not SRPK1, reduced tumor progression in both subcutaneous and caudal vein melanoma induction models. Taken together, these findings suggest different functional roles for SRPK1/2 in metastatic melanoma and highlight the relevance of pursuing selective pharmacological inhibitors of SRPK2.
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Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for Paratuberculosis mainly affecting domestic ruminants. The interaction between MAP and sperm and/or germ cells has not yet been established, however the adherence between MAP and the host cell surface is associated to the 85 complex proteins that bind to the host cell's fibronectin. Therefore, this study aimed to evaluate the binding of MAP to bovine sperm and to verify changes in these cells by the presence of MAP before and after sperm cryopreservation. Polyclonal antibodies to MAP 85 complex proteins were produced and utilized in the analyzes. Two Nelore bulls were used for semen collection and MAP dilutions (103-108 CFU/mL) were inoculated in the samples; sperm motility and vigor were evaluated using light microscopy at different times before and after cryopreservation and in the presence and absence of the antibodies 85A and 85B. Interaction of MAP and sperm, interaction of MAP and sperm in the presence of Ab 85A and in the presence of Ab 85B were analyzed by scanning electron microscopy. The viability of MAP after sperm cryopreservation were evaluated by plating the samples after thawing. It was observed that sperm in the presence of MAP shows a decrease in motility and vigor, and that the higher the MAP concentration, the lower the sperm performance. It was possible to determine the viability of MAP after cryopreservation in samples of higher concentrations, which demonstrates the potential of transmission of this pathogen through artificial insemination. The interaction of MAP with bovine sperm occurs mainly in the midpiece and may be linked to the proteins 85A and 85B present in the MAP membrane.
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Enfermedades de los Bovinos , Mycobacterium avium subsp. paratuberculosis , Paratuberculosis , Animales , Bovinos , Masculino , Motilidad Espermática , EspermatozoidesRESUMEN
The emergence and rapid spread of Zika virus (ZIKV) in the Americas has prompted the development of in vitro and in vivo models to understand several aspects of ZIKV biology and boost the development of vaccines and antivirals. In vitro model studies include reverse genetics systems, two-dimensional (2D) cell models, such as primary cells and cell lines, and ex vivo three-dimensional (3D) models derived from skin, brain and placenta. While these models are cost-effective and allow rigorous control of experimental variables, they do not always recapitulate in vivo scenarios. Thus, a number of in vivo models have been developed, including mosquitoes (Aedes sp. and Culex sp.), embryonated chicken eggs, immunocompetent and immunodeficient mice strains, hamsters, guinea pigs, conventional swine and non-human primates. In this review, we summarize the main research systems that have been developed in recent years and discuss their advantages, limitations and main applications.
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Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Modelos Teóricos , Infección por el Virus Zika/patología , Infección por el Virus Zika/virología , Virus Zika/fisiología , Virus Zika/patogenicidad , Aedes , Animales , Investigación Biomédica/métodos , Pollos , Cricetinae , Culex , Cobayas , Ratones , Primates , PorcinosRESUMEN
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.
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Antineoplásicos/farmacología , Melanoma Experimental/tratamiento farmacológico , Metástasis de la Neoplasia/prevención & control , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Invasividad Neoplásica , Niacinamida/análogos & derivados , Niacinamida/farmacología , Niacinamida/uso terapéutico , Piperidinas/farmacología , Piperidinas/uso terapéutico , Ensayo de Tumor de Célula MadreRESUMEN
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.
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Estructuras Animales/virología , Pollos/virología , Infecciones por Coronavirus/veterinaria , Transmisión Vertical de Enfermedad Infecciosa , Virus de la Bronquitis Infecciosa/aislamiento & purificación , Enfermedades de las Aves de Corral/transmisión , Enfermedades de las Aves de Corral/virología , Animales , Infecciones por Coronavirus/transmisión , Infecciones por Coronavirus/virología , Análisis de Secuencia de ADN , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Niacinamida/análogos & derivados , Piperidinas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/metabolismo , Sitios de Unión , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regulación Leucémica de la Expresión Génica , Células HL-60 , Células HeLa , Humanos , Células Jurkat , Células K562 , Leucemia/genética , Leucemia/metabolismo , Leucemia/patología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Niacinamida/química , Niacinamida/metabolismo , Niacinamida/farmacología , Piperidinas/química , Piperidinas/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de FluorescenciaRESUMEN
Twenty-nine lactic acid bacteria (LAB) isolates were submitted for identification using Biolog, API50CHL, 16S rDNA sequencing, and species-specific PCR reactions. The identification results were compared, and it was concluded that a polyphasic approach is necessary for proper LAB identification, being the molecular analyzes the most reliable.
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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.
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Feline coronavirus (FCoV) is an enveloped single-stranded RNA virus, of the family Coronaviridae and the order Nidovirales. FCoV is an important pathogen of wild and domestic cats and can cause a mild or apparently symptomless enteric infection, especially in kittens. FCoV is also associated with a lethal, systemic disease known as feline infectious peritonitis (FIP). Although the precise cause of FIP pathogenesis remains unclear, some hypotheses have been suggested. In this review we present results from different FCoV studies and attempt to elucidate existing theories on the pathogenesis of FCoV infection.