Development and validation of Mayaro virus with luciferase reporter genes as a tool for antiviral assays.

Arboviruses are etiological agents in an extensive group of emerging diseases with great clinical relevance in Brazil, due to the wide distribution of their vectors and the favorable environmental conditions. Among them, the Mayaro virus (MAYV) has drawn attention since its emergence as the etiologic agent of Mayaro fever, a highly debilitating disease. To study viral replication and identify new drug candidates, traditional antiviral assays based on viral antigens and/or plaque assays have been demonstrating low throughput, making it difficult to carry out larger-scale assays. Therefore, we developed and characterized two DNA-launched infectious clones reporter viruses based on the MAYV strain BeAr 20290 containing the reporter genes of firefly luciferase (FLuc) and nanoluciferase (NLuc), designated as MAYV-firefly and MAYV-nanoluc, respectively. The viruses replicated efficiently with similar properties to the parental wild-type MAYV, and luminescence expression levels reflected viral replication. Reporter genes were also preserved during passage in cell culture, remaining stably expressed for one round of passage for MAYV-firefly and three rounds for MAYV-nanoluc. Employing the infectious clone, we described the effect of Rimantadine, an FDA-approved Alzheimer's drug, as a repurposing agent for MAYV but with a broad-spectrum activity against Zika virus infection. Additionally, we validated MAYV-nanoluc as a tool for antiviral drug screening using the compound EIDD-2749 (4'-Fluorouridine), which acts as an inhibitor of alphavirus RNA-dependent RNA polymerase.

Novel anti-inflammatory diketopiperazine alkaloids from the marine-derived fungus Penicillium brasilianum.

Diketopiperazine alkaloids have proven the most abundant heterocyclic alkaloids up to now, which usually process diverse scaffolds and rich biological activities. In our search for bioactive diketopiperazine alkaloids from marine-derived fungi, two novel diketopiperazine alkaloids, penipiperazine A (1) and its biogenetically related new metabolite (2), together with a known analogue neofipiperzine C (3), were obtained from the strain Penicillium brasilianum. Their planar structures and absolute configurations were elucidated by extensive spectroscopic analyses, 13C NMR calculation, Marfey's, ECD, and ORD methods. Compound 1 featured a unique 6/5/6/6/5 indole-pyrazino-pyrazino-pyrrolo system, and its plausible biogenetic pathway was also proposed. Additionally, compounds 1-3 have been tested for their inflammatory activities. 1 and 2 significantly inhibited the release of NO and the expression of related pro-inflammatory cytokines on LPS-stimulated RAW264.7 cells, suggesting they could be attracting candidate for further development as anti-inflammatory agent. KEY POINTS: • A novel diketopiperazine alkaloid featuring a unique 6/5/6/6/5 indole-pyrazino-pyrazino-pyrrolo system was isolated from the marine fungus Penicillium brasilianum. • The structure of 1 was elucidated by detailed analysis of 2D NMR data, 13C NMR calculation, Marfey's, ECD, and ORD methods. • Compounds 1 and 2 significantly inhibited the release of NO and the expression of related pro-inflammatory cytokines on LPS-stimulated RAW264.7 cells.

Effects of progressive load training on sprinters' cardiorespiratory endurance / Efeitos do treinamento com cargas progressivas na resistência cardiorrespiratória de velocistas / Efectos del entrenamiento con cargas progresivas en la resistencia cardiorrespiratoria de velocistas

ABSTRACT Introduction: The most evident change during exercise is the increase in oxygen demand, and prolonged high-intensity exercises may impact this even more on this demand. Physical training with progressive loads is a method that aims to reach the training goals by adjusting factors such as initial load, stage load, and a load of the athletes and can be a monitor of the aerobic and anaerobic exercise on the athlete with potential benefits for the high demand of oxygenation. Objective: Study the changes in heart rate and indexes of cardiorespiratory endurance of sprinters under training with progressive loads. Methods: By random sampling, the study subjects were 15 male sprinters and 15 non-sportsmen as research volunteers. The study subjects performed a protocol with progressive load training on a platform. All subjects underwent the Italian Kosmai cardiopulmonary function test before and after training. Results: The subjects who did not practice the sport showed VO2 max values at an exercise intensity of 13 km/h. The VO2 max maximum for sprinters was 15 km/h. Individuals who were not sprinters generally showed lower VT levels than sprinters (P <0.05). Average non-speeder individuals demonstrated higher MET scores than sprinters. Conclusion: Cardiopulmonary endurance and energy metabolism levels are strongly related to long-term participation in physical activity and exercise intensity. Incremental load training improved cardiorespiratory fitness in sprinters. The results may provide a specific theoretical basis for the daily training of sprinters in the future. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

RESUMO Introdução: A alteração mais evidente durante o exercício é o aumento da demanda de oxigênio e os exercícios prolongados de alta intensidade podem impactar ainda mais nessa demanda. O treinamento físico com cargas progressivas é um método que visa atingir as metas de treinamento ajustando fatores como carga inicial, carga de estágio e uma carga dos atletas e pode ser um monitor do exercício aeróbico e anaeróbico sobre o atleta com potenciais benefícios para a alta demanda de oxigenação. Objetivo: Estudar as alterações na frequência cardíaca e nos índices de resistência cardiorrespiratória de velocistas sob treinamento com cargas progressivas. Métodos: Selecionou-se 15 velocistas do sexo masculino e 15 homens que não praticavam o esporte como voluntários de pesquisa por amostragem aleatória. Os sujeitos do estudo realizaram um protocolo com treinamento de cargas progressivas em plataforma. Todos os indivíduos foram submetidos ao teste de função cardiopulmonar Italian Kosmai antes e após o treinamento. Resultados: Os indivíduos que não praticavam o esporte apresentaram valores de VO2max em uma intensidade de exercício de 13 km/h. O VO2max máximo para velocistas foi de 15 km/h. Indivíduos que não eram velocistas em geral apresentaram níveis de VT mais baixos do que os velocistas (P <0,05). Os não velocistas médios demonstraram pontuações MET mais altas do que os velocistas. Conclusão: A resistência cardiopulmonar e os níveis de metabolismo energético estão fortemente relacionados com a participação a longo prazo na atividade física e intensidade do exercício. O treinamento de carga incremental melhorou a aptidão cardiorrespiratória em velocistas. Os resultados podem fornecer uma base teórica específica para o treinamento diário de velocistas no futuro. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento.

RESUMEN Introducción: La alteración más evidente durante el ejercicio es el aumento de la demanda de oxígeno y los ejercicios prolongados de alta intensidad pueden incidir aún más en esta demanda. El entrenamiento físico con cargas progresivas es un método que pretende alcanzar los objetivos de entrenamiento ajustando factores como la carga inicial, la carga por etapas y la carga de los atletas y puede ser un monitor del ejercicio aeróbico y anaeróbico en el atleta con beneficios potenciales para la alta demanda de oxigenación. Objetivo: Estudiar las alteraciones en la frecuencia cardíaca y en los índices de resistencia cardiorrespiratoria de los velocistas bajo tratamiento con cargas progresivas. Métodos: Seleccionamos a 15 velocistas masculinos y a 15 hombres no practicantes como voluntarios de la investigación mediante un muestreo aleatorio. Los sujetos se sometieron a un protocolo con entrenamiento de carga progresiva en plataforma. Todos los sujetos fueron sometidos a la prueba de función cardiopulmonar de Kosmai italiano antes y después del entrenamiento. Resultados: Los sujetos que no practicaban el deporte presentaron valores de VO2máx a una intensidad de ejercicio de 13 km/h. El VO2máx de los velocistas era de 15 km/h. Los individuos que no eran velocistas en general mostraron niveles de VT más bajos que los velocistas (P <0,05). La media de los no-principiantes mostró mayores puntuaciones MET que los velocistas. Conclusión: Los niveles de resistencia cardiopulmonar y de metabolismo energético están fuertemente relacionados con la participación a largo plazo en la actividad física y la intensidad del ejercicio. El entrenamiento de carga incremental mejoró la aptitud cardiorrespiratoria de los velocistas. Los resultados pueden proporcionar una base teórica específica para el entrenamiento diario de los velocistas en el futuro. Nivel de evidencia II; Estudios terapéuticos - investigación de los resultados del tratamiento.

Resistance training effects on fatigue in runners / Efeitos do treino de resistência sobre a fadiga dos corredores / Efectos del ejercicio de resistencia en la fatiga de los corredores

ABSTRACT Introduction Start-up belongs to the group of fast combination exercises involving power and speed dominated by the short-term exhaustion of the whole body's power aiming at its displacement at high speed to obtain good results in competition. It is very important to master the technique correctly and to distribute the power reasonably for the acceleration of the start. Objective Explore the effect of aerobic endurance training on speed strength in starts. Methods Taking 16 high-level runners from a university sports team as research objects, 16 athletes were tested according to their test indicators; they were randomly assigned to the experimental group and the control group (8 people each in the experimental group and the control group). Results According to the post-test results of the experiment, the athletic performance of both groups of runners after training was improved, but the improvement in the experimental group was more evident and significant (p<0.05). Conclusion: Start-up aerobic endurance training is very useful for improving endurance and speed in runners. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

RESUMO Introdução O arranque pertence ao grupo de exercício de combinação rápida envolvendo potência e velocidade dominado pela exaustão a curto prazo da potência de todo o corpo objetivando seu deslocamento em alta velocidade a fim de obter bons resultados na competição. É muito importante dominar a técnica corretamente e distribuir a energia razoavelmente para a aceleração do arranque. Objetivo Explorar o efeito do treinamento de resistência aeróbica sobre a força da velocidade em arranques. Métodos Tomando 16 corredores de alto nível de uma equipe esportiva universitária como objetos de pesquisa, 16 atletas foram testados, de acordo com seus indicadores de teste, eles foram designados aleatoriamente ao grupo experimental e ao grupo de controle (8 pessoas no grupo experimental e 8 no grupo de controle). Resultados De acordo com os resultados do pós-teste do experimento, o desempenho atlético dos dois grupos de corredores após o treinamento foi melhorado, mas a melhoria no grupo experimental foi mais evidente e significativa (p<0,05). Conclusão O treinamento de resistência aeróbica para arranque é muito útil para a melhoria da resistência e velocidade dos atletas corredores. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento.

RESUMEN Introducción El arranque pertenece al grupo de ejercicios combinados rápidos de potencia y velocidad dominados por el agotamiento a corto plazo de la potencia de todo el cuerpo con el objetivo de su desplazamiento en alta velocidad para obtener buenos resultados en la competición. Es muy importante dominar la técnica correctamente y distribuir la energía razonablemente para la aceleración de la salida. Objetivo Explorar el efecto del entrenamiento de resistencia aeróbica sobre la fuerza de velocidad en las salidas. Métodos Tomando como objeto de investigación a 16 corredores de alto nivel de un equipo deportivo universitario, se realizaron pruebas a 16 atletas que, según sus indicadores de prueba, fueron asignados aleatoriamente al grupo experimental y al grupo de control (8 personas en el grupo experimental y 8 en el grupo de control). Resultados Según los resultados de la prueba posterior al experimento, el rendimiento atlético de ambos grupos de corredores después del entrenamiento mejoró, pero la mejora en el grupo experimental fue más evidente y significativa (p<0,05). Conclusión El entrenamiento de resistencia aeróbica de arranque es muy útil para mejorar la resistencia y la velocidad de los corredores. Nivel de evidencia II; Estudios terapéuticos - investigación de los resultados del tratamiento.

HBX suppresses PTEN to promote the malignant progression of hepatocellular carcinoma through mi-R155 activation.

INTRODUCTION AND OBJECTIVES: Hepatocellular carcinoma (HCC) is one of the most common and fatal tumors in the world, ranking third in cancer-related mortality. Chronic HBV infection is one of the major risk factors for hepatocellular carcinoma in China, Korea, and Sub-Saharan Africa. The HBx protein encoded by the X gene of HBV is a broadly regulated protein involved in transcriptional activation, epigenetics, apoptosis, DNA repair, and other regulatory processes. This study aimed to investigate the mechanism of HBx regulation of miR-155 and PTEN (Phosphatase and tensin homolog deleted on chromosome ten) in HBV-HCC. METHODS: Exosomal miR-155 quantity was analyzed by sampling serum exosomes of patients with hepatocellular carcinoma and normal subjects. The analysis was divided into different subgroups according to HBV positivity or negativity. At the cellular level, the biological roles of HBX, microRNA-155 and PTEN on hepatocellular carcinoma cells and their regulatory relationships with each other were verified. RESULTS: MicroRNA-155 and PTEN expression in HBV-positive HCC liver cancer tissues were negatively correlated, and HBX and miR-155 expression were positively correlated; microRNA-155 could target and inhibit PTEN expression, thereby promoting hepatocellular carcinoma cell activity, inhibiting apoptosis, and promoting invasion and migration; HBX could upregulate microRNA-155 thereby inhibit PTEN to promote malignant transformation of hepatocellular carcinoma. CONCLUSIONS: HBX could promote malignant transformation of hepatocellular carcinoma cells by upregulating microRNA-155 expression and thereby inhibiting the PTEN/PI3K-AKT pathway. Blocking miR-155 expression could attenuate the proliferation-promoting and invasive effects of HBX.

Effect of Calcium Dobesilate in Preventing Contrast-Induced Nephropathy in Patients with Diabetes and Chronic Kidney Disease.

OBJECTIVES: This study assessed the protective effect of calcium dobesilate against contrast-induced nephropathy (CIN) after coronary angiography (CAG) or percutaneous coronary intervention (PCI) in patients with diabetes and chronic kidney disease (CKD). METHODS: A total of 130 patients with diabetes and CKD estimated glomerular filtration rate: 30-90 mL/min/1.73m2 were enrolled and included in the analysis. They were divided into experimental (n=65) and control groups (n=65). Patients in the experimental group were administered oral calcium dobesilate (500 mg) three times daily for 2 days before and 3 days after the procedure. The serum creatinine (SCr), cystatin C (Cys C), and neutrophil gelatinase-associated lipocalin (NGAL) levels were measured before and after the procedure. RESULTS: The mean SCr level at 24h after the procedure was found to be significantly lower in the experimental group than in the control group (79.1±19.6 µmol/L vs. 87.0±19.3 µmol/L, p=0.023). However, the Cys C and NGAL levels were not significantly different between the two groups at all measurement time points (all p>0.05). The incidence of CIN defined by the SCr level was significantly lower in the experimental group than in the control group (3 [4.6%] vs. 13 [20.0%], p=0.017). However, the incidence of CIN defined by the Cys C level was not statistically different between the two groups (7 [10.8%] vs. 7 [10.8%], p=1.000). CONCLUSIONS: This study revealed that calcium dobesilate has no preventive effect against CIN in patients with diabetes and CKD.

Demethylzeylasteral (T-96) initiates extrinsic apoptosis against prostate cancer cells by inducing ROS-mediated ER stress and suppressing autophagic flux.

BACKGROUND: Demethylzeylasteral (T-96) is a pharmacologically active triterpenoid monomer extracted from Tripterygium wilfordii Hook F (TWHF) that has been reported to exhibit anti-neoplastic effects against several types of cancer cells. However, the potential anti-tumour effects of T-96 against human Prostate cancer (CaP) cells and the possible underlying mechanisms have not been well studied. RESULTS: In the current study, T-96 exerted significant cytotoxicity to CaP cells in vitro and induced cell cycle arrest at S-phase in a dose-dependent manner. Mechanistically, T-96 promoted the initiation of autophagy but inhibited autophagic flux by inducing ROS-mediated endoplasmic reticulum (ER) stress which subsequently activated the extrinsic apoptosis pathway in CaP cells. These findings implied that T-96-induced ER stress activated the caspase-dependent apoptosis pathway to inhibit proliferation of CaP cells. Moreover, we observed that T-96 enhances the sensitivity of CaP cells to the chemotherapeutic drug, cisplatin. CONCLUSIONS: Taken together, our data demonstrated that T-96 is a novel modulator of ER stress and autophagy, and has potential therapeutic applications against CaP in the clinic.

Demethylzeylasteral (T-96) initiates extrinsic apoptosis against prostate cancer cells by inducing ROS-mediated ER stress and suppressing autophagic flux

BACKGROUND: Demethylzeylasteral (T-96) is a pharmacologically active triterpenoid monomer extracted from Tripterygium wilfordii Hook F (TWHF) that has been reported to exhibit anti-neoplastic effects against several types of cancer cells. However, the potential anti-tumour effects of T-96 against human Prostate cancer (CaP) cells and the possible underlying mechanisms have not been well studied. RESULTS: In the current study, T-96 exerted significant cytotoxicity to CaP cells in vitro and induced cell cycle arrest at S-phase in a dose-dependent manner. Mechanistically, T-96 promoted the initiation of autophagy but inhibited autophagic flux by inducing ROS-mediated endoplasmic reticulum (ER) stress which subsequently activated the extrinsic apoptosis pathway in CaP cells. These findings implied that T-96-induced ER stress activated the caspase-dependent apoptosis pathway to inhibit proliferation of CaP cells. Moreover, we observed that T-96 enhances the sensitivity of CaP cells to the chemotherapeutic drug, cisplatin. CONCLUSIONS: Taken together, our data demonstrated that T-96 is a novel modulator of ER stress and autophagy, and has potential therapeutic applications against CaP in the clinic.

Effect of calcium dobesilate in preventing contrast-induced nephropathy in patients with diabetes and chronic kidney disease

OBJECTIVES: This study assessed the protective effect of calcium dobesilate against contrast-induced nephropathy (CIN) after coronary angiography (CAG) or percutaneous coronary intervention (PCI) in patients with diabetes and chronic kidney disease (CKD). METHODS: A total of 130 patients with diabetes and CKD estimated glomerular filtration rate: 30-90 mL/min/1.73m2 were enrolled and included in the analysis. They were divided into experimental (n=65) and control groups (n=65). Patients in the experimental group were administered oral calcium dobesilate (500 mg) three times daily for 2 days before and 3 days after the procedure. The serum creatinine (SCr), cystatin C (Cys C), and neutrophil gelatinase-associated lipocalin (NGAL) levels were measured before and after the procedure. RESULTS: The mean SCr level at 24h after the procedure was found to be significantly lower in the experimental group than in the control group (79.1±19.6 μmol/L vs. 87.0±19.3 μmol/L, p=0.023). However, the Cys C and NGAL levels were not significantly different between the two groups at all measurement time points (all p>0.05). The incidence of CIN defined by the SCr level was significantly lower in the experimental group than in the control group (3 [4.6%] vs. 13 [20.0%], p=0.017). However, the incidence of CIN defined by the Cys C level was not statistically different between the two groups (7 [10.8%] vs. 7 [10.8%], p=1.000). CONCLUSIONS: This study revealed that calcium dobesilate has no preventive effect against CIN in patients with diabetes and CKD.

The Pneumatron: An automated pneumatic apparatus for estimating xylem vulnerability to embolism at high temporal resolution.

Xylem vulnerability to embolism represents an important trait to determine species distribution patterns and drought resistance. However, estimating embolism resistance frequently requires time-consuming and ambiguous hydraulic lab measurements. Based on a recently developed pneumatic method, we present and test the "Pneumatron", a device that generates high time-resolution and fully automated vulnerability curves. Embolism resistance is estimated by applying a partial vacuum to extract air from an excised xylem sample, while monitoring the pressure change over time. Although the amount of gas extracted is strongly correlated with the percentage loss of xylem conductivity, validation of the Pneumatron was performed by comparison with the optical method for Eucalyptus camaldulensis leaves. The Pneumatron improved the precision of the pneumatic method considerably, facilitating the detection of small differences in the (percentage of air discharged [PAD] < 0.47%). Hence, the Pneumatron can directly measure the 50% PAD without any fitting of vulnerability curves. PAD and embolism frequency based on the optical method were strongly correlated (r2 = 0.93) for E. camaldulensis. By providing an open source platform, the Pneumatron represents an easy, low-cost, and powerful tool for field measurements, which can significantly improve our understanding of plant-water relations and the mechanisms behind embolism.

Distribution of serotypes, antimicrobial resistance and virulence genes among Streptococcus agalactiae isolated from bovine in China

Background: Bovine mastitis, a global disease that is responsible for large economic losses each year due to lower milkyield and reduced milk quality. In some countries, especially in China, Streptococcus agalactiae has become one of themost frequently detected pathogen. Antibiotic treatment and vaccine immunization are important strategies for the controlof infectious diseases. The main objective of the present study was to evaluate distribution of bovine mastitis pathogensand antimicrobial resistance of S. agalactiae, and contribute to the treatment of bovine mastitis.Materials, Methods & Results: Clinical mastitis samples (n= 1,122) were collected from 27 dairy farms located in 15different provinces of China during 2012-2018. The pathogens were identified by 16S rDNA method. Antimicrobial susceptibility was assessed by disc diffusion method. Molecular characteristics was distinguished based on PCR. The resultsshowed that the main pathogens were Streptococcus agalactiae (n= 324, 26.2%), Escherichia coli (n= 287, 23.2%), andStaphylococcus aureus (n= 131, 10.6%). The serotypes of Streptococcus agalactiae were serotype II (53.6%), Ia (44 %)and VII (1.2%), respectively. Streptococcus agalactiae were resistant to kanamycin (93.8%), gentamicin (49.4%), vancomycin (49.4%), tetracycline (35.8%), clindamycin (34.6%) and erythromycin (32.1%). The main resistance genes wereermA (53.1%) and ermB (85.2%). Resistance to erythromycin was attributed to the genes ermA (P < 0.05) and resistanceto tetracycline was attributed to the genes tetK, tetM, tetO (P < 0.01). The virulence genes scpB (81.4%), cyl (100%), glnA(76.6%), cfb (98.8%), hylB (98.8%), scaA (69.1%) were detected in almost all isolates.Discussion: In the present study, Streptococcus agalactiae, Escherichia coli and Staphylococcus aureus were the pathogens isolated most frequently from clinical mastitis. In the case of S. agalactiae, we performed capsular serotyping ofisolates...(AU)

Discovery of Bioactive Indole-Diketopiperazines from the Marine-Derived Fungus Penicillium brasilianum Aided by Genomic Information.

Identification and analysis of the whole genome of the marine-derived fungus Penicillium brasilianum HBU-136 revealed the presence of an interesting biosynthetic gene cluster (BGC) for non-ribosomal peptide synthetases (NRPS), highly homologous to the BGCs of indole-diketopiperazine derivatives. With the aid of genomic analysis, eight indole-diketopiperazines (1-8), including three new compounds, spirotryprostatin G (1), and cyclotryprostatins F and G (2 and 3), were obtained by large-scale cultivation of the fungal strain HBU-136 using rice medium with 1.0% MgCl2. The absolute configurations of 1-3 were determined by comparison of their experimental electronic circular dichroism (ECD) with calculated ECD spectra. Selective cytotoxicities were observed for compounds 1 and 4 against HL-60 cell line with the IC50 values of 6.0 and 7.9 µM, respectively, whereas 2, 3, and 5 against MCF-7 cell line with the IC50 values of 7.6, 10.8, and 5.1 µM, respectively.

Large-scale molecular phylogeny, morphology, divergence-time estimation, and the fossil record of advanced caenophidian snakes (Squamata: Serpentes).

Caenophidian snakes include the file snake genus Acrochordus and advanced colubroidean snakes that radiated mainly during the Neogene. Although caenophidian snakes are a well-supported clade, their inferred affinities, based either on molecular or morphological data, remain poorly known or controversial. Here, we provide an expanded molecular phylogenetic analysis of Caenophidia and use three non-parametric measures of support-Shimodaira-Hasegawa-Like test (SHL), Felsentein (FBP) and transfer (TBE) bootstrap measures-to evaluate the robustness of each clade in the molecular tree. That very different alternative support values are common suggests that results based on only one support value should be viewed with caution. Using a scheme to combine support values, we find 20.9% of the 1265 clades comprising the inferred caenophidian tree are unambiguously supported by both SHL and FBP values, while almost 37% are unsupported or ambiguously supported, revealing the substantial extent of phylogenetic problems within Caenophidia. Combined FBP/TBE support values show similar results, while SHL/TBE result in slightly higher combined values. We consider key morphological attributes of colubroidean cranial, vertebral and hemipenial anatomy and provide additional morphological evidence supporting the clades Colubroides, Colubriformes, and Endoglyptodonta. We review and revise the relevant caenophidian fossil record and provide a time-calibrated tree derived from our molecular data to discuss the main cladogenetic events that resulted in present-day patterns of caenophidian diversification. Our results suggest that all extant families of Colubroidea and Elapoidea composing the present-day endoglyptodont fauna originated rapidly within the early Oligocene-between approximately 33 and 28 Mya-following the major terrestrial faunal turnover known as the "Grande Coupure" and associated with the overall climate shift at the Eocene-Oligocene boundary. Our results further suggest that the caenophidian radiation originated within the Caenozoic, with the divergence between Colubroides and Acrochordidae occurring in the early Eocene, at ~ 56 Mya.

Distribution of serotypes, antimicrobial resistance and virulence genes among Streptococcus agalactiae isolated from bovine in China

Background: Bovine mastitis, a global disease that is responsible for large economic losses each year due to lower milkyield and reduced milk quality. In some countries, especially in China, Streptococcus agalactiae has become one of themost frequently detected pathogen. Antibiotic treatment and vaccine immunization are important strategies for the controlof infectious diseases. The main objective of the present study was to evaluate distribution of bovine mastitis pathogensand antimicrobial resistance of S. agalactiae, and contribute to the treatment of bovine mastitis.Materials, Methods & Results: Clinical mastitis samples (n= 1,122) were collected from 27 dairy farms located in 15different provinces of China during 2012-2018. The pathogens were identified by 16S rDNA method. Antimicrobial susceptibility was assessed by disc diffusion method. Molecular characteristics was distinguished based on PCR. The resultsshowed that the main pathogens were Streptococcus agalactiae (n= 324, 26.2%), Escherichia coli (n= 287, 23.2%), andStaphylococcus aureus (n= 131, 10.6%). The serotypes of Streptococcus agalactiae were serotype II (53.6%), Ia (44 %)and VII (1.2%), respectively. Streptococcus agalactiae were resistant to kanamycin (93.8%), gentamicin (49.4%), vancomycin (49.4%), tetracycline (35.8%), clindamycin (34.6%) and erythromycin (32.1%). The main resistance genes wereermA (53.1%) and ermB (85.2%). Resistance to erythromycin was attributed to the genes ermA (P < 0.05) and resistanceto tetracycline was attributed to the genes tetK, tetM, tetO (P < 0.01). The virulence genes scpB (81.4%), cyl (100%), glnA(76.6%), cfb (98.8%), hylB (98.8%), scaA (69.1%) were detected in almost all isolates.Discussion: In the present study, Streptococcus agalactiae, Escherichia coli and Staphylococcus aureus were the pathogens isolated most frequently from clinical mastitis. In the case of S. agalactiae, we performed capsular serotyping ofisolates...

Higher-level phylogenetic affinities of the Neotropical genus Mastigodryas Amaral, 1934 (Serpentes: Colubridae), species-group definition and description of a new genus for Mastigodryas bifossatus

Most Neotropical colubrid snakes belong to a single, well-supported lineage. Relationships between the major constituents of this clade remain. Here, we explore the phylogenetic relationships of Mastigodryas and its affinities to other Neotropical colubrid genera by combining DNA and morphological data. Analyses demonstrate that the concatenation of multiple individuals into a single terminal can mask the detection of new taxa. Further, non-random missing data and/or taxa in some empirical datasets can bias species tree analyses more than concatenation approaches. Our results place Mastigodryas in a strongly supported clade that includes Drymarchon, Rhinobothryum, Drymoluber, Simophis and Leptodrymus. Mastigodryas bifossatus is more closely related to species of Drymoluber and Simophis than to its congeners. Thus, we erect a new genus to accommodate it and recover a monophyletic Mastigodryas. We highlight the importance of the use of morphological characters to diagnose suprageneric clades by showing that some key external and hemipenial characteristics are phylogenetically informative.

Large-scale molecular phylogeny, morphology, divergence-time estimation, and the fossil record of advanced caenophidian snakes (Squamata: Serpentes)

Caenophidian snakes include the file snake genus Acrochordus and advanced colubroidean snakes that radiated mainly during the Neogene. Although caenophidian snakes are a well-supported clade, their inferred affinities, based either on molecular or morphological data, remain poorly known or controversial. Here, we provide an expanded molecular phylogenetic analysis of Caenophidia and use three non-parametric measures of support–Shimodaira-Hasegawa-Like test (SHL), Felsentein (FBP) and transfer (TBE) bootstrap measures–to evaluate the robustness of each clade in the molecular tree. That very different alternative support values are common suggests that results based on only one support value should be viewed with caution. Using a scheme to combine support values, we find 20.9% of the 1265 clades comprising the inferred caenophidian tree are unambiguously supported by both SHL and FBP values, while almost 37% are unsupported or ambiguously supported, revealing the substantial extent of phylogenetic problems within Caenophidia. Combined FBP/TBE support values show similar results, while SHL/TBE result in slightly higher combined values. We consider key morphological attributes of colubroidean cranial, vertebral and hemipenial anatomy and provide additional morphological evidence supporting the clades Colubroides, Colubriformes, and Endoglyptodonta. We review and revise the relevant caenophidian fossil record and provide a time-calibrated tree derived from our molecular data to discuss the main cladogenetic events that resulted in present-day patterns of caenophidian diversification. Our results suggest that all extant families of Colubroidea and Elapoidea composing the present-day endoglyptodont fauna originated rapidly within the early Oligocene–between approximately 33 and 28 Mya–following the major terrestrial faunal turnover known as the "Grande Coupure" and associated with the overall climate shift at the Eocene-Oligocene boundary. Our results further suggest that the caenophidian radiation originated within the Caenozoic, with the divergence between Colubroides and Acrochordidae occurring in the early Eocene, at ~ 56 Mya.

Molecular phylogeny of the New World Dipsadidae (Serpentes: Colubroidea): a reappraisal.

We present a phylogenetic analysis of the New World dipsadids based on an expanded data matrix that includes 246 terminal taxa including 196 dipsadids. The species are sampled for eight genes (12S, 16S, cytb, nd2, nd4, bdnf, c-mos, rag2). The data are explored using two distinct optimality procedures-maximum parsimony and maximum likelihood-and two alignment strategies-dynamic homology and static homology. Two previously unsampled dipsadid genera, Sordellina and Rhachidelus, are now included in the analysis. The definitions of the genera, Erythrolamprus, Clelia, Hypsirhynchus, Philodryas and Phimophis, and the tribes Alsophiini, Echinantherini and Conophiini, are revised. In order to maintain monophyly, the genus Umbrivaga is synonymized with Erythrolamprus, and two new genera are erected to accommodate Phimophis iglesiasi and Clelia rustica, as well as their closely related species. The West Indian genera Schwartzophis, Darlingtonia, Antillophis and Ocyophis are resurrected. © The Willi Hennig Society 2012.

Phylogenetic relationships of the genus Sibynophis (Serpentes: Colubroidea)

We present the results of the first molecular analysis of the phylogenetic affinities of the Asian colubroid genus Sibynophis. We recovered a sister-group relationship between Sibynophis and the New World Scaphiodontophis. Although Liophidium sometimes is associated with these genera, the relationship is distant. Morphological characters that Liophidium shares with Sibynophis and Scaphiodontophis are resolved as homoplasies that probably reflect the similarities of their specialized feeding habits. The traditional subfamily Sibynophiinae is elevated to the family-level, and Scaphiodontophiinae is placed in its synonymy.

Os resultados apresentados correspondem à primeira análise molecular feita acerca das afinidades filogenéticas do gênero Sibynophis, um colubróideo de origem asiática. Sibynophis aparece como sendo o grupo-irmão do gênero Neotropical Scaphiodontophis. Embora Liophidium esteja por vezes associado a estes dois gêneros, a sua relação é apenas distante. Os caracteres morfológicos compartilhados entre Liophidium, Sibynophis e Scaphiodontophis correspondem a homoplasias que refletem provavelmente os hábitos alimentares especializados semelhantes, presentes nos três gêneros em questão. A tradicional subfamília Sibynophiinae é elevada ao nível de família, enquanto que Scaphiodontophiinae passa para sua sinonímia.

Phylogenetic relationships of the genus Sibynophis (Serpentes: Colubroidea)

We present the results of the first molecular analysis of the phylogenetic affinities of the Asian colubroid genus Sibynophis. We recovered a sister-group relationship between Sibynophis and the New World Scaphiodontophis. Although Liophidium sometimes is associated with these genera, the relationship is distant. Morphological characters that Liophidium shares with Sibynophis and Scaphiodontophis are resolved as homoplasies that probably reflect the similarities of their specialized feeding habits. The traditional subfamily Sibynophiinae is elevated to the family-level, and Scaphiodontophiinae is placed in its synonymy.

Os resultados apresentados correspondem à primeira análise molecular feita acerca das afinidades filogenéticas do gênero Sibynophis, um colubróideo de origem asiática. Sibynophis aparece como sendo o grupo-irmão do gênero Neotropical Scaphiodontophis. Embora Liophidium esteja por vezes associado a estes dois gêneros, a sua relação é apenas distante. Os caracteres morfológicos compartilhados entre Liophidium, Sibynophis e Scaphiodontophis correspondem a homoplasias que refletem provavelmente os hábitos alimentares especializados semelhantes, presentes nos três gêneros em questão. A tradicional subfamília Sibynophiinae é elevada ao nível de família, enquanto que Scaphiodontophiinae passa para sua sinonímia.

Evolution of the DRD2 gene haplotype and its association with alcoholism in Mexican Americans.

The human D2 dopamine receptor gene (DRD2) plays a central role in the neuromodulation of appetitive behaviors and is implicated in having a possible role in susceptibility to alcoholism. We genotyped an SNP in DRD2 Exon 8 in 251 nonalcoholic, unrelated, healthy controls and 200 alcoholic Mexican Americans. The DRD2 haplotypes were analyzed using the Exon 8 genotype in combination with five other SNP genotypes, which were obtained from our previous study. The ancestral origins of the DRD2 polymorphisms have been determined by sequencing the homologous region in other higher primates. Twenty DRD2 haplotypes, defined as H1 to H20 based on their frequency from high to low, were obtained in this major minority population. The ancestral haplotype "I-B2-G-C-G-A1" and two one-step mutation haplotypes were absent in our study population. The haplotype H1, "I-B1-T-C-A-A1", with the highest frequency in the population, is a three-step mutation from the ancestral form. The first five or eight major haplotypes make up 87% or 95% of the entire population, respectively. The prevalence of the haplotype H1+ (H1/H1 and H1/Hn genotypes) is significantly higher in alcoholics and alcoholic subgroups, including early onset drinkers and benders, than in their respective control groups. The Promoter -141C allele is in linkage disequilibrium (LD) with five other loci in the nonalcoholic group, but not in the alcoholic group. All of the other five loci are in LD in both the alcoholic and control groups. The DRD2 TaqI B allele is in complete LD with the allele located in intron 6. Five SNPs, Promoter -141C, TaqI B (or Intron 6), Exon 7, Exon 8, and TaqI A, are sufficient to define the DRD2 haplotypes in Mexican Americans. Our data indicate that the DRD2 haplotypes are associated with alcoholism in Mexican Americans.