RESUMEN
Bull fertility is an important trait in breeding as the semen of one bull can, potentially, be used to perform thousands of inseminations. The high number of inseminations needed to obtain reliable measures from Non-Return Rates to oestrus creates difficulties in assessing fertility accurately. Improving molecular knowledge of seminal properties may provide ways to facilitate selection of bulls with good semen quality. In this study, liquid chromatography mass spectrometry (LC-MS/MS) was used to analyze the protein content from the seminal plasma of 20 bulls with Non-Return Rates between 35 and 60%, sampled across three seasons. Overall, 1343 proteins were identified and proteins with consistent correlation to fertility across multiple seasons found. From these, nine protein groups had a significant Pearson correlation (p < 0.1) with fertility in all three seasons and 34 protein groups had a similar correlation in at least two seasons. Among notable proteins showing a high and consistent correlation across seasons were Osteopontin, a lipase (LIPA) and N-acetylglucosamine-1phosphotransferase subunit gamma. Three proteins were combined in a multiple linear regression to predict fertility (r = 0.81). These sets of proteins represent potential markers, which could be used by the breeding industry to phenotype bull fertility. SIGNIFICANCE: The ability of bull spermatozoa to fertilize oocytes is crucial for breeding efficiency. However, the reliability of this trait from field measures is relatively low and the prediction of fertility given by conventional methods to evaluate sperm quality is currently not very accurate. In this work, we identify sets of proteins in bull seminal plasma from repeated samples collected at different times of the year that correlate to fertility in a consistent way. We combined these individual proteins to build a molecular signature predictive of fertility. This study provides an overview of proteins linked to fertility in seminal plasma, thereby increasing knowledge of the bull seminal plasma proteome. Protein signatures from the latter, potentially related to fertility, may be of use to predict fertility for individual bulls.
Asunto(s)
Análisis de Semen , Semen , Animales , Bovinos , Cromatografía Liquida , Fertilidad , Humanos , Masculino , Reproducibilidad de los Resultados , Espermatozoides , Espectrometría de Masas en TándemRESUMEN
Fusarium species are cereal pathogens that cause the Fusarium Head Blight (FHB) disease. FHB can reduce yield, cause mycotoxin accumulation in the grain and reduce germination efficiency of the harvested seeds. Understanding the biochemical interactions between the host plants and the pathogen is crucial for controlling the disease and for the development of cultivars with improved tolerance to FHB. Here, we studied morphological and proteomic differences between the susceptible oat variety Belinda and the more resistant variety Argamak using variety-specific transcriptome assemblies as references. Measurements of deoxynivalenol toxin levels confirmed the partial resistance in Argamak and the susceptibility in Belinda. To jointly investigate the proteomics- and sequence data, we developed an RShiny-based interface for interactive exploration of the dataset using univariate and multivariate statistics. When applying this interface to the dataset, quantitative protein differences between Belinda and Argamak were detected, and eighteen peptides were found uniquely in Argamak during infection, among them several lipoxygenases. Such proteins can be developed as markers for Fusarium resistance breeding. In conclusion, this study provides the first proteogenomic insight on molecular Fusarium-oat interactions at both morphological and molecular levels and the data are openly available through an interactive interface for further inspection. SIGNIFICANCE: Fusarium head blight causes widespread damage to crops, and chronic and acute toxicity to human and livestock due to the accumulation of toxins during infection. In the present study, two oat varieties with differing resistance were challenged with Fusarium to understand the disease better, and studied both at morphological and molecular levels, identifying proteins which could play a role in the defense mechanism. Furthermore, a proteogenomics approach allows joint profiling of expression and sequence level differences to identify potentially functionally differing mutations. Here such analysis is made openly available through an interactive interface which allows other scientists to draw further findings from the data. This study may both serve as a basis for understanding oat disease response and developing breeding markers for Fusarium resistant oat and future proteogenomic studies using the interactive approach described.
Asunto(s)
Fusarium , Proteogenómica , Avena , Humanos , Fitomejoramiento , Enfermedades de las Plantas/genética , Proteómica , TriticumRESUMEN
Therapy directed against oncogenic FLT3 has been shown to induce response in patients with acute myeloid leukemia (AML), but these responses are almost always transient. To address the mechanism of FLT3 inhibitor resistance, we generated two resistant AML cell lines by sustained treatment with the FLT3 inhibitor sorafenib. Parental cell lines carry the FLT3-ITD (tandem duplication) mutation and are highly responsive to FLT3 inhibitors, whereas resistant cell lines display resistance to multiple FLT3 inhibitors. Sanger sequencing and protein mass-spectrometry did not identify any acquired mutations in FLT3 in the resistant cells. Moreover, sorafenib treatment effectively blocked FLT3 activation in resistant cells, whereas it was unable to block colony formation or cell survival, suggesting that the resistant cells are no longer FLT3 dependent. Gene expression analysis of sensitive and resistant cell lines, as well as of blasts from patients with sorafenib-resistant AML, suggested an enrichment of the PI3K/mTOR pathway in the resistant phenotype, which was further supported by next-generation sequencing and phospho-specific-antibody array analysis. Furthermore, a selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation, and induced apoptosis in resistant cell lines. Gedatolisib significantly extended survival of mice in a sorafenib-resistant AML patient-derived xenograft model. Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway in FLT3-ITD-dependent AML results in resistance to drugs targeting FLT3.
Asunto(s)
Resistencia a Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Morfolinas/administración & dosificación , Niacinamida/análogos & derivados , Compuestos de Fenilurea/administración & dosificación , Triazinas/administración & dosificación , Tirosina Quinasa 3 Similar a fms/genética , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Mutación , Niacinamida/administración & dosificación , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/administración & dosificación , Transducción de Señal/efectos de los fármacos , Sorafenib , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The recently described oomycete pathogen Phytophthora pisi causes root rot on pea and faba bean, while the closely related Phytophthora sojae is the causal agent of soybean root and stem rot. Differences in the pathogenicity factor repertoires that enable the two species to have distinct host specificity towards pea and soybean, were studied using tandem mass spectrometry in a global proteome study of hyphae and germinating cysts in P. pisi and P. sojae. In total 2775 proteins from P. pisi and 2891 proteins from P. sojae were identified. Fifty-eight orthologous proteins were more abundant in germinated cysts of both pathogens and thus identified as candidate proteins for the infective stage. Several of these proteins were associated with lipid transport and metabolism, and energy production. Twenty-three orthologous proteins were more abundant in hyphae of both pathogens and thus identified as candidate proteins for vegetative growth. Proteins uniquely present in germinating cysts of either P. pisi or P. sojae were considered as candidates for species-specific pathogenicity factors that may be involved in host specificity. Among these proteins were serine proteases, membrane transporters and a berberine-like protein. These results significantly expand the knowledge of the expressed proteome in P. pisi and P. sojae. BIOLOGICAL SIGNIFICANCE: P. sojae and P. pisi are closely related species that specifically cause root rot on soybean and pea, respectively. The pathogenicity factors contributing to their host specificity remained unknown. We carried out a comparative large-scale proteome analysis of vegetative (hyphae) and infective (germinating cysts) life stages in P. pisi and P. sojae. This study provides knowledge of the common factors and mechanism involved in initiation of infection and species-specific proteins that may contribute to the host specificity of these pathogens. This knowledge will lead to a better understanding of the infection biology of these pathogens, allowing new possibilities towards developing alternative and effective plant protection measures.
Asunto(s)
Phytophthora/metabolismo , Proteoma/metabolismo , ProteómicaRESUMEN
BACKGROUND: Exopolysaccharides (EPSs) produced by lactic acid bacteria are important for the texture of fermented foods and have received a great deal of interest recently. However, the low production levels of EPSs in combination with the complex media used for growth of the bacteria have caused problems in the accurate analysis of the EPS. The purpose of this study was to find a growth medium for physiological studies of the lactic acid bacterium Streptococcus thermophilus, and to develop a simple method for qualitative and quantitative analysis of EPSs produced in this medium. RESULTS: A semi-defined polysaccharide medium was developed and evaluated on six strains of Streptococcus thermophilus. The EPSs were analysed using a novel protocol incorporating ultracentrifugation for the removal of interfering sugars, hydrolysis and analysis of the monomer composition by High Performance Anion-Exchange Chromatography with pulsed amperometric detection. The medium and analysis method allowed accurate quantification and monomer analysis of 0.5 ml samples of EPSs from tube cultures. CONCLUSIONS: The presented medium should be useful for physiological studies of S. thermophilus, and, in combination with the method of analysis of EPS, will allow downscaling of physiological studies and screening for EPSs.
Asunto(s)
Polisacáridos/análisis , Streptococcus/química , Medios de Cultivo , Ácido Láctico/metabolismo , Streptococcus/fisiologíaRESUMEN
A beta-phosphoglucomutase (beta-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of beta-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h(-1), while the deletion of beta-PGM resulted in a maximum specific growth rate of 0.05 h(-1) on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as beta-glucose 1-phosphate in the medium. Furthermore, the beta-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of alpha-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the beta-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded beta-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.
Asunto(s)
Disacáridos/metabolismo , Glucosa/metabolismo , Lactococcus lactis/enzimología , Fosfoglucomutasa/fisiología , Medios de Cultivo , Fermentación , Lactococcus lactis/crecimiento & desarrollo , Lactococcus lactis/ultraestructura , Mutación , Fosfoglucomutasa/genéticaRESUMEN
Lactococcus lactis splits phosphorylated trehalose by the action of inorganic phosphate-dependent trehalose-6-phosphate phosphorylase (TrePP) in a novel catabolic pathway. TrePP was found to catalyze the reversible conversion of trehalose 6-phosphate into beta-glucose 1-phosphate and glucose 6-phosphate by measuring intermediate sugar phosphates in cell extracts from trehalose-cultivated lactococci. According to native PAGE and SDS-PAGE, TrePP was shown to be a monomeric enzyme with a molecular mass of 94 kDa. Reaction kinetics suggested that the enzyme follows a ternary complex mechanism with optimal phosphorolysis at 35 degrees C and pH 6.3. The equilibrium constants were found to be 0.026 and 0.032 at pH 6.3 and 7.0, respectively, favoring the formation of trehalose 6-phosphate. The Michaelis-Menten constants of TrePP for trehalose 6-phosphate, inorganic phosphate, beta-glucose 1-phosphate, and glucose 6-phosphate were determined to be 6, 32, 0.9, and 4 mm, respectively. The TrePP-encoding gene, designated trePP, was localized in a putative trehalose operon of L. lactis. This operon includes the gene encoding beta-phosphoglucomutase in addition to three open reading frames believed to encode a transcriptional regulator and two trehalose-specific phosphotransferase system components. The identity of trePP was confirmed by determining the N-terminal amino acid sequence of TrePP and by its overexpression in Escherichia coli and L. lactis, as well as the construction of a lactococcal trePP knockout mutant. Furthermore, both TrePP and beta-phosphoglucomutase activity were detected in Enterococcus faecalis cell extract, indicating that this bacterium exhibits the same trehalose assimilation route as L. lactis.
Asunto(s)
Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Glucosiltransferasas/fisiología , Lactococcus lactis/enzimología , Lactococcus lactis/metabolismo , Trehalosa/metabolismo , Aminoácidos/química , Southern Blotting , Electroforesis en Gel de Poliacrilamida , Enterococcus faecalis/enzimología , Escherichia coli/metabolismo , Glucosa-6-Fosfato/metabolismo , Glucofosfatos/metabolismo , Glucosiltransferasas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Cinética , Modelos Biológicos , Fosfoglucomutasa/química , Fosfoglucomutasa/genética , Filogenia , Plásmidos/metabolismo , Unión Proteica , Estereoisomerismo , Temperatura , Factores de Tiempo , Transcripción GenéticaRESUMEN
AIMS: To compare galactose-negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs). METHODS AND RESULTS: The structures of the EPSs were determined using nuclear magnetic resonance (NMR) and their genetic relationships determined using restriction endonuclease analysis (REA) and random amplification of polymorphic DNA (RAPD). Similar groupings were apparent by REA and RAPD, and each group produced an EPS with a particular subunit structure. CONCLUSION: Although none of the strains assimilated galactose, all inserted a high proportion of galactose into their EPS when grown in skimmed milk, and fell into three distinct groups. SIGNIFICANCE AND IMPACT OF THE STUDY: This information should help in an understanding of genetic exchanges in lactic acid bacteria.
Asunto(s)
Lactobacillus/metabolismo , Polisacáridos Bacterianos/química , Streptococcus/metabolismo , Secuencia de Carbohidratos , Galactosa/metabolismo , Genotipo , Ácido Láctico/metabolismo , Lactobacillus/genética , Conformación Molecular , Polisacáridos Bacterianos/clasificación , Streptococcus/genética , Transducción GenéticaRESUMEN
To study the influence of phosphoglucomutase (PGM) activity on exopolysaccharide (EPS) synthesis in glucose- and lactose-growing Streptococcus thermophilus, a knockout PGM mutant and a strain with elevated PGM activity were constructed. The pgmA gene, encoding PGM in S. thermophilus LY03, was identified and cloned. The gene was functional in Escherichia coli and was shown to be expressed from its own promoter. The pgmA-deficient mutant was unable to grow on glucose, while the mutation did not affect growth on lactose. Overexpression of pgmA had no significant effect on EPS production in glucose-growing cells. Neither deletion nor overexpression of pgmA changed the growth or EPS production on lactose. Thus, the EPS precursors in lactose-utilizing S. thermophilus are most probably formed from the galactose moiety of lactose via the Leloir pathway, which circumvents the need for a functional PGM.
Asunto(s)
Glucosa/metabolismo , Lactosa/metabolismo , Fosfoglucomutasa/metabolismo , Polisacáridos Bacterianos/biosíntesis , Streptococcus/metabolismo , Galactosa/metabolismo , Genes Bacterianos , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Fosfoglucomutasa/genética , Proteínas Recombinantes/metabolismo , Streptococcus/crecimiento & desarrolloRESUMEN
Alkaliphilic bacteria were isolated from soil and water samples obtained from Ethiopian soda lakes in the Rift Valley area--Lake Shala, Lake Abijata, and Lake Arenguadi. Starch-hydrolyzing isolates were selected on the basis of their activity on starch agar plate assay. Sixteen isolates were chosen, characterized, and subjected to 16S rRNA gene sequence analysis. All the isolates were gram positive and catalase- and beta-galactosidase positive. All isolates except one were motile endospore-forming rods and were found to be closely related to the Bacillus cluster, being grouped with Bacillus pseudofirmus, Bacillus cohnii, Bacillus vedderi, and Bacillus agaradhaerens. The one exception had nonmotile coccoid cells and was closely related to Nesterenkonia halobia. The majority of the isolates showed optimal growth at 37 degrees C and tolerated salinity up to 10% (w/v) NaCl. Both extracellular and cell-bound amylase activity was detected among the isolates. The amylase activity of two isolates, related to B. vedderi and B. cohnii, was stimulated by ethylenediaminetetraacetic acid (EDTA) and inhibited in the presence of calcium ions. Pullulanase activity was expressed by isolates grouped with B. vedderi and also most of the isolates clustered with B. cohnii; cyclodextrin glycosyltransferase was expressed by most of the B. agaradhaerens-related strains. Minor levels of alpha-glucosidase activity were detected in all the strains.