Effect of age and environmental factors on semen quality, glutathione peroxidase activity and oxidative parameters in simmental bulls

Taking into account that semen quality depends on animal age and climate conditions and that oxidative stress has been reported to be a common cause of infertility, the objective of this study was to monitor indicators of oxidative stress and antioxidant protection during four seasonal periods in service bulls of various age to get better insight into the significance of these factors upon evaluating service bull semen. The research was conducted over a year on 19 Simmental service bulls. Animals were divided into two groups according to age; Group I consisted of younger bulls aged two to four yrs (n = 9), and Group II was comprised of older bulls aged five to ten yrs (n = 10). Semen samples were obtained once in the middle of every seasonal period and blood samples for biochemical analysis were collected by jugular venipuncture immediately after ejaculate collection. The activity of total glutathione peroxidase (T-GSH-Px), selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase (non-Se-GSH-Px), together with the intensity of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) and oxidative protein damage (protein carbonyl content (PCC)) were measured in seminal plasma. In samples of spermatozoa and blood serum, the activity of Se-GSH-Px and TBARS and PCC concentrations were determined. Older service bulls had significantly higher ejaculate volume in summer in comparison with younger bulls, whereas the number of spermatozoa and progressive motility percentage did not significantly vary with age. Younger animals had lower progressive motility percentage during summer than in spring, with more intensive oxidative processes observed in seminal plasma (TBARS) and spermatozoa (TBARS and PCC). Based on the results presented here, it can be concluded that younger bulls are more sensitive to elevated ambient temperatures during the summer, when intensified prooxidative processes in semen plasma and spermatozoa eventually led to decreased sperm progressive motility with consequential semen quality deterioration.     

Length and amino acid sequence of peptides substituted for the 5-HT3A receptor M3M4 loop may affect channel expression and desensitization

5-HT3A receptors are pentameric neurotransmitter-gated ion channels in the Cys-loop receptor family. Each subunit contains an extracellular domain, four transmembrane segments (M1, M2, M3, M4) and a 115 residue intracellular loop between M3 and M4. In contrast, the M3M4 loop in prokaryotic homologues is <15 residues. To investigate the limits of M3M4 loop length and composition on channel function we replaced the 5-HT3A M3M4 loop with two to seven alanine residues (5-HT3A-A(n = 2-7)). Mutants were expressed in Xenopus laevis oocytes and characterized using two electrode voltage clamp recording. All mutants were functional. The 5-HT EC(50)'s were at most 5-fold greater than wild-type (WT). The desensitization rate differed significantly among the mutants. Desensitization rates for 5-HT3A-A(2), 5-HT3A-A(4), 5-HT3A-A(6), and 5-HT3A-A(7) were similar to WT. In contrast, 5-HT3A-A(3) and 5-HT3A-A(5) had desensitization rates at least an order of magnitude faster than WT. The one Ala loop construct, 5-HT3A-A(1), entered a non-functional state from which it did not recover after the first 5-HT application. These results suggest that the large M3M4 loop of eukaryotic Cys-loop channels is not required for receptor assembly or function. However, loop length and amino acid composition can effect channel expression and desensitization. We infer that the cytoplasmic ends of the M3 and M4 segments may undergo conformational changes during channel gating and desensitization and/or the loop may influence the position and mobility of these segments as they undergo gating-induced conformational changes. Altering structure or conformational mobility of the cytoplasmic ends of M3 and M4 may be the basis by which phosphorylation or protein binding to the cytoplasmic loop alters channel function.     

Predicting cross-reactive immunological material (CRIM) status in Pompe disease using GAA mutations: lessons learned from 10 years of clinical laboratory testing experience

Enzyme replacement therapy (ERT) for Pompe disease using recombinant acid alpha-glucosidase (rhGAA) has resulted in increased survival although the clinical response is variable. Cross-reactive immunological material (CRIM)-negative status has been recognized as a poor prognostic factor. CRIM-negative patients make no GAA protein and develop sustained high antibody titers to ERT that render the treatment ineffective. Antibody titers are generally low for the majority of CRIM-positive patients and there is typically a better clinical outcome. Because immunomodulation has been found to be most effective in CRIM-negative patients prior to, or shortly after, initiation of ERT, knowledge of CRIM status is important before ERT is begun. We have analyzed 243 patients with infantile Pompe disease using a Western blot method for determining CRIM status and using cultured skin fibroblasts. Sixty-one out of 243 (25.1%) patients tested from various ethnic backgrounds were found to be CRIM-negative. We then correlated the CRIM results with GAA gene mutations where available (52 CRIM-negative and 88 CRIM-positive patients). We found that, in most cases, CRIM status can be predicted from GAA mutations, potentially circumventing the need for invasive skin biopsy and time wasted in culturing cells in the future. Continued studies in this area will help to increase the power of GAA gene mutations in predicting CRIM status as well as possibly identifying CRIM-positive patients who are at risk for developing high antibody titers.     

Induction of betaine: Homocysteine methyltransferase in some murine cells cultured in vitro

Betaine when present in the culture medium could induce the activity of betaine: homocysteine methyltransferase (EC 2.1.1.5) in mouse L-cells, and leukemic L1210 cells, as well as in mouse embryo fibroblasts grown in vitro. We found this process to be time- and concentration-dependent. A persisting contact of the cells with betaine was indispensible for expressing and maintaining the enzyme activity. The treatment of cells with cycloheximide or actinomycin D abolished the process of induction. Methionine as well as homocysteine, when present either in the culture medium or in the reaction mixture, strongly depressed the activity of this enzyme. The L-cells with the induced betaine: homocysteine methyltransferase survived but did not multiply in the methionine-deficient medium, therefore, they did not become prototrophs with respect to methionine.     

Effect on lycopene, β-carotene,ascorbic acid and phenolic content in tomato fruits infected by Alternaria alternata and its toxins (TeA,AOH and AME)

Tomato is considered as one of the most important sources of nutrients such as lycopene, β-carotene, flavonoids, ascorbic acid (vitamin C) and hydroxyl-cinnamic acid derivatives. The quality and quantity of nutrients in tomato fruits were decreased during the severe infection of Alternaria alternata. The present study deals with the estimation of lycopene, β-carotene, phenolic and ascorbic acid content in tomato fruits which were infected with A. alternata and its toxins such as tenuazonic acid (TeA), alternariol (AOH) and alternariol monomethyl ether (AME). The lycopene, β-carotene, ascorbic acid and phenolic content were found lowest in pathogen-infected fruits i.e. (0.66 ± 0.03 mg/g), (0.14 ± 0.01 mg/g), (1.89 ± 0.2 mg/g) and (0.58 ± 0.05 mg/g), respectively, followed by toxins-treated samples as compared to the control. The results concluded that A. alternata mostly affects the nutritional values of tomato fruits due to the combined effect of the toxins.     

Chemical–Genetic Profiling of Imidazo[1,2-a]pyridines and -Pyrimidines Reveals Target Pathways Conserved between Yeast and Human Cells

Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines compose a class of compounds that target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo[1,2-a]pyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo[1,2-a]pyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo[1,2-a]pyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical–genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure–activity relationships.     

Cell type discovery and representation in the era of high-content single cell phenotyping

Background

A fundamental characteristic of multicellular organisms is the specialization of functional cell types through the process of differentiation. These specialized cell types not only characterize the normal functioning of different organs and tissues, they can also be used as cellular biomarkers of a variety of different disease states and therapeutic/vaccine responses. In order to serve as a reference for cell type representation, the Cell Ontology has been developed to provide a standard nomenclature of defined cell types for comparative analysis and biomarker discovery. Historically, these cell types have been defined based on unique cellular shapes and structures, anatomic locations, and marker protein expression. However, we are now experiencing a revolution in cellular characterization resulting from the application of new high-throughput, high-content cytometry and sequencing technologies. The resulting explosion in the number of distinct cell types being identified is challenging the current paradigm for cell type definition in the Cell Ontology.

Results

In this paper, we provide examples of state-of-the-art cellular biomarker characterization using high-content cytometry and single cell RNA sequencing, and present strategies for standardized cell type representations based on the data outputs from these cutting-edge technologies, including “context annotations” in the form of standardized experiment metadata about the specimen source analyzed and marker genes that serve as the most useful features in machine learning-based cell type classification models. We also propose a statistical strategy for comparing new experiment data to these standardized cell type representations.

Conclusion

The advent of high-throughput/high-content single cell technologies is leading to an explosion in the number of distinct cell types being identified. It will be critical for the bioinformatics community to develop and adopt data standard conventions that will be compatible with these new technologies and support the data representation needs of the research community. The proposals enumerated here will serve as a useful starting point to address these challenges.

     

Probable advanced hydroid from the Early Ordovician of China

A fossil of a colonial organism with pyritized soft tissues of elongated fusiform zooids, found in the middle part of the early Floian Fenxiang Formation in Hubei Province of China, probably represents the oldest reliable record of a hydroid cnidarian. The preservation of the fossil is sufficiently different from that of associated carbonized skeletons of graptolites to exclude affinities with this group. The fossil is unlikely to be a bryozoan because of the mode of budding from proximal, not distal, parts of parent zooids, which is typical rather of hydroids. Although no thecae are preserved, the fossil, named Sinobryon elongatum gen. et sp. nov., is suggested to be a thecate hydroid, possibly related to the Haleciidae. The apparent presence of an advanced member of the thecaphoran Macrocolonia clade in strata 470 Ma old means that much of the hydroid (and cnidarian) diversification preceded the Middle Ordovician.