An in silico mining for simple sequence repeats from expressed sequence tags of zebrafish, medaka, Fundulus, and Xiphophorus

Teleost fish genome projects involving model species are resulting in a rapid accumulation of genomic and expressed DNA sequences in public databases. The expressed sequence tags (ESTs) collected in the databases can be mined for the analysis of both structural and functional genomics. In this study, we in silico analyzed 49,430 unigenes representing a total of 692,654 ESTs from four model fish for their potential use in developing simple sequence repeats (SSRs), or microsatellites. After bioinformatical mining, a total of 3,018 EST derived SSRs (EST-SSRs) were identified for 2,335 SSR containing ESTs (SSR-ESTs). The frequency of identified SSR-ESTs ranged from 1.5% for Xiphophorus to 7.3% for zebrafish. The dinucleotide repeat motif is the most abundant SSR, accounting for 47%, 52%, 64%, and 78% for medaka, Fundulus, zebrafish, and Xiphophorus, respectively. Simulation analysis suggests that a majority of these EST-SSRs have sufficient flanking sequences for polymerase chain reaction (PCR) primer design. Comparative DNA sequence analyses of SSR-ESTs identified several cross-species SSRs and sequences that may be used as cross-reference genes in comparative studies. For example, the flanking sequences of one SSR (CTG)n within the pituitary tumor-transforming gene (PTTG) 1 interacting protein (PTTGIP), showed conservation spanning the medaka, Fundulus, human, and mouse genomes. This study provides a large body of information on EST-SSRs that can be useful for the development of polymorphic markers, gene mapping, and comparative genome analysis. Functional analysis of these SSR-ESTs may reveal their role in metabolism and gene evolution of these model species.     

Environmental DNA metabarcoding provides enhanced detection of the European eel Anguilla anguilla and fish community structure in pumped river catchments

The European eel Anguilla anguilla (eel hereafter) is critically endangered and has a catadromous life cycle, which means adult eels that live in pumped catchments must pass through pumps during their downstream spawning migration. Policy makers are currently lacking detailed site-by-site eel distribution information to estimate the overall impact of individual pumping stations on eel escapement, and as such lack the data to enable informed prioritisation of pumping station management and targeted mitigation. This study investigated whether environmental DNA (eDNA) metabarcoding can provide increased detection sensitivity for eel and fish community structure in highly regulated pumped catchments, when compared directly to current standard practice fish survey protocols (seine netting/electric fishing). Eels were detected in 14 of 17 sites (82.4%) using eDNA metabarcoding in contrast to 3 of 17 sites (17.6%) using traditional catch methods. In addition, when using eDNA monitoring, species richness was higher in 16 of 17 sites (94.1%), and site occupancy was greater than or equal to traditional methods for 23 of 26 of the fish species detected (88.5%). Although eDNA methods presented significantly higher average species richness and species site occupancy overall, eDNA and catch methods were positively correlated in terms of species richness and site occupancy. It was therefore found that eDNA metabarcoding was a high-sensitivity method for detecting eels in pumped catchments while also increasing the detection of overall fish community structure compared to traditional catch methods. In addition, this study highlights how eDNA monitoring is especially suited to increase the detection of particular species, with traditional methods sufficient for others. This high sensitivity, coupled with the ability to sample multiple sites in a short time frame, suggests that eDNA metabarcoding workflows could be invaluable tools when prioritising pumping station management.     

Insulin action in AgRP-expressing neurons is required for suppression of hepatic glucose production

Insulin action in the central nervous system regulates energy homeostasis and glucose metabolism. To define the insulin-responsive neurons that mediate these effects, we generated mice with selective inactivation of the insulin receptor (IR) in either pro-opiomelanocortin (POMC)- or agouti-related peptide (AgRP)-expressing neurons of the arcuate nucleus of the hypothalamus. While neither POMC- nor AgRP-restricted IR knockout mice exhibited altered energy homeostasis, insulin failed to normally suppress hepatic glucose production during euglycemic-hyperinsulinemic clamps in AgRP-IR knockout (IR^ΔAgRP) mice. These mice also exhibited reduced insulin-stimulated hepatic interleukin-6 expression and increased hepatic expression of glucose-6-phosphatase. These results directly demonstrate that insulin action in POMC and AgRP cells is not required for steady-state regulation of food intake and body weight. However, insulin action specifically in AgRP-expressing neurons does play a critical role in controlling hepatic glucose production and may provide a target for the treatment of insulin resistance in type 2 diabetes.     

Influence of sex, high-fat diet, and exercise training on potassium currents of swine coronary smooth muscle

Potassium channels in vascular smooth muscle (VSM) control vasodilation and are potential regulatory targets. This study evaluated effects of sex differences, exercise training (EX), and high-fat diet (HF) on K(+) currents (I(K)) of coronary VSM cells. Yucatan male and female swine were assigned to either sedentary confinement (SED), 16 wk of EX, 20 wk of HF, or 20 wk of HF with 16 wk of EX (HF-EX). VSM cells of normal-diet SED animals exhibited three components of I(K): 4-aminopyridine-sensitive I(K(KV)), TEA-sensitive I(K(BK)), and 4-aminopyridine + TEA-insensitive I(K). Females exhibited significantly higher basal I(K) than males in the same group. EX increased basal I(K) in males and females. HF reduced I(K) in males and females and nullified effects of EX. Endothelin-1 increased I(K) significantly in males but not in females. In the presence of endothelin-1, 1) I(K(KV)) was similar in SED males and females and EX increased I(K(KV)) to a greater extent in males than in females and 2) I(K(BK)) was greater in SED females than in males and EX increased I(K(BK)) to a greater extent in males, resulting in I(K(BK)) similar to EX females. Importantly, HF nullified effects of EX on I(K(KV)) and I(K(BK)). These data indicate that basal I(K) of SED female swine is inherently greater than that shown in SED males and that males require EX to achieve comparable levels of I(K). Importantly, HF reduced I(K) in males and females and nullified effects of EX, suggesting HF abrogates beneficial effects of EX on coronary smooth muscle.     

A rapid survival assay to measure drug-induced cytotoxicity and cell cycle effects

We describe a rapid method to accurately measure the cytotoxicity of mammalian cells upon exposure to various drugs. Using this assay, we obtain survival data in a fraction of the time required to perform the traditional clonogenic survival assay, considered the gold standard. The dynamic range of the assay allows sensitivity measurements on a multi-log scale allowing better resolution of comparative sensitivities. Moreover, the results obtained contain additional information on cell cycle effects of the drug treatment. Cell survival is obtained from a quantitative comparison of proliferation between drug-treated and untreated cells. During the assay, cells are treated with a drug and, following a recovery period, allowed to proliferate in the presence of bromodeoxyuridine (BrdU). Cells that synthesize DNA in the presence of BrdU exhibit quenched Hoechst fluorescence, easily detected by flow cytometry; quenching is used to determine relative proliferation in treated vs. untreated cells. Finally, this assay can be used in high-throughput format to simultaneously screen multiple cell lines and drugs for accurate measurements of cell survival and cell cycle effects after drug treatment.     

Effect of lantibiotic gallidermin against biogenic amine-producing faecal staphylococci from ostriches and pheasants

In ostriches and pheasants, there is still limited information relating to staphylococci and their properties. Biogenic amines (BAs) are nitrogenous low-molecular-weight substances with biological functions in animals, plants and microorganisms. In this study, we focused on BA production by targeted faecal staphylococci from ostriches and pheasants and their sensitivity to lantibiotic bacteriocin gallidermin. Gallidermin belongs in a group of polycyclic proteinaceous antimicrobial substances. Thirty-six faecal staphylococci (24 strains from 140 ostriches, 12 from 60 pheasants) comprising different species were tested. Staphylococci from ostriches and pheasants did not produce tryptamine-TRYP, putrescine–PUT, cadaverine–CAD or histamine–HIS. Production of tyramine-TYM, phenylethylamine–PEA was high or very high (100–1000 mg/L). Production of spermine–SPM and spermidine–SPD by staphylococci was very low or low although in the case of staphylococci from pheasants medium production of SPM was found. Because of the risk posed by BAs for consumers, the control of BA-producing bacteria is important from the points of view not only of safety assessment of food-producing animals but also of human health safety. The sensitivity to gallidermin in biogenic amine-producing staphylococci from ostriches and pheasants detected here is the most promising indication for further application of gallidermin for veterinary purposes. The novelty of our study lies in testing the ability of faecal staphylococci from ostriches and pheasants to produce BAs and in their treatment with gallidermin which has so far not been tested in this way.     

Cryptococcus Strains with Different Pathogenic Potentials Have Diverse Protein Secretomes

Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.     

Absence of age effect on meiotic recombination between human X and Y chromosomes

Recombination between the X and Y chromosomes is limited to the pseudoautosomal region and is necessary for proper segregation of the sex chromosomes during spermatogenesis. Failure of the sex chromosomes to disjoin properly during meiosis can result in individuals with a 47,XXY constitution, and approximately one-half of these result from paternal nondisjunction at meiosis I. Analysis of individuals with paternally derived 47,XXY has shown that the majority are the result of meiosis in which the X and Y chromosomes have failed to recombine. Our studies of sperm have demonstrated that aneuploid 24,XY sperm have a decreased recombination frequency, compared with that of normal sperm. Some studies have indicated a relationship of increased paternal age with 47,XXY offspring and with the production of XY disomic sperm, whereas others have failed to find such relationships. To determine whether there is a relationship between paternal age and recombination in the pseudoautosomal region, single-sperm genotyping was performed to measure the frequency of recombination between a sex-specific locus, STS/STS pseudogene, and a pseudoautosomal locus, DXYS15, in younger men (age < or =30 years) compared with older men (age > or =50 years). A total of 2,329 sperm cells were typed by single-sperm PCR in 20 men who were heterozygous for the DXYS15 locus (1,014 sperm from 10 younger men and 1,315 sperm from 10 older men). The mean recombination frequency was 39.2% in the younger men and 37.8% in the older men. There was no heterogeneity in the frequency of recombination rates. There was no significant difference between the recombination frequencies among the younger men and those among the older men, when analyzed by the clustered binomial Z test (Z=.69, P=.49). This result suggests that paternal age has no effect on the recombination frequency in the pseudoautosomal region.     

Looking for sex in the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii

Why are we interested in understanding the mode of reproduction being used by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii? Empirical evidence has finally supported the long-held assumption that, by increasing the rate of adaptive evolution, sex increases the chances of long-term survival. Understanding the ability of pathogenic organisms to adapt to diagnostic and treatment regimes is also important in the fight against the diseases caused by these organisms. This review looks at the different approaches used to identify population structure in C. neoformans and C. gattii. These are sexual species; however, recombination in natural populations has only recently been found. We highlight the importance of population selection and the value of both indirect molecular analysis and direct biological evidence for sexual recombination, when looking for the mode of reproduction in these fungal pathogens.