Headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) for the determination of 5alpha-androst-2-en-17-one and -17beta-ol in the female Asian elephant: application for reproductive monitoring and prediction of parturition

Asian elephants are not self-sustaining in captivity. The main reasons for this phenomenon are a low birth rate, an aging population, and poor calf-rearing. Therefore, it is essential that reproductive rates had to be improved and there is need for rapid quantitative measures to monitor reproductive functions focussing on estrous detection and the prediction of the period of parturition. The objective of this study was to develop a method which combines headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) for analyses of 5alpha-androst-2-en-17beta-ol and -17-one to prognose estrous and to predict the period of parturition. SPME was carried out with a CTC Combi Pal system.The course of the luteal phase-specific substance 5alpha-androst-2-en-17beta-ol and -17-one followed a cyclic pattern in which the follicular and luteal phases could be clearly distinguished (mean estrous cycle length, 15+/-1.4 weeks). Based on daily urine samples, estrous prognosis might be possibly based on the initial 5alpha-androst-2-en-17beta-o1 increase at the end of the follicular phase. Parturition prognosis was performed in three elephant cows based on the 5alpha-androst-2-en-17beta-o1 drop to baseline levels 5-4 days prior parturition. Experiments revealed that 5alpha-androst-3alpha-ol-17-one and probably 5alpha-androst-3alpha-ol-17beta-ol are generated from sulfate conjugates by a thermal process.     

Overt fatal and chronic subclinical Encephalitozoon cuniculi microsporidiosis in a colony of captive emperor tamarins (Saguinus imperator)

The course of an infection with the microsporidian Encephalitozoon cuniculi in a colony of captive emperor tamarins (Saguinus imperator) is described. In two litters, the infection was associated with overt disease and death of all infants. Immunohistochemistry for E. cuniculi showed generalized infections, and histopathologic evaluation revealed systemic vasculitis and disseminated mixed inflammatory cell infiltration with and without necrosis in several organs. Serologically, some of the juvenile animals presented with high titres for Encephalitozoon, while the adults had low titres. The E. cuniculi "dog strain" was identified by molecular means for the first time in Europe. The origin of the infection appeared to be a pair of breeding adults that originated from the US. Our data suggest that the organism persisted over years in the colony, and that subclinically infected animals most likely were involved in perpetuating the infection. Efforts should be made to ascertain if this microorganism is present in other captive populations of this endangered monkey species and to prevent its further spreading.     

Intensive allochthonous inputs along the Ganges River and their effect on microbial community composition and dynamics

Little is known about microbial communities in the Ganges River, India and how they respond to intensive anthropogenic inputs. Here we applied shotgun metagenomics sequencing to study microbial community dynamics and function in planktonic samples collected along an approximately 700 km river transect, including urban cities and rural settings in upstream waters, before and after the monsoon rainy season. Our results showed that 11%–32% of the microbes represented terrestrial, sewage and human inputs (allochthonous). Sewage inputs significantly contributed to the higher abundance, by 13-fold of human gut microbiome (HG) associated sequences and 2-fold of antibiotic resistance genes (ARGs) in the Ganges relative to other riverine ecosystems in Europe, North and South America. Metagenome-assembled genome sequences (MAGs) representing allochthonous populations were detectable and tractable across the river after 1–2 days of (downstream) transport (> 200 km apart). Only approximately 8% of these MAGs were abundant in U.S. freshwater ecosystems, revealing distinct biodiversity for the Ganges. Microbial communities in the rainy season exhibited increased alpha-diversity and spatial heterogeneity and showed significantly weaker distance-decay patterns compared with the dry season. These results advance our understanding of the Ganges microbial communities and how they respond to anthropogenic pollution.     

Catchment urbanisation and increased benthic algal biomass in streams: linking mechanisms to management

1. Urbanisation is an important cause of eutrophication in waters draining urban areas. We determined whether benthic algal biomass in small streams draining urban areas was explained primarily by small‐scale factors (benthic light, substratum type and nutrient concentrations) within a stream, or by catchment‐scale variables that incorporate the interacting multiple impacts of urbanisation (i.e. variables that describe urban density and the intensity of drainage or septic tank systems). 2. Benthic algal biomass was assessed as chlorophyll a density (chl a) in 16 streams spanning a rural–urban gradient, with both a wide range of urban density and of piped stormwater infrastructure intensity on the eastern fringe of metropolitan Melbourne, Australia. The gradient of urban density among streams was broadly correlated with catchment imperviousness, drainage connection (proportion of impervious areas connected to streams by stormwater pipes), altitude, longitude and median phosphorus concentration. Catchment area, septic tank density, median nitrogen concentration, benthic light (photosynthetically active radiation) and substratum type were not strongly correlated with the urban gradient. 3. Variation in benthic light and substratum type within streams explained a relatively small amount of variation in log chl a (3–11 and 1–13%, respectively) compared with between‐site variation (39–54%). 4. Median chl a was positively correlated with catchment urbanisation, with a large proportion of variance explained jointly (as determined by hierarchical partitioning) by those variables correlated with urban density. Independent of this correlation, the contributions of drainage connection and altitude to the explained variance in chl a were significant. 5. The direct connection of impervious surfaces to streams by stormwater pipes is hypothesised as the main determinant of algal biomass in these streams through its effect on the supply of phosphorus, possibly in interaction with stormwater‐related impacts on grazing fauna. Management of benthic algal biomass in streams of urbanised catchments is likely to be most effective through the application of stormwater management approaches that reduce drainage connection.     

Physicochemical properties of cartilage proteoglycans extracted by lanthanum chloride

Bovine nasal cartilage was extracted with 0.5 M LaCl₃ and the extract then diluted with nine volumes of water. The resulting precipitate (P_LaCl3) contained the proteoglycan subunits, together with minor protein components, but was essentially free from hyaluronic acid. The properties of P_LaCl3 were investigated by chemical analysis, electrophoresis, viscometry and analytical ultracentrifugation, and the results compared with those for proteoglycan obtained by caesium chloride density gradient centrifugation of 2 M CaCl₂ cartilage extracts. Proteoglycan subunits (A1D1) prepared from P_LaCl3 showed identical properties to those obtained from other high ionic strength cartilage extracts.     

Partial Agonism of Taurine at Gamma-Containing Native and Recombinant GABAA Receptors

Taurine is a semi-essential sulfonic acid found at high concentrations in plasma and mammalian tissues which regulates osmolarity, ion channel activity and glucose homeostasis. The structural requirements of GABA_A-receptors (GABA_AR) gated by taurine are not yet known. We determined taurine potency and efficacy relative to GABA at different types of recombinant GABA_AR occurring in central histaminergic neurons of the mouse hypothalamic tuberomamillary nucleus (TMN) which controls arousal. At binary α_1/2β_1/3 receptors taurine was as efficient as GABA, whereas incorporation of the γ_1/2 subunit reduced taurine efficacy to 60–90% of GABA. The mutation γ_2F77I, which abolishes zolpidem potentiation, significantly reduced taurine efficacy at recombinant and native receptors compared to the wild type controls. As taurine was a full- or super- agonist at recombinant α_xβ₁δ-GABA_AR, we generated a chimeric γ₂ subunit carrying the δ subunit motif around F77 (MTVFLH). At α_1/2β₁γ_2(MTVFLH) receptors taurine became a super-agonist, similar to δ-containing ternary receptors, but remained a partial agonist at β₃-containing receptors. In conclusion, using site-directed mutagenesis we found structural determinants of taurine’s partial agonism at γ-containing GABA_A receptors. Our study sheds new light on the β₁ subunit conferring the widest range of taurine-efficacies modifying GABA_AR function under (patho)physiological conditions.     

G protein-coupled receptor signaling via Src kinase induces endogenous human transient receptor potential vanilloid type 6 (TRPV6) channel activation

Ca(2+) homeostasis plays a critical role in a variety of cellular processes. We showed previously that stimulation of the prostate-specific G protein-coupled receptor (PSGR) enhances cytosolic Ca(2+) and inhibits proliferation of prostate cells. Here, we analyzed the signaling mechanisms underlying the PSGR-mediated Ca(2+) increase. Using complementary molecular, biochemical, electrophysiological, and live-cell imaging techniques, we found that endogenous Ca(2+)-selective transient receptor potential vanilloid type 6 (TRPV6) channels are critically involved in the PSGR-induced Ca(2+) signal. Biophysical characterization of the current activated by PSGR stimulation revealed characteristic properties of TRPV6. The molecular identity of the involved channel was confirmed using RNA interference targeting TrpV6. TRPV6-mediated Ca(2+) influx depended on Src kinase activity. Src kinase activation occurred independently of G protein activation, presumably by direct interaction with PSGR. Taken together, we report that endogenous TRPV6 channels are activated downstream of a G protein-coupled receptor and present the first physiological characterization of these channels in situ.