Resilience of Xanthoria parietina under Mars-like conditions: photosynthesis and oxidative stress response.

MAIN CONCLUSION: Xanthoria parietina survivability in Mars-like conditions was supported by water-lysis efficiency recovery and antioxidant content balancing with ROS production after 30 days of exposure. Xanthoria parietina (L.) Th. Fr. is a widespread lichen showing tolerance against air pollutants and UV-radiation. It has been tested under space-like and Mars-like conditions resulting in high recovery performances. Hereby, we aim to assess the mechanisms at the basis of the thalli resilience against multiple space stress factors. Living thalli of X. parietina were exposed to simulated Martian atmospheric conditions (Dark Mars) and UV radiation (Full Mars). Then, we monitored as vitality indicator the photosynthetic efficiency, assessed by in vivo chlorophyll emission fluorescence measurements (FM; FV/F0). The physiological defense was evaluated by analyzing the thalli antioxidant capacity. The drop of FM and FV/F0 immediately after the exposure indicated a reduction of photosynthesis. After 24 h from exposure, photosynthetic efficiency began to recover suggesting the occurrence of protective mechanisms. Antioxidant concentrations were higher during the exposure, only decreasing after 30 days. The recovery of photosynthetic efficiency in both treatments suggested a strong resilience by the photosynthetic apparatus against combined space stress factors, likely due to the boosted antioxidants at the beginning and their depletion at the end of the exposure. The overall results indicated that the production of antioxidants, along with the occurrence of photoprotection mechanisms, guarantee X. parietina survivability in Mars-like environment.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay.

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

Survivability of the lichen Xanthoria parietina in simulated Martian environmental conditions.

Xanthoria parietina (L.) Th. Fr. is a widely spread foliose lichen showing high tolerance against UV-radiation thanks to parietin, a secondary lichen substance. We exposed samples of X. parietina under simulated Martian conditions for 30 days to explore its survivability. The lichen's vitality was monitored via chlorophyll a fluorescence that gives an indication for active light reaction of photosynthesis, performing in situ and after-treatment analyses. Raman spectroscopy and TEM were used to evaluate carotenoid preservation and possible variations in the photobiont's ultrastructure respectively. Significant differences in the photo-efficiency between UV irradiated samples and dark-kept samples were observed. Fluorescence values correlated with temperature and humidity day-night cycles. The photo-efficiency recovery showed that UV irradiation caused significant effects on the photosynthetic light reaction. Raman spectroscopy showed that the carotenoid signal from UV exposed samples decreased significantly after the exposure. TEM observations confirmed that UV exposed samples were the most affected by the treatment, showing chloroplastidial disorganization in photobionts' cells. Overall, X. parietina was able to survive the simulated Mars conditions, and for this reason it may be considered as a candidate for space long-term space exposure and evaluations of the parietin photodegradability.

Comparison of sensitivity between real-time detection of a TaqMan assay for Batrachochytrium dendrobatidis and conventional detection.

A sensitive and quantitative TaqMan assay for the causative agent of chytridiomycosis in amphibians (Batrachochytrium dendrobatidis) has been developed and is routinely used in diagnostic laboratories. We assessed whether the real time detection of the TaqMan assay was as sensitive as the detection of the PCR product by agarose gel electrophoresis and ethidium bromide staining. We found, for practical purposes, that gel-based detection of the diagnostic fragment produced by means of the TaqMan assay or by conventional PCR that used a different polymerase and reaction mix was as sensitive as the real-time detection of the TaqMan assay. We recommend the qualified use of conventional PCR amplification combined with agarose gel electrophoresis and ethidium bromide staining for studies where only prevalence data are required, funding for equipment is limited or the acquisition of a real-time system is not cost effective.

Polymorphic repetitive loci of the amphibian pathogen Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd), the cause of a fatal fungal skin disease of amphibians that has led to massive die-offs, global declines and extinctions, has spread internationally as a pandemic clone with low genetic diversity. A need exists to develop highly polymorphic markers to determine centers of origin and patterns of spread to assist in the development of management strategies. Comparison of paralogous sequences, obtained from the 2 sequenced Bd genomes, indicates useful levels of inter-strain polymorphism in repetitive fragments. We assessed 6 repetitive loci for variation within and among Australian isolates using standard fragment analysis and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) analysis. Confirmation of inter-isolate polymorphism was achieved for 2 marker systems, highlighting the potential of repetitive loci for the development of polymorphic markers in Bd. In addition, we found that repetitive loci in Bd include possible orthologs of virulence-related genes from pathogenic fungi.

Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74.

The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1.

Potent oxadiazole CGRP receptor antagonists for the potential treatment of migraine.

A pharmacophore model was built, based on known CGRP receptor antagonists, and this was used to aid the identification of novel leads. Analogues were designed, modelled and synthesised which incorporated alternative 'LHS' fragments linked via either an amide or urea to a privileged 'RHS' fragment commonly found in CGRP receptor antagonists. As a result a novel series of oxadiazole CGRP receptor antagonists has been identified and the subsequent optimisation to enhance both potency and bioavailability is presented.

The identification of structurally novel, selective, orally bioavailable positive modulators of mGluR2.

The optimisation of an HTS hit series (1) leading to the identification of structurally novel, selective, orally bioavailable mGluR2 positive modulators GSK1331258 and GSK1331268 is described. Structure-activity relationships, attenuation of dopaminergic activity, and potentiation of mGluR2 responses in rat hippocampal MPP-DG synapses are also reported.

BSA reduces inhibition in a TaqMan assay for the detection of Batrachochytrium dendrobatidis.

A TaqMan assay for the causative agent of chytridiomycosis in amphibians (Batrachochytrium dendrobatidis) can be inhibited by phenolic compounds, including humic and tannic acids, resulting in false negatives. Bovine serum albumin (BSA) is known to reduce inhibition of PCR when samples are contaminated with these inhibitors. We assessed the effect of BSA in reducing inhibition of the TaqMan assay when analyzing skin swabs for B. dendrobatidis. We found that the addition of BSA to the TaqMan reaction reduced inhibition to insignificant levels. BSA did not appreciably affect the efficiency or analytical sensitivity of the TaqMan reaction in the analysis of standard DNA solutions free from environmental inhibitors. We recommend the addition of 400 ng microl(-1) of BSA to the standard TaqMan assay to reduce inhibition associated with sampling wild amphibians.

Experimental infection of self-cured Leiopelma archeyi with the amphibian chytrid Batrachochytrium dendrobatidis.

The susceptibility of Archey's frog Leiopelma archeyi to Batrachochytrium dendrobatidis (Bd) is unknown, although one large population is thought to have declined sharply due to chytridiomycosis. As primary infection experiments were not permitted in this endangered New Zealand species, 6 wild-caught L. archeyi that naturally cleared infections with Bd while in captivity were exposed again to Bd to assess their immunity. These frogs were from an infected population at Whareorino, which has no known declines. All 6 L. archeyi became reinfected at low intensities, but rapidly self cured, most by 2 wk. Six Litoria ewingii were used as positive controls and developed heavier infections and clinical signs by 3 wk, demonstrating that the zoospore inoculum was virulent. Six negative controls of each species remained uninfected and healthy. Our results show that L. archeyi that have self cured have resistance to chytridiomycosis when exposed. The pattern is consistent with innate or acquired immunity to Bd, and immunological studies are needed to confirm this.

'Priming' exercise and O2 uptake kinetics during treadmill running.

We tested the hypothesis that priming exercise would speed V(O2) kinetics during treadmill running. Eight subjects completed a square-wave protocol, involving two bouts of treadmill running at 70% of the difference between the running speeds at lactate threshold (LT) and V(O2) max, separated by 6-min of walking at 4 km h(-1), on two occasions. Oxygen uptake was measured breath-by-breath and subsequently modelled using non-linear regression techniques. Heart rate and blood lactate concentration were significantly elevated prior to the second exercise bout compared to the first. However, V(O2) kinetics was not significantly different between the first and second exercise bouts (mean+/-S.D., phase II time constant, Bout 1: 16+/-3s vs. Bout 2: 16+/-4s; V(O2) slow component amplitude, Bout 1: 0.24+/-0.10 L min(-1)vs. Bout 2: 0.20+/-0.12 L min(-1); mean response time, Bout 1: 34+/-4s vs. Bout 2: 34+/-6s; P>0.05 for all comparisons). These results indicate that, contrary to previous findings with other exercise modalities, priming exercise does not alter V(O2) kinetics during high-intensity treadmill running, at least in physically active young subjects. We speculate that the relatively fast V(O2) kinetics and the relatively small V(O2) slow component in the control ('un-primed') condition negated any enhancement of V(O2) kinetics by priming exercise in this exercise modality.

The effects of contrast bathing and compression therapy on muscular performance.

UNLABELLED: Contrast bathing (CB) and compression garments (CG) are widely used to promote recovery. PURPOSE: To evaluate CB and CG as regeneration strategies after exercise-induced muscle damage (EIMD). METHODS: Baseline values of muscle soreness, serum creatine kinase (CK) and myoglobin (Mb), joint range of motion, limb girth, 10- or 30-m sprint, countermovement jump (CMJ), and five repetition maximum squat were completed by 26 young men who then undertook a resistance exercise challenge (REC) to induce EIMD: 6 x 10 parallel squats at 100% body weight with 5-s one repetition maximum eccentric squat superimposed onto each set. After the REC, subjects were separated into three intervention groups: CB, CG, and control (CONT). Forty-eight hours after REC, the subjects exercise performance was reassessed. CK and Mb were also measured +1, +24, and +48 h post-REC. RESULTS: CK was elevated at +24 h ( upward arrow140%; upward arrow161%; upward arrow270%), and Mb was elevated at +1 h ( upward arrow523%; upward arrow458%; upward arrow682%) in CB, CG, and CONT. Within-group large effect sizes for loge[CK] were found for CB at +24 h (0.80) and +48 h (0.84). Area under the [Mb] curve was lower in CB compared with CG and CONT (P < or = 0.05). At +48 h, significant differences from baseline were found in all groups for CMJ (CG, downward arrow5.1%; CB, downward arrow4.4%; CONT, downward arrow8.5%) and soreness ( upward arrow213%; upward arrow284%; upward arrow284%). Soreness transiently fell at +1 h compared with post-REC in the CB group. At +48 h, midthigh girth increased in CB ( upward arrow1.4%) and CONT ( upward arrow1.6%), whereas 30-m sprint time increased in CG ( upward arrow2%). CONCLUSION: No hierarchy of recovery effects was found. Neither contrast bathing nor compression acted to promote acute recovery from EIMD any more effectively than passive conditions, although contrast bathing may transiently attenuate postexercise soreness.

Increased Numbers of Culturable Inhibitory Bacterial Taxa May Mitigate the Effects of Batrachochytrium dendrobatidis in Australian Wet Tropics Frogs.

Symbiotic bacterial communities resident on amphibian skin can benefit their hosts. For example, antibiotic production by community members can control the pathogen Batrachochytrium dendrobatidis (Bd) and it is possible for these community members to be used as probiotics to reduce infection levels. In the early 1990s, the emergence of Bd caused declines and disappearances of frogs in the Australian Wet Tropics; the severity of its effects varied among species and sites. Some species have since recolonized despite enzootic Bd within their populations. This variation in history among species and sites provided an opportunity to investigate the role of anti-fungal cutaneous bacteria in protecting frogs against Bd infection. We collected cutaneous swab samples from three species of frogs at two upland and two lowland sites in the Wet Tropics, and used in vitro challenge assays to identify culturable Bd-inhibitory bacterial isolates for further analysis. We sequenced DNA from cultured inhibitory isolates to identify taxa, resulting in the classification of 16 Bd-inhibitory OTUs, and determined whether inhibitory taxa were associated with frog species, site, or intensity of infection. We present preliminary results showing that the upper limit of Bd infection intensity was negatively correlated with number of inhibitory OTUs present per frog indicating that increased numbers of Bd-inhibiting taxa may play a role in reducing the intensity of Bd infections, facilitating frog coexistence with enzootic Bd. One upland site had a significantly lower prevalence of Bd infection, a significantly higher proportion of frogs with one or more culturable Bd-inhibitory OTUs, a greater number of inhibitory bacterial genera present per frog, and statistically significant clustering of individual frogs with similar Bd-inhibitory signatures when compared to all other sites. This suggests that Bd-inhibitory taxa are likely to be particularly important to frogs at this site and may have played a role in their ability to recolonize following population declines. Our findings suggest that the use of multi-taxon Bd-inhibitory probiotics to support at-risk amphibian populations may be more effective than single-taxon alternatives.

Determining the Accuracy and Reliability of Indirect Calorimeters Utilizing the Methanol Combustion Technique.

BACKGROUND: Several indirect calorimetry (IC) instruments are commercially available, but comparative validity and reliability data are lacking. Existing data are limited by inconsistencies in protocols, subject characteristics, or single-instrument validation comparisons. The aim of this study was to compare accuracy and reliability of metabolic carts using methanol combustion as the cross-laboratory criterion. METHODS: Eight 20-minute methanol burn trials were completed on 12 metabolic carts. Respiratory exchange ratio (RER) and percent O2 and CO2 recovery were calculated. RESULTS: For accuracy, 1 Omnical, Cosmed Quark CPET (Cosmed), and both Parvos (Parvo Medics trueOne 2400) measured all 3 variables within 2% of the true value; both DeltaTracs and the Vmax Encore System (Vmax) showed similar accuracy in measuring 1 or 2, but not all, variables. For reliability, 8 instruments were shown to be reliable, with the 2 Omnicals ranking best (coefficient of variation [CV] < 1.26%). Both Cosmeds, Parvos, DeltaTracs, 1 Jaeger Oxycon Pro (Oxycon), Max-II Metabolic Systems (Max-II), and Vmax were reliable for at least 1 variable (CV ≤ 3%). For multiple regression, humidity and amount of combusted methanol were significant predictors of RER (R2 = 0.33, P < .001). Temperature and amount of burned methanol were significant predictors of O2 recovery (R2 = 0.18, P < .001); only humidity was a predictor for CO2 recovery (R2 = 0.15, P < .001). CONCLUSIONS: Omnical, Parvo, Cosmed, and DeltaTrac had greater accuracy and reliability. The small number of instruments tested and expected differences in gas calibration variability limits the generalizability of conclusions. Finally, humidity and temperature could be modified in the laboratory to optimize IC conditions.

The creation and characterisation of a National Compound Collection: the Royal Society of Chemistry pilot.

We present a summary of the National Compound Collection (NCC) pilot; which harvested chemical structure data from 746 publicly-available PhD theses to create an enhanced database of diverse and interesting (largely organic) molecular entities. The database comprised ∼75 000 structure entries, of which 70% were new to ChemSpider at the time of upload. The dataset was evaluated for structural uniqueness by twelve external drug discovery groups from the pharmaceutical, biotech, academic and not-for-profit sectors. These partners generated data reported here comparing the NCC pilot with their in-house compound collections. The proportion of NCC structures considered to be useful for drug discovery ranged from 5-80% depending on the strictness of the filters used; most interestingly from a drug discovery standpoint ∼13k NCC compounds (18% of the NCC) passed the filters and were of good diversity. These compounds are quite different from those that are already present in the screening collections but not so different that they are no longer considered to be drug-like. In general, the drug discovery teams would consider these compounds to be high value molecules for inclusion in their screening collections. This pilot addressed the potential value of unpublished data and explored the practicalities of large-scale data extraction, to inform both retrospective and prospective extraction of chemical data from theses.

Defects in host immune function in tree frogs with chronic chytridiomycosis.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused mass mortality leading to population declines and extinctions in many frog species worldwide. The lack of host resistance may be due to fungal immunosuppressive effects that have been observed when Bd is incubated with cultured lymphocytes, but whether in vivo host immunosuppression occurs is unknown. We used a broad range of hematologic and protein electrophoresis biomarkers, along with various functional tests, to assess immune competence in common green (Litoria caerulea) and white-lipped (L. infrafrenata) tree frogs experimentally infected with Bd. Compared with uninfected frogs, Bd infection in L. caerulea caused a reduction in immunoglobulin and splenic lymphocyte responses to antigenic stimulation with sheep red blood cells, along with decreased white blood cell and serum protein concentrations, indicating possible impaired immune response capability of Bd-infected frogs. This is the first in vivo study suggesting that infection with Bd causes multiple defects in systemic host immune function, and this may contribute to disease development in susceptible host species. Although L. infrafrenata failed to maintain Bd infection after exposure, white blood cell and serum globulin concentrations were lower in recovered frogs compared with unexposed frogs, but antigen-specific serum and splenic antibody, and splenic cellular, responses were similar in both recovered and unexposed frogs. This may indicate potential systemic costs associated with infection clearance and/or redirection of host resources towards more effective mechanisms to overcome infection. No clear mechanism for resistance was identified in L. infrafrenata, suggesting that localized and/or innate immune defense mechanisms may be important factors involved in disease resistance in this species.

Are GPCRs still a source of new targets?

G-protein-coupled receptors (GPCRs) still offer enormous scope for new therapeutic targets. Currently marketed agents are dominated by those with activity at aminergic receptors and yet they account for only ~10% of the family. Progress up until now with other subfamilies, notably orphans, Family A/peptide, Family A/lipid, Family B, Family C, and Family F, has been, at best, patchy. This may be attributable to the heterogeneous nature of GPCRs, their endogenous ligands, and consequently their binding sites. Our appreciation of receptor similarity has arguably been too simplistic, and screening collections have not necessarily been well suited to identifying leads in new areas. Despite the relative shortage of high-quality tool molecules in a number of cases, there is an emerging, and increasingly substantial, body of evidence associating many as yet "undrugged" receptors with a very wide range of diseases. Significant advances in our understanding of receptor pharmacology and technical advances in screening, protein X-ray crystallography, and ligand design methods are paving the way for new successes in the area. Exploitation of allosteric mechanisms; alternative signaling pathways such as G12/13, Gßγ, and ß-arrestin; the discovery of "biased" ligands; and the emergence of GPCR-protein complexes as potential drug targets offer scope for new and much improved drugs.

Whether the weather drives patterns of endemic amphibian chytridiomycosis: a pathogen proliferation approach.

The pandemic amphibian disease chytridiomycosis often exhibits strong seasonality in both prevalence and disease-associated mortality once it becomes endemic. One hypothesis that could explain this temporal pattern is that simple weather-driven pathogen proliferation (population growth) is a major driver of chytridiomycosis disease dynamics. Despite various elaborations of this hypothesis in the literature for explaining amphibian declines (e.g., the chytrid thermal-optimum hypothesis) it has not been formally tested on infection patterns in the wild. In this study we developed a simple process-based model to simulate the growth of the pathogen Batrachochytrium dendrobatidis (Bd) under varying weather conditions to provide an a priori test of a weather-linked pathogen proliferation hypothesis for endemic chytridiomycosis. We found strong support for several predictions of the proliferation hypothesis when applied to our model species, Litoria pearsoniana, sampled across multiple sites and years: the weather-driven simulations of pathogen growth potential (represented as a growth index in the 30 days prior to sampling; GI30) were positively related to both the prevalence and intensity of Bd infections, which were themselves strongly and positively correlated. In addition, a machine-learning classifier achieved ~72% success in classifying positive qPCR results when utilising just three informative predictors 1) GI30, 2) frog body size and 3) rain on the day of sampling. Hence, while intrinsic traits of the individuals sampled (species, size, sex) and nuisance sampling variables (rainfall when sampling) influenced infection patterns obtained when sampling via qPCR, our results also strongly suggest that weather-linked pathogen proliferation plays a key role in the infection dynamics of endemic chytridiomycosis in our study system. Predictive applications of the model include surveillance design, outbreak preparedness and response, climate change scenario modelling and the interpretation of historical patterns of amphibian decline.

A ligand's view of target similarity: chemogenomic binding site-directed techniques for drug discovery.

GPCR binding site-directed techniques are rapidly evolving into powerful tools for modern drug discovery. Many of these approaches bridge chemistry and biology, which are inseparable concepts in nature but are often treated as separate worlds in drug discovery and science in general. This review shows with several examples how focusing on the binding site(s) has a clear advantage when it comes to establishing sequence-correlated pharmacological profiles. By organizing and comparing sequence and structural data it is possible to "borrow" SAR from similar targets to increase the speed of lead-finding and, potentially, to produce ligands for previously intractable receptors. Sequence motifs correlated with ligands can be applied in the design of target-specific focused libraries that are both efficient and cost-effective and should provide increased hit-rates over diversity screening. Furthermore, in the optimization phase, the binding motif approach offers the possibility to identify quickly the most likely off-target candidates to be chosen for selectivity screening, as well as potentially characterizing those pockets which may best be exploited for improved selectivity.