Characterization of a High-Affinity Copper Transporter in the White-Nose Syndrome Causing Fungal Pathogen Pseudogymnoascus destructans.

Copper is an essential micronutrient and the ability to scavenge tightly bound or trace levels of copper ions at the host-pathogen interface is vital for fungal proliferation in animal hosts. Recent studies suggest that trace metal ion acquisition is critical for the establishment and propagation of Pseudogymnoascus destructans, the fungal pathogen responsible for white-nose syndrome (WNS), on their bat host. However, little is known about these metal acquisition pathways in P. destructans. In this study, we report the characterization of the P. destructans high-affinity copper transporter VC83_00191 (PdCTR1a), which is implicated as a virulence factor associated with the WNS disease state. Using Saccharomyces cerevisiae as a recombinant expression host, we find that PdCTR1a localizes to the cell surface plasma membrane and can efficiently traffic Cu-ions into the yeast cytoplasm. Complementary studies in the native P. destructans fungus provide evidence that PdCTR1a transcripts and protein levels are dictated by Cu-bioavailability in the growth media. Our study demonstrates that PdCTR1a is a functional high-affinity copper transporter and is relevant to Cu-homeostasis pathways in P. destructans.

Developments of Recent Applications for Early Diagnosis of Diseases Using Electronic-Nose and Other VOC-Detection Devices.

This Editorial provides summaries and an overview of research and review articles published in the Sensors journal, volumes 21 (2021), 22 (2022), and 23 (2023), within the biomedical Special Issue "Portable Electronic-Nose Devices for Noninvasive Early Disease Detection", which focused on recent sensors, biosensors, and clinical instruments developed for noninvasive early detection and diagnosis of human and animal diseases. The ten articles published in this Special Issue provide new information associated with recent electronic-nose (e-nose) and related volatile organic compound (VOC)-detection technologies developed to improve the effectiveness and efficiency of diagnostic methodologies for early disease detection prior to symptom development. For review purposes, the summarized articles were placed into three broad groupings or topic areas, including veterinary-wildlife pathology, human clinical pathology, and the detection of dietary effects on VOC emissions. These specified categories were used to define sectional headings devoted to related research studies with a commonality based on a particular disease being investigated or type of analytical instrument used in analyses.

Mitigation of benzoic acid-driven autotoxicity in waste nutrient solution using O3 and O3/H2O2 treatments: Seed germination and root growth of Lactuca sativa L.

Benzoic acid (BA), a secondary metabolite released through root exudates, is considered to be the most common inhibitor that leads to plant autotoxicity, even at low concentrations in closed hydroponic systems. In this study, to mitigate BA-driven autotoxicity, the effects of O3 and O3/H2O2 oxidation treatment (O3 concentration: 1, 2, 4, 8 mg L-1, H2O2 concentration: 4, 8 mg L-1) on waste nutrient solution (WNS) were investigated in terms of BA degradation, the rate of germination inhibition (GI), and the rate of root growth inhibition (RI). In the case of O3 treatment, the BA degradation rate improved up to 14.1% as the O3 concentration increased, while alleviation of GI was insignificant (94.6-100%), confirming that a single O3 treatment was unsuitable for mitigating autotoxicity. On the other hand, O3/H2O2 treatment increased BA degradation by up to 24.8%, thereby significantly reducing GI (up to 7.69%) and RI (up to 0.88%). Both the highest BA mineralization rate and phytotoxicity mitigation was observed at BA125 (4-4) (BA mineralization: 16.7%, GI: 12.82%, RI: 11.69%) and BA125 (1-8) (BA mineralization: 17.7%, GI: 7.69%, RI: 0.88%) at each H2O2 concentration. In addition, the operating costs were evaluated by a chemical and electricity cost analysis at the different treatments. As a result, the operating costs of BA125 (4-4) and BA125 (1-8) were calculated to be 0.40 and 0.42 $ L-1 mg-1 of mineralized BA, respectively. After consideration of the mineralization rate, autotoxicity mitigation, and operating cost, BA125 (1-8) was suggested for the optimal treatment condition and our findings would contribute to the alleviation of BA-driven autotoxicity.

COULD WHITE-NOSE SYNDROME MANIFEST DIFFERENTLY IN MYOTIS LUCIFUGUS IN WESTERN VERSUS EASTERN REGIONS OF NORTH AMERICA? A REVIEW OF FACTORS.

White-nose syndrome (WNS) has notably affected the abundance of Myotis lucifugus (little brown myotis) in North America. Thus far, substantial mortality has been restricted to the eastern part of the continent where the cause of WNS, the invasive fungus Pseudogymnoascus destructans, has infected bats since 2006. To date, the state of Washington is the only area in the Western US or Canada (the Rocky Mountains and further west in North America) with confirmed cases of WNS in bats, and there the disease has spread more slowly than it did in Eastern North America. Here, we review differences between M. lucifugus in western and eastern parts of the continent that may affect transmission, spread, and severity of WNS in the West and highlight important gaps in knowledge. We explore the hypothesis that western M. lucifugus may respond differently to WNS on the basis of different hibernation strategies, habitat use, and greater genetic structure. To document the effect of WNS on M. lucifugus in the West most effectively, we recommend focusing on maternity roosts for strategic disease surveillance and monitoring abundance. We further recommend continuing the challenging work of identifying hibernation and swarming sites to better understand the microclimates, microbial communities, and role in disease transmission of these sites, as well as the ecology and hibernation physiology of bats in noncavernous hibernacula.

Characterization of Pseudogymnoascus destructans conidial adherence to extracellular matrix: Association with fungal secreted proteases and identification of candidate extracellular matrix binding proteins.

Pseudogymnoascus destructans is the etiological agent of white-nose syndrome (WNS), a fungal skin infection of hibernating bats. Pathophysiology of the disease involves disruption of bat metabolism and hibernation patterns, which subsequently causes premature emergence and mortality. However, information on the mechanism(s) and virulence factors of P. destructans infection is minimally known. Typically, fungal adherence to host cells and extracellular matrix (ECM) is the critical first step of the infection. It allows pathogenic fungi to establish colonization and provides an entry for invasion in host tissues. In this study, we characterized P. destructans conidial adherence to laminin and fibronectin. We found that P. destructans conidia adhered to laminin and fibronectin in a dose-dependent, time-dependent and saturable manner. We also observed changes in the gene expression of secreted proteases, in response to ECM exposure. However, the interaction between fungal conidia and ECM was not specific, nor was it facilitated by enzymatic activity of secreted proteases. We therefore further investigated other P. destructans proteins that recognized ECM and found glyceraldehyde-3-phosphate dehydrogenase and elongation factor 1-alpha among the candidate proteins. Our results demonstrate that P. destructans may use conidial surface proteins to recognize laminin and fibronectin and facilitate conidial adhesion to ECM. In addition, other non-specific interactions may contribute to the conidial adherence to ECM. However, the ECM binding protein candidates identified in this study highlight additional potential fungal virulence factors worth investigating in the P. destructans mechanism of infection in future studies.

Disease-related population declines in bats demonstrate non-exchangeability in generalist predators.

The extent to which persisting species may fill the functional role of extirpated or declining species has profound implications for the structure of biological communities and ecosystem functioning. In North America, arthropodivorous bats are threatened on a continent-wide scale by the spread of white-nose syndrome (WNS), a disease caused by the fungus Pseudogymnoascus destructans. We tested whether bat species that display lower mortality from this disease can partially fill the functional role of other bat species experiencing population declines. Specifically, we performed high-throughput amplicon sequencing of guano from two generalist predators: the little brown bat (Myotis lucifugus) and big brown bat (Eptesicus fuscus). We then compared changes in prey consumption before versus after population declines related to WNS. Dietary niches contracted for both species after large and abrupt declines in little brown bats and smaller declines in big brown bats, but interspecific dietary overlap did not change. Furthermore, the incidence and taxonomic richness of agricultural pest taxa detected in diet samples decreased following bat population declines. Our results suggest that persisting generalist predators do not necessarily expand their dietary niches following population declines in other predators, providing further evidence that the functional roles of different generalist predators are ecologically distinct.

Evaluation of Virus-Free and Wild-Type Isolates of Pseudogymnoascus destructans Using a Porcine Ear Model.

White-nose syndrome (WNS), responsible for the mass mortality of North American bats, lacks economically viable and practical in vitro models for Pseudogymnoascus destructans infection, the causative agent of WNS. Not only are many susceptible North American insectivorous bats nearing extinction and, thus, scarce for experimental studies, but they are difficult to care for and maintain in captivity because of their specialized habitats and diets. In this study, we explored porcine ears as a potential substrate for studying infection development and the dynamics of P. destructans growth in the laboratory. Porcine ear skin shares many tissue-level similarities with bat skin and is a readily available resource. We found the porcine ear model provided a substrate faithfully mimicking external P. destructans colony morphology and internal histology similar to what is seen with P. destructans infections in bat wing membranes. This model provided a major advance by distinguishing virulence attributes between a wild-type Pseudogymnoascus destructans strain harboring a partitivirus common to all North American strains of the fungus and an isogenic virus-cured P. destructans strain. ImageJ analysis showed that the cured P. destructans strain was reduced significantly in ability to produce hyphal cover and showed less spore production on porcine skin. Taken together, these results strengthen our previous finding that the partitivirus infection has a role in WNS and provides a valuable model host tool in understanding P. destructans virulence factors for therapeutic application. IMPORTANCE This work describes an important insight into the role of Pseudogymnoascus destructans partitivirus in fungal biology and provides a model system for studying white-nose syndrome in bats, which has decimated North American populations.

Trans-2-hexenal downregulates several pathogenicity genes of Pseudogymnoascus destructans, the causative agent of white-nose syndrome in bats.

White-nose syndrome is an emergent wildlife disease that has killed millions of North American bats. It is caused by Pseudogymnoascus destructans, a cold-loving, invasive fungal pathogen that grows on bat tissues and disrupts normal hibernation patterns. Previous work identified trans-2-hexenal as a fungistatic volatile compound that potentially could be used as a fumigant against P. destructans in bat hibernacula. To determine the physiological responses of the fungus to trans-2-hexenal exposure, we characterized the P. destructans transcriptome in the presence and absence of trans-2-hexenal. Specifically, we analyzed the effects of sublethal concentrations (5 µmol/L, 10 µmol/L, and 20 µmol/L) of gas-phase trans-2-hexenal of the fungus grown in liquid culture. Among the three treatments, a total of 407 unique differentially expressed genes (DEGs) were identified, of which 74 were commonly affected across all three treatments, with 44 upregulated and 30 downregulated. Downregulated DEGs included several probable virulence genes including those coding for a high-affinity iron permease, a superoxide dismutase, and two protein-degrading enzymes. There was an accompanying upregulation of an ion homeostasis gene, as well as several genes involved in transcription, translation, and other essential cellular processes. These data provide insights into the mechanisms of action of trans-2-hexenal as an anti-fungal fumigant that is active at cold temperatures and will guide future studies on the molecular mechanisms by which six carbon volatiles inhibit growth of P. destructans and other pathogenic fungi.

Dr. Dwight Parkinson: a Canadian neurosurgical pioneer.

In 1950, Dwight Parkinson was the first qualified neurosurgeon to arrive in Winnipeg, Manitoba. He played a monumental role in developing one of the earliest neurosurgical training programs in Western Canada. Parkinson was a pioneering neurosurgeon who served as the first president of the Canadian Neurosurgical Society in 1965. He was the epitome of the skull base neurosurgeon, which was not recognized as a distinct discipline at that time. He contributed to its development through detailed neuroanatomical study of the lateral sellar compartment (housing the parasellar venous plexus, a term he emphasized as more accurate than "cavernous sinus"). Parkinson also made seminal contributions to the management of cerebrovascular disease and offered new insights on cerebral concussion. Parkinson's dedication to clinical excellence and education laid a cornerstone for the development of neurosurgery and the neurosciences in Manitoba, making him a key figure in Canadian neurosurgery. Using published materials, online resources, hospital archives, and personal interviews, the authors conducted a systematic review of Parkinson's formative years, his development of the Section of Neurosurgery at the University of Manitoba, his achievements, and his legacy. This updated biography captures the exploits of this remarkable, and at times strictly disciplinarian, neurosurgeon-anatomist.

HD/FD and DF/AF with Fixed-Gain or Variable-Gain Protocol Switching Mechanism over Cooperative NOMA for Green-Wireless Networks.

This article studied the application of multiple protocol switching mechanism (PSM) over cooperating Non-Orthogonal Multiple Access (NOMA) networks to minimize the probability of outage and maximize the system throughput and energy efficiency (EE). This study investigated six scenarios: (1) a cooperative NOMA system with half-duplex (HD) and decode-and-forward (DF) protocols at the relay; (2) a cooperative NOMA system with full-duplex (FD) and DF protocols at the relay; (3) a cooperative NOMA system with HD and amplification amplify-and-forward (AF) with fixed-gain (FG) protocols at the relay; (4) a cooperative NOMA system with HD and amplification AF with variable-gain (VG) protocols at the relay; (5) a cooperative NOMA system with FD and amplification AF with FG protocols at the relay; (6) a cooperative NOMA system with FD and amplification AF with VG protocols at the relay. Based on the results of analysis and simulations, the study determined the transmission scenario for best system performance. This paper also proposed a mechanism to switch between HD/FD and DF/AF with FG/VG protocols in order to improve the quality of service (QoS) for users with a weak conditional channel. This mechanism can be deployed in future 5G wireless network sensors. Finally, EE was also assessed in relation to future green-wireless networks (G-WNs).

Experimental Infection of Tadarida brasiliensis with Pseudogymnoascus destructans, the Fungus That Causes White-Nose Syndrome.

White-nose syndrome (WNS) is causing significant declines in populations of North American hibernating bats, and recent western and southern expansions of the disease have placed additional species at risk. Understanding differences in species susceptibility and identifying management actions to reduce mortality of bats from WNS are top research priorities. However, the use of wild-caught susceptible bats, such as Myotis lucifugus, as model species for WNS research is problematic and places additional pressure on remnant populations. We investigated the feasibility of using Tadarida brasiliensis, a highly abundant species of bat that tolerates captivity, as the basis for an experimental animal model for WNS. Using methods previously established to confirm the etiology of WNS in M. lucifugus, we experimentally infected 11 T. brasiliensis bats with Pseudogymnoascus destructans in the laboratory under conditions that induced hibernation. We detected P. destructans on all 11 experimentally infected bats, 7 of which exhibited localized proliferation of hyphae within the epidermis, dermis, and subcutaneous tissue, similar to invasive cutaneous ascomycosis observed in M. lucifugus bats with WNS. However, the distribution of lesions across wing membranes of T. brasiliensis bats was limited, and only one discrete "cupping erosion," diagnostic for WNS, was identified. Thus, the rarity of lesions definitive for WNS suggests that T. brasiliensis does not likely represent an appropriate model for studying the pathophysiology of this disease. Nonetheless, the results of this study prompt questions concerning the potential for free-ranging, migratory T. brasiliensis bats to become infected with P. destructans and move the fungal pathogen between roost sites used by species susceptible to WNS.IMPORTANCE White-nose syndrome (WNS) is a fungal disease that is causing severe declines of bat populations in North America. Identifying ways to reduce the impacts of this disease is a priority but is inhibited by the lack of an experimental animal model that does not require the use of wild-caught bat species already impacted by WNS. We tested whether Tadarida brasiliensis, one of the most abundant species of bats in the Americas, could serve as a suitable animal model for WNS research. While T. brasiliensis bats were susceptible to experimental infection with the fungus under conditions that induced hibernation, the species exhibited limited pathology diagnostic for WNS. These results indicate that T. brasiliensis is not likely a suitable experimental model for WNS research. However, the recovery of viable WNS-causing fungus from experimentally infected bats indicates a potential for this species to contribute to the spread of the pathogen where it coexists with other species of bats affected by WNS.

Green Fluorescent Protein Expression in Pseudogymnoascus destructans to Study Its Abiotic and Biotic Lifestyles.

Pseudogymnoascus destructans (Pd) is the etiologic agent of bat White-nose syndrome, a disease that has caused the unprecedented reduction in the hibernating bat populations across eastern North America. The Pd pathogenesis appears to be a complex adaptation of fungus in its abiotic (caves and mines) and biotic (bats) environments. There is a general lack of experimental tools for the study of Pd biology. We described the successful expression of codon-optimized synthetic green fluorescent protein sGFP in Pd. The sGFP(S65T) gene was first fused in frame with the Aspergillus nidulans promoter in the tumor-inducing plasmid pRF-HUE, and the resulting plasmid pHUE-sGFP(S65T) was transformed into Pd by Agrobacterium tumefaciens-mediated transformation system. The integration of sGFP(S65T) in Pd genome was analyzed by PCR, and single integration frequency of approximately 66% was confirmed by Southern hybridization. Fluorescent microscopy and flow cytometric analyses of two randomly selected transformants with single integration revealed high expression of sGFP in both spores and hyphal structures. The biology of mutants as judged by sporulation, growth rate, and urease production was not altered indicating sGFP is not toxic to Pd. Thus, we have generated a valuable tool that will facilitate the elucidation of Pd biology, ecology, and pathogenicity in real time.

Determinants of Pseudogymnoascus destructans within bat hibernacula: implications for surveillance and management of white-nose syndrome.

1. Fungal diseases are an emerging global problem affecting human health, food security and biodiversity. Ability of many fungal pathogens to persist within environmental reservoirs can increase extinction risks for host species and presents challenges for disease control. Understanding factors that regulate pathogen spread and persistence in these reservoirs is critical for effective disease management. 2. White-nose syndrome (WNS) is a disease of hibernating bats caused by Pseudogymnoascus destructans (Pd), a fungus that establishes persistent environmental reservoirs within bat hibernacula, which contribute to seasonal disease transmission dynamics in bats. However, host and environmental factors influencing distribution of Pd within these reservoirs are unknown. 3. We used model selection on longitudinally collected field data to test multiple hypotheses describing presence-absence and abundance of Pd in environmental substrates and on bats within hibernacula at different stages of WNS. 4. First detection of Pd in the environment lagged up to one year after first detection on bats within that hibernaculum. Once detected, the probability of detecting Pd within environmental samples from a hibernaculum increased over time and was higher in sediment compared to wall surfaces. Temperature had marginal effects on the distribution of Pd. For bats, prevalence and abundance of Pd were highest on Myotis lucifugus and on bats with visible signs of WNS. 5. Synthesis and applications. Our results indicate that distribution of Pseudogymnoascus destructans (Pd) within a hibernaculum is driven primarily by bats with delayed establishment of environmental reservoirs. Thus, collection of samples from Myotis lucifugus, or from sediment if bats cannot be sampled, should be prioritized to improve detection probabilities for Pd surveillance. Long-term persistence of Pd in sediment suggests that disease management for white-nose syndrome should address risks of sustained transmission from environmental reservoirs.

Pseudogymnoascus destructans: Causative Agent of White-Nose Syndrome in Bats Is Inhibited by Safe Volatile Organic Compounds.

White-nose syndrome (WNS) is caused by Pseudogymnoascus destructans, a psychrophilic fungus that infects hibernating bats and has caused a serious decline in some species. Natural aroma compounds have been used to control growth of fungal food storage pathogens, so we hypothesized that a similar strategy could work for control of P. destructans. The effectiveness of exposure to low concentrations of the vapor phase of four of these compounds was tested on mycelial plugs and conidiospores at temperatures of 5, 10 and 15 °C. Here we report the efficacy of vapor phase mushroom alcohol (1-octen-3-ol) for inhibiting mycelial and conidiospore growth of P. destructans at 0.4 and 0.8 µmol/mL and demonstrate that the R enantiomer of this compound is more effective than the S enantiomer, supporting the finding that biological systems can be sensitive to stereochemistry. Further, we report that vapor phase leaf aldehyde (trans-2-hexenal), a common aroma compound associated with cut grass odors and also the major volatile compound in extra virgin olive oil, is more effective than mushroom alcohol. At 0.05 µmol/mL, trans-2-hexenal is fungicidal to both conidiospores and mycelia of P. destructans.

Body temperatures of hibernating little brown bats reveal pronounced behavioural activity during deep torpor and suggest a fever response during white-nose syndrome.

Hibernating animals use torpor [reduced body temperature (T b) and metabolic rate] to reduce energy expenditure during winter. Periodic arousals to normal T b are energetically expensive, so hibernators trade off arousal benefits against energetic costs. This is especially important for bats with white-nose syndrome (WNS), a fungal disease causing increased arousal frequency. Little brown bats (Myotis lucifugus) with WNS show upregulation of endogenous pyrogens and sickness behaviour. Therefore, we hypothesized that WNS should cause a fever response characterized by elevated T b. Hibernators could also accrue some benefits of arousals with minimal T b increase, thus avoiding full arousal costs. We compared skin temperature (T sk) of captive Myotis lucifugus inoculated with the WNS-causing fungus to T sk of sham-inoculated controls. Infected bats re-warmed to higher T sk during arousals which is consistent with a fever response. Torpid T sk did not differ. During what we term "cold arousals", bats exhibited movement following T sk increases of only 2.2 ± 0.3 °C, compared to >20 °C increases during normal arousals. Cold arousals occurred in both infected and control bats, suggesting they are not a pathophysiological consequence of WNS. Fever responses are energetically costly and could exacerbate energy limitation and premature fat depletion for bats with WNS. Cold arousals could represent an energy-saving mechanism for both healthy and WNS-affected bats when complete arousals are unnecessary or too costly. A few cold arousals were observed mid-hibernation, typically in response to disturbances. Cold arousals may, therefore, represent a voluntary restriction of arousal temperature instead of loss of thermoregulatory control.

Skin and fur bacterial diversity and community structure on American southwestern bats: effects of habitat, geography and bat traits.

Microorganisms that reside on and in mammals, such as bats, have the potential to influence their host's health and to provide defenses against invading pathogens. However, we have little understanding of the skin and fur bacterial microbiota on bats, or factors that influence the structure of these communities. The southwestern United States offers excellent sites for the study of external bat bacterial microbiota due to the diversity of bat species, the variety of abiotic and biotic factors that may govern bat bacterial microbiota communities, and the lack of the newly emergent fungal disease in bats, white-nose syndrome (WNS), in the southwest. To test these variables, we used 16S rRNA gene 454 pyrosequencing from swabs of external skin and fur surfaces from 163 bats from 13 species sampled from southeastern New Mexico to northwestern Arizona. Community similarity patterns, random forest models, and generalized linear mixed-effects models show that factors such as location (e.g., cave-caught versus surface-netted) and ecoregion are major contributors to the structure of bacterial communities on bats. Bats caught in caves had a distinct microbial community compared to those that were netted on the surface. Our results provide a first insight into the distribution of skin and fur bat bacteria in the WNS-free environment of New Mexico and Arizona. More importantly, it provides a baseline of bat external microbiota that can be explored for potential natural defenses against pathogens.

DISPERSAL HAZARDS OF PSEUDOGYMNOASCUS DESTRUCTANS BY BATS AND HUMAN ACTIVITY AT HIBERNACULA IN SUMMER.

Bats occupying hibernacula during summer are exposed to Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome (WNS), and may contribute to its dispersal. Furthermore, equipment and clothing exposed to cave environments are a potential source for human-assisted spread of Pd. To explore dispersal hazards for Pd during the nonhibernal season, we tested samples that were collected from bats, the environment, and equipment at hibernacula in the eastern US between 18 July-22 August 2012. Study sites included six hibernacula known to harbor bats with Pd with varying winter-count impacts from WNS and two hibernacula (control sites) without prior history of WNS. Nucleic acid from Pd was detected from wing-skin swabs or guano from 40 of 617 bats (7% prevalence), including males and females of five species at five sites where WNS had previously been confirmed as well as from one control site. Analysis of guano collected during summer demonstrated a higher apparent prevalence of Pd among bats (17%, 37/223) than did analysis of wing-skin swabs (1%, 4/617). Viable Pd cultured from wing skin (2%, 1/56) and low recapture rates at all sites suggested bats harboring Pd during summer could contribute to pathogen dispersal. Additionally, Pd DNA was detected on clothing and trapping equipment used inside and near hibernacula, and Pd was detected in sediment more readily than in swabs of hibernaculum walls. Statistically significant differences in environmental abundance of Pd were not detected among sites, but prevalence of Pd differed between sites and among bat species. Overall, bats using hibernacula in summer can harbor Pd on their skin and in their guano, and demonstration of Pd on clothing, traps, and other equipment used at hibernacula during summertime within the WNS-affected region indicates risk for pathogen dispersal during the nonhibernal season.

Conservation implications of physiological carry-over effects in bats recovering from white-nose syndrome.

Although it is well documented that infectious diseases can pose threats to biodiversity, the potential long-term consequences of pathogen exposure on individual fitness and its effects on population viability have rarely been studied. We tested the hypothesis that pathogen exposure causes physiological carry-over effects with a pathogen that is uniquely suited to this question because the infection period is specific and time limited. The fungus Pseudogymnoascus destructans causes white-nose syndrome (WNS) in hibernating bats, which either die due to the infection while hibernating or recover following emergence from hibernation. The fungus infects all exposed individuals in an overwintering site simultaneously, and bats that survive infection during hibernation clear the pathogen within a few weeks following emergence. We quantified chronic stress during the active season, when bats are not infected, by measuring cortisol in bat claws. Free-ranging Myotis lucifugus who survived previous exposure to P. destructans had significantly higher levels of claw cortisol than naïve individuals. Thus, cryptic physiological carry-over effects of pathogen exposure may persist in asymptomatic, recovered individuals. If these effects result in reduced survival or reproductive success, they could also affect population viability and even act as a third stream in the extinction vortex. For example, significant increases in chronic stress, such as those indicated here, are correlated with reduced reproductive success in a number of species. Future research should directly explore the link between pathogen exposure and the viability of apparently recovered populations to improve understanding of the true impacts of infectious diseases on threatened populations.

Model-Driven Approach for Body Area Network Application Development.

This paper introduces the sensor-networked IoT model as a prototype to support the design of Body Area Network (BAN) applications for healthcare. Using the model, we analyze the synergistic effect of the functional requirements (data collection from the human body and transferring it to the top level) and non-functional requirements (trade-offs between energy-security-environmental factors, treated as Quality-of-Service (QoS)). We use feature models to represent the requirements at the earliest stage for the analysis and describe a model-driven methodology to design the possible BAN applications. Firstly, we specify the requirements as the problem domain (PD) variability model for the BAN applications. Next, we introduce the generative technology (meta-programming as the solution domain (SD)) and the mapping procedure to map the PD feature-based variability model onto the SD feature model. Finally, we create an executable meta-specification that represents the BAN functionality to describe the variability of the problem domain though transformations. The meta-specification (along with the meta-language processor) is a software generator for multiple BAN-oriented applications. We validate the methodology with experiments and a case study to generate a family of programs for the BAN sensor controllers. This enables to obtain the adequate measure of QoS efficiently through the interactive adjustment of the meta-parameter values and re-generation process for the concrete BAN application.

Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi Pseudogymnoascus destructans and P. pannorum.

The mechanisms of cold adaptation by fungi remain unknown. This topic is of high interest due to the emergence of white-nose syndrome (WNS), a skin infection of hibernating bats caused by Pseudogymnoascus destructans (Pd). Recent studies indicated that apart from Pd, there is an abundance of other Pseudogymnoascus species in the hibernacula soil. We developed an Agrobacterium tumefaciens-mediated transformation (ATMT) system for Pd and a related fungus Pseudogymnoascus pannorum (Pp) to advance experimental studies. URE1 gene encoding the enzyme urease was used as an easy to screen marker to facilitate molecular genetic analyses. A Uracil-Specific Excision Reagent (USER) Friendly pRF-HU2 vector containing Pd or Pp ure1::hygromycin (HYG) disruption cassette was introduced into A. tumefaciens AGL-1 cells by electroporation and the resulting strains were co-cultivated with conidia of Pd or Pp for various durations and temperatures to optimize the ATMT system. Overall, 680 Pd (0.006%) and 1800 Pp (0.018%) transformants were obtained from plating of 10(7) conidia; their recoveries were strongly correlated with the length of the incubation period (96h for Pd; 72h for Pp) and with temperature (15-18°C for Pd; 25°C for Pp). The homologous recombination in transformants was 3.1% for Pd and 16.7% for Pp. The availability of a standardized ATMT system would allow future molecular genetic analyses of Pd and related cold-adapted fungi.