Disease recovery in bats affected by white-nose syndrome.

Processes associated with recovery of survivors are understudied components of wildlife infectious diseases. White-nose syndrome (WNS) in bats provides an opportunity to study recovery of disease survivors, understand implications of recovery for individual energetics, and assess the role of survivors in pathogen transmission. We documented temporal patterns of recovery from WNS in little brown bats (Myotis lucifugus) following hibernation to test the hypotheses that: (1) recovery of wing structure from WNS matches a rapid time scale (i.e. approximately 30 days) suggested by data from free-ranging bats; (2) torpor expression plays a role in recovery; (3) wing physiological function returns to normal alongside structural recovery; and (4) pathogen loads decline quickly during recovery. We collected naturally infected bats at the end of hibernation, brought them into captivity, and quantified recovery over 40 days by monitoring body mass, wing damage, thermoregulation, histopathology of wing biopsies, skin surface lipids and fungal load. Most metrics returned to normal within 30 days, although wing damage was still detectable at the end of the study. Torpor expression declined overall throughout the study, but bats expressed relatively shallow torpor bouts - with a plateau in minimum skin temperature - during intensive healing between approximately days 8 and 15. Pathogen loads were nearly undetectable after the first week of the study, but some bats were still detectably infected at day 40. Our results suggest that healing bats face a severe energetic imbalance during early recovery from direct costs of healing and reduced foraging efficiency. Management of WNS should not rely solely on actions during winter, but should also aim to support energy balance of recovering bats during spring and summer.

Bats are not squirrels: Revisiting the cost of cooling in hibernating mammals.

Many species use stored energy to hibernate through periods of resource limitation. Hibernation, a physiological state characterized by depressed metabolism and body temperature, is critical to winter survival and reproduction, and therefore has been extensively quantified and modeled. Hibernation consists of alternating phases of extended periods of torpor (low body temperature, low metabolic rate), and energetically costly periodic arousals to normal body temperature. Arousals consist of multiple phases: warming, euthermia, and cooling. Warming and euthermic costs are regularly included in energetic models, but although cooling to torpid body temperature is an important phase of the torpor-arousal cycle, it is often overlooked in energetic models. When included, cooling cost is assumed to be 67% of warming cost, an assumption originally derived from a single study that measured cooling cost in ground squirrels. Since this study, the same proportional value has been assumed across a variety of hibernating species. However, no additional values have been derived. We derived a model of cooling cost from first principles and validated the model with empirical energetic measurements. We compared the assumed 67% proportional cooling cost with our model-predicted cooling cost for 53 hibernating mammals. Our results indicate that using 67% of warming cost only adequately represents cooling cost in ground squirrel-sized mammals. In smaller species, this value overestimates cooling cost and in larger species, the value underestimates cooling cost. Our model allows for the generalization of energetic costs for multiple species using species-specific physiological and morphometric parameters, and for predictions over variable environmental conditions.

Minimization of drug-drug interaction risk and candidate selection in a natural product-based class of gamma-secretase modulators.

Early lead compounds in this gamma secretase modulator series were found to potently inhibit CYP3A4 and other human CYP isoforms increasing their risk of causing drug-drug-interactions (DDIs). Using structure-activity relationships and CYP3A4 structural information, analogs were developed that minimized this DDI potential. Three of these new analogs were further characterized by rat PK, rat PK/PD and rat exploratory toxicity studies resulting in selection of SPI-1865 (14) as a preclinical development candidate.

A protocol and calibration method for accurate multi-camera field videography.

Stereo videography is a powerful technique for quantifying the kinematics and behavior of animals, but it can be challenging to use in an outdoor field setting. We here present a workflow and associated software for performing calibration of cameras placed in a field setting and estimating the accuracy of the resulting stereoscopic reconstructions. We demonstrate the workflow through example stereoscopic reconstructions of bat and bird flight. We provide software tools for planning experiments and processing the resulting calibrations that other researchers may use to calibrate their own cameras. Our field protocol can be deployed in a single afternoon, requiring only short video clips of light, portable calibration objects.

Interannual Survival of Myotis lucifugus (Chiroptera: Vespertilionidae) near the Epicenter of White-Nose Syndrome.

Reduced populations of Myotis lucifugus (Little Brown Myotis) devastated by white-nose syndrome (WNS) persist in eastern North America. Between 2009 and 2013, we recaptured 113 marked individuals that survived between 1 and 6 winters in New England since the arrival of WNS. We also observed signs of reproductive success in 57 recaptured bats.

Sebaceous lipid profiling of bat integumentary tissues: quantitative analysis of free Fatty acids, monoacylglycerides, squalene, and sterols.

White-nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans and is devastating North American bat populations. Sebaceous lipids secreted from host integumentary tissues are implicated in the initial attachment and recognition of host tissues by pathogenic fungi. We are interested in determining if ratios of lipid classes in sebum can be used as biomarkers to diagnose severity of fungal infection in bats. To first establish lipid compositions in bats, we isolated secreted and integral lipid fractions from the hair and wing tissues of three species: big brown bats (Eptesicus fuscus), Eastern red bats (Lasiurus borealis), and evening bats (Nycticeius humeralis). Sterols, FFAs, MAGs, and squalene were derivatized as trimethylsilyl esters, separated by gas chromatography, and identified by mass spectrometry. Ratios of sterol to squalene in different tissues were determined, and cholesterol as a disease biomarker was assessed. Free sterol was the dominant lipid class of bat integument. Squalene/sterol ratio is highest in wing sebum. Secreted wing lipid contained higher proportions of saturated FFAs and MAGs than integral wing or secreted hair lipid. These compounds are targets for investigating responses of P. destructans to specific host lipid compounds and as biomarkers to diagnose WNS.

Discovery of potent and selective matrix metalloprotease 12 inhibitors for the potential treatment of chronic obstructive pulmonary disease (COPD).

Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease associated with irreversible progressive airflow limitation. Matrix metalloproteinase-12 (MMP-12) has been characterized to be one of the major proteolytic enzymes to induce airway remodeling, destruction of elastin and the aberrant remodeling of damaged alveoli in COPD and asthma. The goal of this project is to develop and identify an orally potent and selective small molecule inhibitor of MMP-12 for treatment of COPD and asthma. Syntheses and structure-activity relationship (SAR) studies of a series of dibenzofuran (DBF) sulfonamides as MMP-12 inhibitors are described. Potent inhibitors of MMP-12 with excellent selectivity against other MMPs were identified. Compound 26 (MMP118), which exhibits excellent oral efficacy in the MMP-12 induced ear-swelling inflammation and lung inflammation mouse models, had been successfully advanced into Development Track status.

Evidence for competition between Ixodes scapularis and Dermacentor albipictus feeding concurrently on white-tailed deer.

Competition among ticks, and among ectoparasites generally, has rarely been demonstrated. Ixodes scapularis and Dermacentor albipictus are both hard ticks commonly found feeding on deer harvested at Letterkenny Army Depot, in south central Pennsylvania, USA. The two species have contrasting life histories resulting in D. albipictus spending notably more time on the shared host. We hypothesized that this would give D. albipictus an advantage in locating and occupying optimal attachment sites (highly vascularized areas like the head and ears). Ticks were collected from 224 hunter-killed deer in December 2005 and November 2006 to determine if there is evidence of competition for attachment sites when these two species concurrently infest deer. A timed sample (3 min per region) of representative ticks was collected from the head (ears, face and neck regions) and body (axillae regions). Ixodes scapularis was more abundant and prevalent overall than D. albipictus. Dermacentor albipictus was found almost exclusively on the head, whereas I. scapularis was more evenly distributed, but somewhat more abundant on the body than on the head. The proportion of I. scapularis on the head was reduced at high D. albipictus abundances, but I. scapularis abundance did not alter the distribution of D. albipictus. This study supports the hypothesis of competition for preferred attachment sites between these two species of ticks, and suggests that D. albipictus may be competitively dominant over I. scapularis on the head region of concurrently infested white-tailed deer.

Lean Mass Dynamics in Hibernating Bats and Implications for Energy and Water Budgets.

AbstractHibernation requires balancing energy and water demands over several months. Many studies have noted the importance of fat for hibernation energy budgets, but protein catabolism in hibernation has received less attention, and whole-animal changes in lean mass have not previously been considered. We used quantitative magnetic resonance body composition analysis to measure deposition of fat and lean mass of cave myotis (Myotis velifer) during the prehibernation period and decreases in fat and lean mass of Townsend's big-eared bats (Corynorhinus townsendii) during hibernation. For cave myotis, lean mass represented 25% and 38% (female and male, respectively) of prehibernation mass gain. In hibernating Townsend's big-eared bats, lean mass decrease was similar for females and males. We used values for Townsend's big-eared bats to explore the functional implications of lean mass change for water and energy budgets. Lean mass accounted for a substantial proportion of mass change during hibernation (female: 18%, male: 35%), and although not accounting for a large proportion of the energy budget (female: 3%, male: 7%), lean mass catabolism represented an important contribution to water production (female: 14%, male: 29%). Although most mammals cannot rely on protein catabolism for metabolic water production because of the water cost of excreting urea, we propose a variation of the protein-for-water strategy whereby hibernators could temporally compartmentalize the benefits of protein catabolism to periods of torpor and the water cost to periodic arousals when free drinking water is typically available. Combined, our analyses demonstrate the importance of considering changes in lean mass during hibernation.

Similar hibernation physiology in bats across broad geographic ranges.

Species with broad geographic ranges may experience varied environmental conditions throughout their range leading to local adaptation. Variation among populations reflects potential adaptability or plasticity, with implications for populations impacted by disease, climate change, and other anthropogenic influences. However, behavior may counteract divergent selection among populations. We studied intraspecific variation in hibernation physiology of Myotis lucifugus (little brown myotis) and Corynorhinus townsendii (Townsend's big-eared bat), two species of bats with large geographic ranges. We studied M. lucifugus at three hibernacula which spanned a latitudinal gradient of 1500 km, and C. townsendii from 6 hibernacula spread across 1200 km latitude and 1200 km longitude. We found no difference in torpid metabolic rate among populations of either species, nor was there a difference in the effect of ambient temperature among sites. Evaporative water loss was similar among populations of both species, with the exception of one C. townsendii pairwise site difference and one M. lucifugus site that differed from the others. We suggest the general lack of geographic variation is a consequence of behavioral microhabitat selection. As volant animals, bats can travel relatively long distances in search of preferred microclimates for hibernation. Despite dramatic macroclimate differences among populations, hibernating bats are able to find preferred microclimate conditions within their range, resulting in similar selection pressures among populations spread across wide geographic ranges.

Toward a total synthesis of the stemofoline alkaloids: Advancement of a 1,3-dipolar cycloaddition strategy.

Novel, intramolecular 1,3-dipolar cycloadditions of azomethine ylides have been applied to the synthesis of functionalized core structures of the stemofoline alkaloids. In an effort to maximize the efficiency of this key transformation in the context of an eventual total synthesis of these complex natural products, a number of strategic modifications to the cycloaddition substrate were investigated. These collective efforts have provided useful insights into the operative, regiochemical control elements for 1,3-dipolar cycloadditions leading to stemofoline alkaloids. A potential intermediate in the synthesis of these alkaloids was prepared.

Interspecific variation in evaporative water loss and temperature response, but not metabolic rate, among hibernating bats.

Hibernation is widespread among mammals in a variety of environmental contexts. However, few experimental studies consider interspecific comparisons, which may provide insight into general patterns of hibernation strategies. We studied 13 species of free-living bats, including populations spread over thousands of kilometers and diverse habitats. We measured torpid metabolic rate (TMR) and evaporative water loss (two key parameters for understanding hibernation energetics) across a range of temperatures. There was no difference in minimum TMR among species (i.e., all species achieved similarly low torpid metabolic rate) but the temperature associated with minimum TMR varied among species. The minimum defended temperature (temperature below which TMR increased) varied from 8 °C to < 2 °C among species. Conversely, evaporative water loss varied among species, with species clustered in two groups representing high and low evaporative water loss. Notably, species that have suffered population declines due to white-nose syndrome fall in the high evaporative water loss group and less affected species in the low evaporative water loss group. Documenting general patterns of physiological diversity, and associated ecological implications, contributes to broader understanding of biodiversity, and may help predict which species are at greater risk of environmental and anthropogenic stressors.

Body mass and hibernation microclimate may predict bat susceptibility to white-nose syndrome.

In multihost disease systems, differences in mortality between species may reflect variation in host physiology, morphology, and behavior. In systems where the pathogen can persist in the environment, microclimate conditions, and the adaptation of the host to these conditions, may also impact mortality. White-nose syndrome (WNS) is an emerging disease of hibernating bats caused by an environmentally persistent fungus, Pseudogymnoascus destructans. We assessed the effects of body mass, torpid metabolic rate, evaporative water loss, and hibernaculum temperature and water vapor deficit on predicted overwinter survival of bats infected by P. destructans. We used a hibernation energetics model in an individual-based model framework to predict the probability of survival of nine bat species at eight sampling sites across North America. The model predicts time until fat exhaustion as a function of species-specific host characteristics, hibernaculum microclimate, and fungal growth. We fit a linear model to determine relationships with each variable and predicted survival and semipartial correlation coefficients to determine the major drivers in variation in bat survival. We found host body mass and hibernaculum water vapor deficit explained over half of the variation in survival with WNS across species. As previous work on the interplay between host and pathogen physiology and the environment has focused on species with narrow microclimate preferences, our view on this relationship is limited. Our results highlight some key predictors of interspecific survival among western bat species and provide a framework to assess impacts of WNS as the fungus continues to spread into western North America.

What is winter? Modeling spatial variation in bat host traits and hibernation and their implications for overwintering energetics.

White-nose syndrome (WNS) has decimated hibernating bat populations across eastern and central North America for over a decade. Disease severity is driven by the interaction between bat characteristics, the cold-loving fungal agent, and the hibernation environment. While we further improve hibernation energetics models, we have yet to examine how spatial heterogeneity in host traits is linked to survival in this disease system. Here, we develop predictive spatial models of body mass for the little brown myotis (Myotis lucifugus) and reassess previous definitions of the duration of hibernation of this species. Using data from published literature, public databases, local experts, and our own fieldwork, we fit a series of generalized linear models with hypothesized abiotic drivers to create distribution-wide predictions of prehibernation body fat and hibernation duration. Our results provide improved estimations of hibernation duration and identify a scaling relationship between body mass and body fat; this relationship allows for the first continuous estimates of prehibernation body mass and fat across the species' distribution. We used these results to inform a hibernation energetic model to create spatially varying fat use estimates for M. lucifugus. These results predict WNS mortality of M. lucifugus populations in western North America may be comparable to the substantial die-off observed in eastern and central populations.

Mechanistic pathways in CF3COOH-mediated deacetalization reactions.

It has been widely accepted that both the protection of carbonyls and the deprotection of acetals and ketals involve the participation of a water molecule: formation of acetals and ketals is a dehydration process, whereas the deprotection is often referred to as hydrolysis, which, as implied by its name, always requires the presence of water. Herein, we report experimental evidence and mechanistic investigations that provide an alternative view to this process. We have demonstrated that water is not required to convert acetals and ketals to the corresponding carbonyls. The (1)H NMR experimental results revealed that the TFA-mediated transformation of acetal to aldehyde occurs via a hemiacetal TFA ester intermediate, which differentiates itself from the classic acid-catalyzed hydrolysis, where the hemiacetal is the putative intermediate responsible for the formation of the aldehyde. More interestingly, alcohols are not the final byproducts as they are in the classical hydrolysis, rather, the two alcohol molecules are converted to two TFA esters under the reaction conditions. On the basis of the NMR evidence, we have proposed that the two TFA esters are formed in two separate steps via a different mechanism along the reaction pathway. Formation of the TFA esters renders the reaction irreversible. To the best of our knowledge, the cascade reaction pathway presented by the TFA-mediated conversion of acetals and ketals to carbonyls has never been previously postulated.

Fragment-based Differential Targeting of PPI Stabilizer Interfaces.

Stabilization of protein-protein interactions (PPIs) holds great potential for therapeutic agents, as illustrated by the successful drugs rapamycin and lenalidomide. However, how such interface-binding molecules can be created in a rational, bottom-up manner is a largely unanswered question. We report here how a fragment-based approach can be used to identify chemical starting points for the development of small-molecule stabilizers that differentiate between two different PPI interfaces of the adapter protein 14-3-3. The fragments discriminately bind to the interface of 14-3-3 with the recognition motif of either the tumor suppressor protein p53 or the oncogenic transcription factor TAZ. This X-ray crystallography driven study shows that the rim of the interface of individual 14-3-3 complexes can be targeted in a differential manner with fragments that represent promising starting points for the development of specific 14-3-3 PPI stabilizers.

CoREST Complex-Selective Histone Deacetylase Inhibitors Show Prosynaptic Effects and an Improved Safety Profile To Enable Treatment of Synaptopathies.

Synaptic dysfunction is a pathological feature in many neurodegenerative disorders, including Alzheimer's disease, and synaptic loss correlates closely with cognitive decline. Histone deacetylases (HDACs) are involved in chromatin remodeling and gene expression and have been shown to regulate synaptogenesis and synaptic plasticity, thus providing an attractive drug discovery target for promoting synaptic growth and function. To date, HDAC inhibitor compounds with prosynaptic effects are plagued by known HDAC dose-limiting hematological toxicities, precluding their application to treating chronic neurologic conditions. We have identified a series of novel HDAC inhibitor compounds that selectively inhibit the HDAC-co-repressor of repressor element-1 silencing transcription factor (CoREST) complex while minimizing hematological side effects. HDAC1 and HDAC2 associate with multiple co-repressor complexes including CoREST, which regulates neuronal gene expression. We show that selectively targeting the CoREST co-repressor complex with the representative compound Rodin-A results in increased spine density and synaptic proteins, and improved long-term potentiation in a mouse model at doses that provide a substantial safety margin that would enable chronic treatment. The CoREST-selective HDAC inhibitor Rodin-A thus represents a promising therapeutic strategy in targeting synaptic pathology involved in neurologic disorders.

Incorporating evaporative water loss into bioenergetic models of hibernation to test for relative influence of host and pathogen traits on white-nose syndrome.

Hibernation consists of extended durations of torpor interrupted by periodic arousals. The 'dehydration hypothesis' proposes that hibernating mammals arouse to replenish water lost through evaporation during torpor. Arousals are energetically expensive, and increased arousal frequency can alter survival throughout hibernation. Yet we lack a means to assess the effect of evaporative water loss (EWL), determined by animal physiology and hibernation microclimate, on torpor bout duration and subsequent survival. White-nose syndrome (WNS), a devastating disease impacting hibernating bats, causes increased frequency of arousals during hibernation and EWL has been hypothesized to contribute to this increased arousal frequency. WNS is caused by a fungus, which grows well in humid hibernaculum environments and damages wing tissue important for water conservation. Here, we integrated the effect of EWL on torpor expression in a hibernation energetics model, including the effects of fungal infection, to determine the link between EWL and survival. We collected field data for Myotis lucifugus, a species that experiences high mortality from WNS, to gather parameters for the model. In saturating conditions, we predicted healthy bats experience minimal mortality. Infected bats, however, suffer high fungal growth in highly saturated environments, leading to exhaustion of fat stores before spring. Our results suggest that host adaptation to humid environments leads to increased arousal frequency from infection, which drives mortality across hibernaculum conditions. Our modified hibernation model provides a tool to assess the interplay between host physiology, hibernaculum microclimate, and diseases such as WNS on winter survival.

Stable isotope tracing of trout hatchery carbon to sediments and foodwebs of limestone spring creeks.

Limestone springs support productive ecosystems and fisheries, yet aquaculture may modify or impair these ecosystems. We determined trout hatchery organic contribution to spring creek sediments and foodwebs with natural abundance stable isotope methods. Hatchery feed, waste, and trout were significantly enriched in delta(13)C relative to autotrophs and wild fish. Spring creek sediments were enriched in delta(13)C toward the hatchery endmember relative to reference streams without hatcheries and relative to a larger larger-order, spring-influenced stream. Contribution of hatchery C to spring creek sediments was greatest during March and associated with greatest sediment %C. Contribution of hatchery C to pollution-tolerant isopod diet was 39-51% in a stream receiving limestone spring water via hatchery effluent. Isopods of one spring creek also relied on hatchery-derived C within one month of hatchery closure. Four years later, less pollution pollution-tolerant amphipods dominated and consumed non-vascular over vascular autotrophs (86%). Isopods of a second spring creek with an active hatchery did not appear to be using hatchery matter directly, but were enriched in delta(34)S relative to a spring creek tributary with no hatchery influence. Isopods in both of these streams were relatively enriched in delta(15)N, indicating general nutrient enrichment from surrounding agricultural land use. The contribution of hatchery vs. wild fish in diet of herons and egrets was traced with delta(13)C of guano. These birds were strongly dependent on stocked trout in a spring creek with a recently closed state trout hatchery, and also near another large, state-run hatchery. Heron dependence on hatchery fish in the spring creek decreased with time since hatchery closure. Use of stable isotope natural abundance techniques in karst spring creeks can reveal stream impairment due to aquaculture, specific C sources to bio-indicating consumers, losses of farmed fish to predation, and potential exposure of higher order consumers to contaminants associated with aquaculture.

Development of a Novel B-Cell Lymphoma 6 (BCL6) PROTAC To Provide Insight into Small Molecule Targeting of BCL6.

B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.