Is the future female for turtles? Climate change and wetland configuration predict sex ratios of a freshwater species.

Climate change and land-use change are leading drivers of biodiversity decline, affecting demographic parameters that are important for population persistence. For example, scientists have speculated for decades that climate change may skew adult sex ratios in taxa that express temperature-dependent sex determination (TSD), but limited evidence exists that this phenomenon is occurring in natural settings. For species that are vulnerable to anthropogenic land-use practices, differential mortality among sexes may also skew sex ratios. We sampled the spotted turtle (Clemmys guttata), a freshwater species with TSD, across a large portion of its geographic range (Florida to Maine), to assess the environmental factors influencing adult sex ratios. We present evidence that suggests recent climate change has potentially skewed the adult sex ratio of spotted turtles, with samples following a pattern of increased proportions of females concomitant with warming trends, but only within the warmer areas sampled. At intermediate temperatures, there was no relationship with climate, while in the cooler areas we found the opposite pattern, with samples becoming more male biased with increasing temperatures. These patterns might be explained in part by variation in relative adaptive capacity via phenotypic plasticity in nest site selection. Our findings also suggest that spotted turtles have a context-dependent and multi-scale relationship with land use. We observed a negative relationship between male proportion and the amount of crop cover (within 300 m) when wetlands were less spatially aggregated. However, when wetlands were aggregated, sex ratios remained consistent. This pattern may reflect sex-specific patterns in movement that render males more vulnerable to mortality from agricultural machinery and other threats. Our findings highlight the complexity of species' responses to both climate change and land use, and emphasize the role that landscape structure can play in shaping wildlife population demographics.

Evaluating Mechanisms of Short-term Woodland Salamander Response to Forest Management.

Contemporary forest management often requires meeting diverse ecological objectives including maintaining ecosystem function and promoting biodiversity through timber harvesting. Wildlife are essential in this process by providing ecological services that can facilitate forest resiliency in response to timber harvesting. However, the mechanisms driving species' responses remain ambiguous. The goal of this study was to assess mechanisms influencing eastern red-backed salamander (RBS; Plethodon cinereus) response to overstory cover removal. We evaluated two mitigation strategies for the RBS in response to overstory removal. We used a before-after-control-impact design to study how (1) retaining residual trees or (2) eliminating soil compaction affected RBS surface counts and body condition index (BCI) up to two-years post-treatment. Additionally, we assessed how surface counts of RBS were influenced by overstory tree cover. Surface counts of RBS were not strongly influenced by overstory removal when tree residuals were retained. Body condition index increased in treatments where harvest residuals were retained. In treatments where soil compaction was eliminated, surface counts and BCI were inversely related. Finally, surface counts from both mitigation strategies were not strongly influenced by overstory cover. Overall, both mitigation techniques appeared to ameliorate impacts of overstory removal on RBS. These results highlight the importance of understanding mechanisms driving species' responses to forest management. To reduce the perceived negative effects of overstory removal on RBS, incorporating these mitigation measures may contribute to the viability and stability of RBS populations. Incorporating species' life history traits into management strategies could increase continuity of ecological function and integrity through harvesting.

Evaluating the effect of expert elicitation techniques on population status assessment in the face of large uncertainty.

Population projection models are important tools for conservation and management. They are often used for population status assessments, for threat analyses, and to predict the consequences of conservation actions. Although conservation decisions should be informed by science, critical decisions are often made with very little information to support decision-making. Conversely, postponing decisions until better information is available may reduce the benefit of a conservation decision. When empirical data are limited or lacking, expert elicitation can be used to supplement existing data and inform model parameter estimates. The use of rigorous techniques for expert elicitation that account for uncertainty can improve the quality of the expert elicited values and therefore the accuracy of the projection models. One recurring challenge for summarizing expert elicited values is how to aggregate them. Here, we illustrate a process for population status assessment using a combination of expert elicitation and data from the ecological literature. We discuss the importance of considering various aggregation techniques, and illustrate this process using matrix population models for the wood turtle (Glyptemys insculpta) to assist U.S. Fish and Wildlife Service decision-makers with their Species Status Assessment. We compare estimates of population growth using data from the ecological literature and four alternative aggregation techniques for the expert-elicited values. The estimate of population growth rate based on estimates from the literature (λmean = 0.952, 95% CI: 0.87-1.01) could not be used to unequivocally reject the hypotheses of a rapidly declining population nor the hypothesis of a stable, or even slightly growing population, whereas our results for the expert-elicited estimates supported the hypothesis that the wood turtle population will decline over time. Our results showed that the aggregation techniques used had an impact on model estimates, suggesting that the choice of techniques should be carefully considered. We discuss the benefits and limitations associated with each method and their relevance to the population status assessment. We note a difference in the temporal scope or inference between the literature-based estimates that provided insights about historical changes, whereas the expert-based estimates were forward looking. Therefore, conducting an expert-elicitation in addition to using parameter estimates from the literature improved our understanding of our species of interest.

Nonlinear optical crosslinking (NLO CXL) for correcting refractive errors.

Ultraviolet A (UVA) light-based photoactivation of riboflavin (Rf) to induce corneal crosslinking (CXL) and mechanical stiffening is now a well-known treatment for corneal ectasia and Keratoconus that is being used in a topographically guided photorefractive intrastromal CXL (PiXL) procedure to treat low degrees of refractive errors. Alternative approaches for non-invasive treatment of refractive errors have also been proposed that use femtosecond lasers (FS) that provide much faster, more precise, and safer results than UVA CXL. One such treatment, nonlinear optical crosslinking (NLO CXL), has been able to replicate the effects of UVA CXL, while producing a smaller area of cellular damage and requiring a shorter procedure time. Unlike UVA CXL, the treatment volume of NLO CXL only occurs within the focal volume of the laser, which can be placed at any depth and scanned into any pattern for true topographically guided refractive correction. This review presents our experience with using FS lasers to photoactivate Rf and perform highly controlled corneal CXL that leads to mechanical stiffening and changes in corneal shape.

Collagen fiber crimping following in vivo UVA-induced corneal crosslinking.

The purpose of this study was to measure collagen fiber crimping (CFC) using nonlinear optical imaging of second harmonic generated (SHG) signals to determine the effects of UVA-riboflavin induced corneal collagen crosslinking (UVA CXL) on collagen structure. Two groups, four rabbits each, were treated in the right eye with standard UVA CXL. In vivo confocal microscopy was performed at 1, 2, and 4 weeks after treatment for the first group and up to three months for the second group to measure epithelial/stromal thickness and corneal haze during recovery. Rabbits were sacrificed at one and three months, respectively, and their corneas fixed under pressure. Regions of crosslinking were identified by the presence of collagen autofluorescence (CAF) and then collagen structure was imaged using SHG microscopy. The degree of CFC was determined by measuring the percentage difference between the length of the collagen fiber and the linear distance traveled. CFC was measured in the central anterior and posterior CXL region, the peripheral non-crosslinked region in the same cornea, and the central cornea of the non-crosslinked contralateral eye. No change in corneal thickness was detected after one month, however the stromal thickness surpassed its original baseline thickness at three months by 25.9 µm. Corneal haze peaked at one month and then began to clear. Increased CAF was detected in all CXL corneas, localized to the anterior stroma and extending to 42.4 ±â€¯3.4% and 47.7 ±â€¯7.6% of the corneal thickness at one and three months. There was a significant (P < 0.05) reduction in CFC in the CAF region in all eyes averaging 1.007 ±â€¯0.006 and 1.009 ±â€¯0.005 in one and three month samples compared to 1.017 ±â€¯0.04 and 1.016 ±â€¯0.06 for controls. These results indicate that there is a significant reduction in collagen crimping following UVA CXL of approximately 1%. One possible explanation for this loss of crimping could be shortening of the collagen fibers over the CXL region.

Axial mechanical and structural characterization of keratoconus corneas.

PURPOSE: Previous studies indicate that there is an axial gradient of collagen lamellar branching and anastomosing leading to regional differences in corneal tissue stiffness that may control corneal shape. To further test this hypothesis we have measured the axial material stiffness and quantified the collagen lamellar complexity in ectatic and mechanically weakened keratoconus corneas (KC). METHODS: Acoustic radiation force elastic microscopy (ARFEM) was used to probe the axial mechanical properties of the cone region of three donor KC buttons. 3 Dimensional second harmonic generation microscopy (3D-SHG) was used to qualitatively evaluate lamellar organization in 3 kC buttons and quantitatively measure lamellar branching point density (BPD) in a separate KC button that had been treated with epikeratophakia (Epi-KP). RESULTS: The mean elastic modulus for the KC corneas was 1.67 ±â€¯0.44 kPa anteriorly and 0.970 ±â€¯0.30 kPa posteriorly, substantially below that previously measured for normal human cornea. 3D-SHG of KC buttons showed a simplified collagen lamellar structure lacking noticeable angled lamellae in the region of the cone. BPD in the anterior, posterior, central and paracentral regions of the KC cornea were significantly lower than in the overlying Epi-KP lenticule. Additionally, BPD in the cone region was significantly lower than the adjacent paracentral region in the KC button. CONCLUSIONS: The KC cornea exhibits an axial gradient of mechanical stiffness and a BPD that appears substantially lower in the cone region compared to normal cornea. The findings reinforce the hypothesis that collagen architecture may control corneal mechanical stiffness and hence corneal shape.

Meibocyte differentiation and renewal: Insights into novel mechanisms of meibomian gland dysfunction (MGD).

This paper reviews our current understanding of age-related meibomian gland dysfunction (MGD) and the role of the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ), in the regulation of meibomian gland function, meibocyte differentiation and lipid synthesis. The studies suggest that PPARγ is a master regulator of meibocyte differentiation and function, whose expression and nuclear signaling coupled with meibocyte renewal is altered during aging, potentially leading to atrophy of the meibomian gland as seen in clinical MGD. Study of meibomian gland stem cells also suggest that there is a limited number of precursor meibocytes that provide progeny to the acini, that may be susceptible to exhaustion as occurs during aging and other environmental factors. Further study of pathways regulating PPARγ expression and function as well as meibocyte stem cell maintenance may provide clues to establishing cellular and molecular mechanisms underlying MGD and the development of novel therapeutic strategies to treating this disease.

Transcriptome analysis of aging mouse meibomian glands.

PURPOSE: Dry eye disease is a common condition associated with age-related meibomian gland dysfunction (ARMGD). We have previously shown that ARMGD occurs in old mice, similar to that observed in human patients with MGD. To begin to understand the mechanism underlying ARMGD, we generated transcriptome profiles of eyelids excised from young and old mice of both sexes. METHODS: Male and female C57BL/6 mice were euthanized at ages of 3 months or 2 years and their lower eyelids removed, the conjunctival epithelium scrapped off, and the tarsal plate, containing the meibomian glands, dissected from the overlying muscle and lid epidermis. RNA was isolated, enriched, and transcribed into cDNA and processed to generate four non-stranded libraries with distinct bar codes on each adaptor. The libraries were then sequenced and mapped to the mm10 reference genome, and expression results were gathered as reads per length of transcript in kilobases per million mapped reads (RPKM) values. Differential gene expression analyses were performed using CyberT. RESULTS: Approximately 55 million reads were generated from each library. Expression data indicated that about 15,000 genes were expressed in these tissues. Of the genes that showed more than twofold significant differences in either young or old tissue, 698 were identified as differentially expressed. According to the Gene Ontology (GO) analysis, the cellular, developmental, and metabolic processes were found to be highly represented with Wnt function noted to be altered in the aging mouse. CONCLUSIONS: The RNA sequencing data identified several signaling pathways, including fibroblast growth factor (FGF) and Wnt that were altered in the meibomian glands of aging mice.

From nano to macro: studying the hierarchical structure of the corneal extracellular matrix.

In this review, we discuss current methods for studying ocular extracellular matrix (ECM) assembly from the 'nano' to the 'macro' levels of hierarchical organization. Since collagen is the major structural protein in the eye, providing mechanical strength and controlling ocular shape, the methods presented focus on understanding the molecular assembly of collagen at the nanometre level using X-ray scattering through to the millimetre to centimetre level using non-linear optical (NLO) imaging of second harmonic generated (SHG) signals. Three-dimensional analysis of ECM structure is also discussed, including electron tomography, serial block face scanning electron microscopy (SBF-SEM) and digital image reconstruction. Techniques to detect non-collagenous structural components of the ECM are also presented, and these include immunoelectron microscopy and staining with cationic dyes. Together, these various approaches are providing new insights into the structural blueprint of the ocular ECM, and in particular that of the cornea, which impacts upon our current understanding of the control of corneal shape, pathogenic mechanisms underlying ectatic disorders of the cornea and the potential for corneal tissue engineering.

Meibomian gland dysfunction: hyperkeratinization or atrophy?

Meibomian gland dysfunction (MGD) is the major cause of evaporative dry eye disease (EDED) and dysfunction is widely thought to mechanistically involve ductal hyperkeratinization, plugging and obstruction. This review re-evaluates the role of hyperkeratinization in MGD based on more recent findings from mouse models. In these studies, eyelids from normal young and old mice or mice exposed to desiccating stress were evaluated by immunofluorescent tomography and 3-dimensional reconstruction to evaluate gland volume, expression of hyperkeratinization markers and cell proliferation or stimulated Raman scattering (SRS) microscopy to assess lipid quality. Results indicate that aging mice show dropout of meibomian glands with loss of gland volume and a forward migration of the mucocutaneous junction anterior to the gland orifice; similar age-related changes that are detected in human subjects. Atrophic glands also showed evidence of epithelial plugging of the orifice without the presence of hyperkeratinization. Mice exposed to desiccating stress showed hyperproliferation of the meibomian gland and ductal dilation suggesting a marked increase in lipid synthesis. Lipid quality was also affected in EDED mice with an increase in the protein content of lipid within the duct of the gland. Overall, age-related changes in the mouse show similar structural and functional correlates with that observed in clinical MGD without evidence of hyperkeratinization suggesting that gland atrophy may be a major cause of EDED. The response of the meibomian gland to desiccating stress also suggest that environmental conditions may accelerate or potentiate age-related changes.

A microfabricated, optically accessible device to study the effects of mechanical cues on collagen fiber organization.

As the primary structural protein of our bodies, fibrillar collagen and its organizational patterns determine the biomechanics and shape of tissues. While the molecular assembly of individual fibrils is well understood, the mechanisms determining the arrangement of fibers and thus the shape and form of tissues remain largely unknown. We have developed a cell culture model that successfully recapitulates early tissue development and the de novo deposition of collagen fibers to investigate the role of mechanical cues on collagen fiber alignment. The devices used a thin, collagen-coated deformable PDMS membrane inside a tissue culture well built on microscope-grade coverslips. Deformations and strains in the PDMS membrane were quantified by tracking fluorescent bead displacement and through the use of a COMSOL model. Cyclical strains were applied to serum-cultured rabbit corneal cells at 0.5 Hz for 24-48 h and showed a preferred alignment after 36 h of cyclical loading. Cells cultured with ascorbic acid under methylcellulose serum-free conditions deposited a collagenous matrix that was visible under live second harmonic generation microscopy at week 4. Our microfabricated tissue culture system allows for the controllable application of a wide range of stress profiles to cells, and for the observation and quantification of cells and de novo collagen formation in vitro. Future studies will involve the fabrication of models to study the formation and organization of collagen in ocular diseases.

Modeling meibum secretion: Alternatives for obstructive Meibomian Gland Dysfunction (MGD).

PURPOSE: While changes in meibum quality are correlated with severity of meibomian gland dysfunction (MGD) and dry eye disease, little is known regarding the mechanics of meibum secretion. The purpose of this study was to develop a finite element model of meibum secretion and evaluate the effect of various factors that might impact meibum delivery to the ocular surface. METHODS: A finite element analysis in COMSOL 6.0 was used to simulate the flow of meibum within the gland's terminal excretory duct. Historical normal human meibum rheology data taken over the meibum melting range from fluid (35-40 °C) to solid (25-30 °C) were then used to calculate the minimum yield stress and plastic viscosity of meibum. The effects of meibum melting state, eyelid pressure and terminal duct diameter on meibum flow rates were then systematically investigated. RESULTS: The melting state of meibum from liquid to solid was associated with an increase in the minimum yield stress and plastic viscosity that caused an exponential decrease in meibum flow. Modeling also established that there was a linear correlation between meibum flow rate and eyelid pressure needed to express meibum and the 4th power of the terminal duct radius. CONCLUSIONS: Our results suggest that changes in the melting state of meibum from fluid to solid, as well as changes in the radius of the terminal excretory duct and the force exerted by the eyelid can lead to dramatic decreases in the flow of meibum. Together these findings suggest alternative mechanisms for meibomian gland obstruction.

Influence of temporary emigration on wood turtle (Glyptemys insculpta) detectability, with implications for abundance estimation.

Reliable population estimates are important for making informed management decisions about wildlife species. Standardized survey protocols have been developed for monitoring population trends of the wood turtle (Glyptemys insculpta), a semi-aquatic freshwater turtle species of conservation concern throughout its distribution in east-central North America. The protocols use repeated active search surveys of defined areas, allowing for estimation of survey-specific detection probability (p) and site-specific abundance. These protocols assume population closure within the survey area during the survey period, which is unlikely to be met as wood turtles are a highly mobile species. Additionally, current protocols use a single-pass design that does not allow for separation of availability (pa) and detectability (pd). If there are systematic influences on pa or pd that are not accounted for in the survey design or data analysis, then resulting abundance estimates could be biased. The objectives of this study were to determine if pa is a random process and if pa and pd are influenced by demographic characteristics. We modified the wood turtle survey protocol used in the upper Midwest to include a double-pass design, allowing us to estimate pa and pd using a robust design capture-recapture model. The modified protocol was implemented at 14 wood turtle monitoring sites in Minnesota and Wisconsin between 2017 and 2022. Our results indicated that pa was non-random and that pd increased with turtle carapace length. Our study suggests that model assumptions for current wood turtle population models may be violated, likely resulting in an overestimation of abundance. We discuss possible protocol and modeling modifications that could result in more accurate wood turtle abundance estimates.

Pharmacokinetics and bioavailability of plant lignan 7-hydroxymatairesinol and effects on serum enterolactone and clinical symptoms in postmenopausal women: a single-blinded, parallel, dose-comparison study.

OBJECTIVE: 7-Hydroxymaitairesinol (7-HMR) is a naturally occurring plant lignan found in whole grains and the Norway spruce (Piciea abies). The purpose of this study was to evaluate the bioavailability of a proprietary 7-HMR product (HMRlignan, Linnea SA, Locarno, Switzerland) through measurement of lignan metabolites and metabolic precursors. METHODS: A single-blind, parallel, pharmacokinetic and dose-comparison study was conducted on 22 postmenopausal females not receiving hormone replacement therapy. Subjects were enrolled in either a 36 mg/d (low-dose) or 72 mg/d dose (high-dose) regimen for 8 weeks. Primary measured outcomes included plasma levels of 7-HMR and enterolactone (ENL), and single-dose pharmacokinetic analysis was performed on a subset of subjects in the low-dose group. Safety data and adverse event reports were collected as well as data on hot flash frequency and severity. RESULTS: Pharmacokinetic studies demonstrated 7-HMR C max = 757.08 ng/ml at 1 hour and ENL C max = 4.8 ng/ml at 24 hours. From baseline to week 8, plasma 7-HMR levels increased by 191% in the low-dose group (p < 0.01) and by 1238% in the high-dose group (p < 0.05). Plasma ENL levels consistently increased as much as 157% from baseline in the low-dose group and 137% in the high-dose group. Additionally, the mean number of weekly hot flashes decreased by 50%, from 28.0/week to 14.3/week (p < 0.05) in the high-dose group. No significant safety issues were identified in this study. CONCLUSION: The results demonstrate that HMRlignan is quickly absorbed into the plasma and is metabolized to ENL in healthy postmenopausal women. Clinically, the data demonstrate a statistically significant improvement in hot flash frequency. Doses up to 72 mg/d HMRlignan for 8 weeks were safe and well tolerated in this population.

PREVALENCE OF RANAVIRUS IN SPOTTED SALAMANDER (AMBYSTOMA MACULATUM) LARVAE FROM CREATED VERNAL POOLS IN WEST VIRGINIA, USA.

Ranavirosis is a disease of high concern for amphibians due to widespread documentation of its lethal and sublethal impacts and its high transmission potential across populations and species. We investigated whether spotted salamander (Ambystoma maculatum) ranavirus prevalence and viral load were associated with habitat characteristics, genetic diversity, corticosterone levels, and body size. In 2015 and 2016, we sampled 34 recently created vernal pools in the Monongahela National Forest, West Virginia, USA. We collected tail clippings from 1,128 spotted salamander larvae and waterborne hormone samples from 436 of those larvae, along with eight environmental characteristics of the pools. Over the 2-yr period, we detected ranavirus in 62% of pools, with prevalence ranging from 0% to 63% (mean, 7.68%). Spotted salamander size was positively correlated with ranavirus presence and viral load; however, we did not find associations between ranavirus prevalence or viral load and habitat characteristics, spotted salamander genetic diversity, relatedness, effective number of breeders, or corticosterone levels. The widespread occurrence of ranavirus in the vernal pools illustrates the potential for rapid natural introduction of the pathogen to created wetlands. Managers could consider monitoring local distributions of ranavirus before creation of new vernal pools to guide strategic placement of the wetlands to minimize occurrence and prevalence of this pathogen.

Quiescent keratocytes fail to repair MMC induced DNA damage leading to the long-term inhibition of myofibroblast differentiation and wound healing.

PURPOSE: The purpose of this study was to determine the acute and long-term effects of mitomycin C (MMC) on quiescent rabbit corneal keratocytes regarding cell proliferation, myofibroblast differentiation and DNA repair. METHODS: Quiescent keratocytes cultured in serum-free media were exposed to various concentrations of MMC and then treated with transforming growth factor-ß (TGFß). DNA damage was evaluated in both cultured keratocytes and live rabbit eyes following treatment with MMC. The long-term ability of quiescent keratocytes to repair MMC induced damage in vivo was evaluated in rabbits treated with MMC 2 months before 100 µm deep lamellar keratectomy (LK) injury. RESULTS: MMC significantly blocked TGFß-induced cell proliferation and myofibroblast differentiation in cultured quiescent keratocytes and altered the transcriptional regulation of macrophage chemotactic protein-1 (MCP-1) and alpha smooth muscle actin (αSMA). MMC also induced phosphorylation of the nuclear histone marker of DNA damage, γH2AX (a member of the H2A histone family), without induction of cell cycle entry or immediate DNA repair measured by Comet assay. In live rabbits, 0.2 mg/ml MMC significantly induced γH2AX nuclear immunostaining (p<0.05) throughout the cornea and corneas receiving 0.2 mg/ml MMC treatment 2 months before LK injury showed complete absence of any corneal scarring. CONCLUSIONS: MMC induces DNA damage to quiescent corneal keratocytes, which remains unrepaired, resulting in abnormal cell replication and gene transcription that leads to long-term effects on corneal repair. Overall these findings suggest that there may be long-term and perhaps permanent consequences to the application of MMC as an anti-fibrotic therapy.

Amyloid Precursor Protein in Abusive Head Trauma Suspects.

PURPOSE: To study orthograde axonal transport with amyloid precursor protein (APP-A4) immunohistochemistry (IHC) in the retina and lamina cribrosa (LC) portion of the optic nerve in abusive head trauma (AHT) suspects. DESIGN: Retrospective, case-control study. METHODS: Seventy-two eyes from suspected AHT victims referred by the Los Angeles Coroner and control eyes from nontraumatized infants were included. IHC was conducted using University of California, Irvine (UCI), Lab Medicine Department's standard protocol and results analyzed by light microcopy after paraffin processing. Quantitation of LC APP-A4 block was estimated in 21 cases with known survival using MetaMorph, a proprietary biomicroscopy imaging software. RESULTS: The presence or absence of APP-A4 label accumulations in retinal ganglion cells, nerve fiber layer at the disc margin, and in LC axonal bundles were compared to matching tissues from nontraumatized control eyes with only background staining. Among the globes from AHT suspects with nerve heads available for study, 94% were positive for LC accumulation of marker. Among suspect AHT cases with known survival after injury of 1 to 1588 days, most demonstrated LC APP-A4 accumulations. CONCLUSIONS: Our findings reinforce a recent publication based on APP-A4 IHC that demonstrated similar orthograde axonal transport block in the LC in children with AHT and recommend that intraocular pressures be recorded and addressed in these patients.

Immuno Tomography (IT) and Imaging Mass Cytometry (IMC) for constructing spatially resolved, multiplexed 3D IMC data sets.

PURPOSE: We have previously used Immuno Tomography (IT) to identify label-retaining stem cell populations in the cornea and meibomian gland. While this method provides the unique ability to quantify stem cell populations comprised of 1-4 cells, the number of antigens that can be sequentially used to characterize these unique cells is limited by antigen stability after antibody stripping and re-probing. To address this deficiency, we have evaluated the capability of Imaging Mass Cytometry™ (IMC™) to generate multiplexed images using metal-conjugated antibodies to label IT plastic sections and generate 3-dimensional IMC data sets (3D IMC). METHODS: K5-H2B-GFP mice, 56 days after doxycycline chase, were sacrificed and eyelid tissue processed for IT. A total of 400 serial, plastic sections, 2 µm thick, were then probed using metal-tagged antibodies specific for sox 9, collagen type I, E-cadherin, Ki67, GFP, αSMA, vimentin, and DNA intercalator. Multiplexed images were then generated using an Imaging Mass Cytometry system (Fluidigm®), and 3D reconstructions were assembled. RESULTS: All 8 metal-labeled tags were detected and their images were successfully assembled into 3D IMC data sets. GFP-labeled nuclei were identified within the meibomian glands in comparable numbers to those previously reported for slow-cycling meibomian gland stem cells. CONCLUSIONS: These findings demonstrate that IMC can be used on plastic sections to generate multiplexed, 3D data sets that can be reconstructed to show the spatial localization of meibomian gland stem cells. We propose that 3D IMC might prove valuable in more fully characterizing stem cell populations in different tissues.

Expression of Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2) by human and rabbit meibomian glands and meibocytes.

PURPOSE: Previously, we showed that Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), an essential enzyme required for meibum wax ester synthesis, was not expressed by immortalized human meibomian gland epithelial cells (hMGEC) in culture. To begin to understand the mechanisms controlling AWAT2 expression, we have analyzed its expression in human and rabbit meibomian glands and cultured meibocytes. METHODS: Rabbit meibocyte progenitor cells (rMPC) were first grown in Cnt-BM.1 basal medium (Cellntec) supplemented with rhEGF, FGF10, and ROCK inhibitor (Y-27632 dihydrochloride), and then passed at 70-80% confluency with Accutase. Differentiation of rMPC to meibocytes (rMC) was induced by removal of Y-27632 and addition of 1 mM calcium with and without PPARγ agonists. RNA from the tissue, primary, passaged rMPC and differentiated rMC were obtained for AWAT2 qPCR analysis. Proteins and cells were evaluated for western blotting and neutral lipid synthesis, respectively. For comparison, human meibomian glands were separated for RNA and protein analysis. hMGEC was cultured to collect RNA and protein. RESULTS: Rabbit rMPCs were successfully grown, passaged, and differentiated, showing a significant increase in lipid droplet accumulation. AWAT2 RNA was highly expressed in tissue but showed a -16.9 log2 fold decrease in primary and passaged rMPCs and was not induced by differentiation to rMC. By comparison, human meibomian glands showed high expression of AWAT2, and hMGEC expressed non-detectable levels of AWAT2 transcripts or protein. CONCLUSIONS: AWAT2 expression is lost in cultured rMPC and rMC suggesting that cells in culture do not undergo complete meibocyte differentiation and require yet to be identified culture conditions.

Long-term changes in occurrence, relative abundance, and reproductive fitness of bat species in relation to arrival of White-nose Syndrome in West Virginia, USA.

White-nose syndrome (WNS) is a disease caused by the fungus Pseudogymnoascus destructans which has resulted in the deaths of millions of bats across eastern North America. To date, hibernacula counts have been the predominant means of tracking the spread and impact of this disease on bat populations. However, an understanding of the impacts of WNS on demographic parameters outside the winter season is critical to conservation and recovery of bat populations impacted by this disease. We used long-term monitoring data to examine WNS-related impacts to summer populations in West Virginia, where WNS has been documented since 2009. Using capture data from 290 mist-net sites surveyed from 2003 to 2019 on the Monongahela National Forest, we estimated temporal patterns in presence and relative abundance for each bat species. For species that exhibited a population-level response to WNS, we investigated post-WNS changes in adult female reproductive state and body mass. Myotis lucifugus (little brown bat), M. septentrionalis (northern long-eared bat), and Perimyotis subflavus (tri-colored bat) all showed significant decreases in presence and relative abundance during and following the introduction of WNS, while Eptesicus fuscus (big brown bat) and Lasiurus borealis (eastern red bat) responded positively during the WNS invasion. Probability of being reproductively active was not significantly different for any species, though a shift to earlier reproduction was estimated for E. fuscus and M. septentrionalis. For some species, body mass appeared to be influenced by the WNS invasion, but the response differed by species and reproductive state. Results suggest that continued long-term monitoring studies, additional research into impacts of this disease on the fitness of WNS survivors, and a focus on providing optimal nonwintering habitat may be valuable strategies for assessing and promoting recovery of WNS-affected bat populations.