To maintain normal functionality, it is necessary for a multicellular organism to generate robust responses to external temporal signals. However, the underlying mechanisms to coordinate the collective dynamics of cells remain poorly understood. Here, we study the calcium activity of biological neuron networks excited by periodic ATP stimuli. We use micropatterning to control the cells' physical connectivity. We find that whereas isolated cells become more synchronized in their calcium activity at long driving periods, connected cells become less synchronized, despite expressing more gap junctions which enable calcium exchange. To understand this result, we use a mathematical model in which a bifurcation analysis has previously shown coupling-induced desynchronization in an oscillatory network. Using parameters close to this bifurcation but in the excitable regime, we find that this desynchronization persists and can explain the experimental observations. The model further predicts that co-culturing with gap-junction-deficient cells should restore synchronization, which experiments confirm. Combining quantitative experiments, the physical and biological manipulation of cells, and mathematical modeling, our results suggest that cell-to-cell connectivity significantly affects how populations encode an external temporal signal as it slows down: Sparse networks synchronize due to longer entrainment, whereas highly connected networks can desynchronize due to dynamic frustration.

Abstract Disclosure: R. Toyoizumi: None. C.Y. Markgraf: None. A. Starman: None. E. Rodriguez: None. B.L. Lee: None. R. Bahn-Bales: None. O. Stricklin: None. C. Estill: None. C.E. Roselli: None. P.E. Chappell: None. The multiphenotypic hypothalamic KNDy neurons play a critical role in the neuroendocrine regulation of reproduction, and via their stimulation of GnRH release, are requisite for fertility and pubertal progression. In vivo evidence has accumulated identifying this more caudal population of Kisspeptin neurons in mediating predominantly negative feedback effects of gonadal steroids, and their ability to synthesize and secrete kisspeptin (KP), neurokinin B (NKB/Tac3), and dynorphin (Dyn) implicates them as crucial regulators of GnRH pulse generation. We have previously generated murine immortalized arcuate KNDy neuronal cell lines to explore molecular mechanisms underlying the steroid hormone regulation of these cells, and in the current study, we characterize a novel immortalized ovine neuronal cell line model of KNDy (oKNDy) cells derived from fetal female sheep brain (∼133 days of fetal development). Clonal lines were derived from isolated mediobasal hypothalamic explants using lentiviral infection of neurons with plasmids encoding SV40 large T-antigen. These neurons express ovine kiss1, tac3, and pdyn, as well as steroid receptors esr1, esr2, and pgr. Similar to KNDy neurons in vivo, receptors for Tac3 (tacr3) and Dyn (oprk1) were also found to be expressed in our oKNDy cells. Further, low physiological concentrations (5-50pM) of 17β-estradiol (E2) were found to significantly repress kiss1 and tac3 expression, while having no effect on tacr3. Concentrations of E2 and progesterone (P4) approximating proestrous levels maximally repressed kiss1 and tac3 after 24h, with elevations in pdyn noted under the same conditions. While cells exposed to higher E2 levels exhibited greater kiss1/tac3 repression, P4 exposure alone revealed stimulation of expression with increasing P4. Since these neurons were derived from the short-day seasonally breeding sheep, they can also be used to explore how circannual timing cues derived from the pituitary pars tuberalis may exert gating signals on the reproductive axis as previously modeled in vivo. Consistent with this, we have found high levels of thyroid hormone receptor α (thra) expression in our oKNDy neurons, suggesting they can respond to locally-synthesized T3. We are continuing to characterize these oKNDy neuronal cells in culture in order to provide insight into GnRH pulse generation and construct molecular models of neuronal responsiveness to steroids and other regulatory inputs. Presentation: Friday, June 16, 2023

Abstract Disclosure: R.N. Tanori: None. J.E. Barboza Sagrero: None. K. Noll-Bader: None. S. Chandu: None. P.E. Chappell: None. C.V. Bishop: Consulting Fee; Self; Oviva, Oxford Pharmaceuticals Inc. There are several rodent models for polycystic ovarian syndrome (PCOS), but unlike primates, most rodents will not form a long-lived corpus luteum (CL) unless they experience cervico-vaginal stimulation/mating. Our research group has begun to determine if guinea pigs (Cavia porcellus), rodents with long-lived CL, are a suitable model for mild hyperandrogenemia, a risk factor for PCOS. The objective of this analysis was to determine the relative abundance of each follicle type within a cross-section of 20-week old guinea pig ovarian tissue from control and T-treated females after 3 months of androgen exposure. Ovarian tissues were collected from six guinea pigs treated with cholesterol implants (vehicle, n=4) or testosterone implants (n=3), which elevated androgen levels ∼2-fold above control females. Tissues were embedded in paraffin wax and cut into serial sections (5 μm) and placed onto glass slides (1 section/slide) for hematoxylin and eosin staining to identify follicle morphology within the ovarian tissue. Every fifth section was stained and analyzed using an ECHO Revolve microscope and follicles counted using ImageJ. Follicles of interest were primordial, primary, multi-layer, antral, and corpus luteum, and counts were normalized to ovarian area. All follicle types were found in all the ovaries imaged, as well as some corpora lutea. Multilayer follicles were significantly reduced by chronic exposure to mild hyperandrogenemia (p=0.01). These should provide needed baseline analyses for future experiments employing guinea pigs as a model of PCOS. In addition to histological analysis, we have begun quantifying gene expression changes for hormones and receptors throughout the reproductive axis. Preliminary studies using qPCR illustrate significantly elevated expression levels of hypothalamic kiss1, pituitary gnrhr, and ovarian fshr in T-treated females. No discernable differences were observed between treatment groups for kiss1r, gnrh, or fshb. Ongoing studies will explore differences in estrogen and androgen receptor expression throughout the reproductive axis, as well as any changes in metabolic regulators in liver and skeletal muscle that are associated with mild hyperandrogenemia in these animals. Presentation Date: Saturday, June 17, 2023

Abstract In female animals, ovarian estradiol (E2) acts as negative feedback inhibition of GnRH secretion and positive feedback stimulation at ovulation. Both E2-regulated mechanisms work via stimulation or repression of distinct neuronal populations of Kisspeptin (KP)-synthesizing neurons. While E2 clearly stimulates AVPV KP neurons to increase kiss1 expression during the preovulatory surge, mechanisms required for optimal potentiation of maximal GnRH surge release remain incompletely characterized. We used two immortalized KP cell lines demonstrating increased kiss1 expression under high E2 (KTaV-3), or kiss1 suppression under low E2 (KTaR-1), to probe GnRH receptor (GnRHR) expression levels under E2 conditions of varying doses and times of exposure. Additionally, we have begun to investigate transcriptional mechanisms underlying gnrhr expression in KP neurons in vitro. AVPV-derived KTaV-3 cells were treated with either E2 alone (5-100pM) or E2 with progesterone (4-40nM) for varying durations (4-96h), either constitutively or under modulating levels approximating changes found during the murine estrous cycle. All treatments were undertaken in serum-free (SF) media (DMEM) following a 50% serum synchronization. After total RNA isolation, cDNAs were probed with mouse gnrhr primers. Results reveal that gnrhr expression, not observed in typical growth media conditions, is induced in KTaV-3 cells to varying degrees dependent on prior E2/P4 exposure. Following shorter treatment times (12-24h), proestrus levels (100pM E2; 4nM P4) of sex steroids noticeably induced gnrhr, while exposure of KTaV-3 cells to a 96h estrous cycle revealed maximal gnrhr expression at the diestrus II→proestrus transition, with exposure cycles concluding with declining E2/rising P4 exhibiting no to minimal gnrhr expression. These results suggest that maximal KP neuronal expression of gnrhr may occur prior to the E2 peak on proestrus, potentially preparing AVPV KP neurons for GnRH surge release 12-16h later. Similar steroid conditions also increased dlx3 expression, a transcription factor required for GnRHR in pituitary, only in KTaV-3 cells. In KTaR-1 cells, gnrhr expression was observed only following E2 decreases, while dlx3 was constitutively elevated. Since cFos/JunB complex formation on the gnrhr promoter has been implicated in gonadotrope GnRHR levels, we explored how transfection of these transcription factors might affect gnrhr expression in KTaV-3 cells. Preliminary results reveal that fos/jun co-transfection stimulates gnrhr expression maximally at intermediate (DII) E2 levels, while higher E2 levels are required with fos or jun expression alone. While reciprocal GnRH-Kisspeptin connections have not yet been observed in vivo, these results suggest KP neurons may possess the capacity to respond to intrahypothalamic GnRH only in certain estrous cycle stages, by differentially modulating GnRH receptivity in the AVPV. These findings supplement the prevailing model of feed-forward KP regulation of GnRH with potential short-loop KP-GnRH positive feedback regulation, providing an increased understanding of feedback mechanisms required for normal neuroendocrine regulation of reproduction. Presentation: Sunday, June 12, 2022 11:30 a.m. - 11:45 a.m.

Abstract Previous work in our laboratory explored the proteomic contents of exosome-like extracellular vesicles (EVs) released into the media in vitro from immortalized Kisspeptin (KP) neuronal cell lines KTaV-3 (derived from female mouse AVPV) and KTaR-1 (derived from KNDy neurons in the ARC). EVs were isolated from conditioned media via ultracentrifugation or filtration kit and validated using a NanoCyte, and LCMS-MS analysis revealed that relative abundance of exosomal cargo varied dependent upon estrogen (E2) exposure in vitro, with ∼150-170 proteins up-regulated and ∼200-220 proteins downregulated by E2 in EVs of KTaR-1 and KTaV-3 KP neurons. Since E2-regulated KP exosomal proteins included candidates implicated in the regulation of synaptic plasticity and signaling (i.e. annexins, semaphorins, connexins), we investigated the effects of exposure to purified EVs on gene expression in immortalized GnRH neurons (GT1-7 cells). Notably, EVs from 24h E2-treated KTaV-3 neurons induced increased expression of kiss1r in GT1-7 cells, in contrast to KTaV-3 neurons not exposed to E2, suggesting that AVPV KP neurons may signal an increase in KP receptivity in GnRH neurons in vivo via non-neuronal communication. Additionally, increases in expression of the synaptic scaffolding protein PSD-95 (dlg4) were seen in GT1-7 cells treated with E2-treated KTaV-3 EVs, suggesting AVPV KP neurons may use extracellular vesicles to modulate GnRH neuronal synaptic plasticity over the estrous cycle. While EVs isolated from KTaR-1 conditioned media did not alter GT1-7 kiss1r or dlg4 levels, treatment (24h) resulted in induction of selective gap junction hemichannel expression in GT1-7 cells. KTaR-1 (but not KTaV-3) EVs increased expression of Cx26 (gjb2) irrespective of E2 exposure, while induction of Cx43 (gja1) expression in GT1-7 cells was only observed following treatment with E2-deprived KTaR-1 EVs. Further, we found that KTaR-1 media and EVs can affect osteoblast function in vitro, including increases in sp7 and runx2 expression, E2-dependent modulation of wnt10b, and formation of bone matrix (evaluated by Alizarin Red assay) in cultured osteoblast lines, supporting recent studies implicating ARC KP neurons in bone remodeling. Lastly, we found significant levels of immunomodulatory pentraxins (PTX3) in KP EVs, with abundance dependent upon prior E2 exposure in KTaR-1 KP neurons, as revealed by ELISA. Together, results from these studies suggest that EVs may represent additional intercellular communication pathways utilized by Kiss-1 neurons to elicit changes in nearby neuronal populations and potentially even in the periphery, affecting inflammation and bone remodeling. Future studies will address mechanisms involved in the E2 regulation of exosomal cargo in these critical neuronal populations. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Coordinated responses to environmental stimuli are critical for multicellular organisms. To overcome the obstacles of cell-to-cell heterogeneity and noisy signaling dynamics within individual cells, cells must effectively exchange information with peers. However, the dynamics and mechanisms of collective information transfer driven by external signals is poorly understood. Here we investigate the calcium dynamics of neuronal cells that form confluent monolayers and respond to cyclic ATP stimuli in microfluidic devices. Using Granger inference to reconstruct the underlying causal relations between the cells, we find that the cells self-organize into spatially decentralized and temporally stationary networks to support information transfer via gap junction channels. The connectivity of the causal networks depend on the temporal profile of the external stimuli, where short periods, or long periods with small duty fractions, lead to reduced connectivity and fractured network topology. We build a theoretical model based on communicating excitable units that reproduces our observations. The model further predicts that connectivity of the causal network is maximal at an optimal communication strength, which is confirmed by the experiments. Together, our results show that information transfer between neuronal cells is externally regulated by the temporal profile of the stimuli, and internally regulated by cell-cell communication.

Conventional veterinary training emphasizes correct methodologies, potentially failing to exploit learning opportunities that arise as a result of errors. Error management training (EMT) encourages mistakes during low-stakes training, with the intention of modifying perceptions toward errors and using them to improve performance in unfamiliar scenarios (adaptive transfer). Herein, we aimed to determine the efficacy of EMT, supplemented by a metacognitive module, for veterinary students learning blood smear preparation and interpretation. Our hypothesis was that EMT and metacognition are associated with improved adaptive transfer performance, as compared with error avoidance training (EAT). A total of 26 students were prospectively enrolled in this double-blind study. Performance was evaluated according to monolayer area, smear quality, cell identification, calculated white blood cell differential counts, and overall application/interpretation. Students were trained with normal canine blood and static photomicrographs. Participants tested 72 hours after training demonstrated improved performance in a test that directly recapitulated training (Wilcoxon matched-pairs signed-rank test; two-tailed p all ≤ .001). There were no significant differences between EAT and EMT in this test (Mann-Whitney U test and Welch's t-test; two-tailed p ≥ .26) or in short- and long-term adaptive transfer tests (p ≥ .22). Survey data indicate that participants found errors to be a valuable element of training, and that many felt capable of accurately reflecting on their own performance. These data suggest that EMT might produce outcomes comparable to EAT as it relates to blood smear analysis.

Abstract Estrogen (E2) is essential for multiple physiological effects in females, ensuring maximum reproductive fitness and maintaining skeletal homeostasis. E2 has been shown to stimulate cancellous bone formation via activation of estrogen receptor alpha (ERα), an effect widely accepted to be mediated directly at bone. A recent landmark study (Herber et al., Nat Commun 2019) demonstrated bone density increases in female mice harboring ERα-deletions specifically in arcuate Kiss-1 neurons. In this study, bone from transgenic females showed higher osteoblast functioning and increases in the expression of sp7 and runx2, positing a direct neural-bone regulatory axis altered by circulating E2 acting in brain. Our laboratory has used two immortalized Kisspeptin (Kiss1)-expressing and -secreting cell lines, KTaR-1 (representative of female arcuate Kiss-1 neurons) and KTaV-3 cells (representative of female AVPV Kiss-1 neurons) as models to explore the role of Kiss-1 in multiple physiological regulatory contexts. We recently determined that factors in the media of female ARC-derived KTaR-1 cells can affect parameters of osteoblast function in vitro, including increases in sp7 and runx2 expression, and formation of bone matrix (evaluated by Alizarin Red assay). Exposure of canine osteosarcoma cells to conditioned media from KTaR-1 cells led to increases in sp7 expression in an E2-dependent manner, and 24h E2-deprivation of these neurons stimulated secretion of osteogenic factors. In this current study, we have used LCMS-MS proteomic analysis to determine the contents of exosomes isolated from Kisspeptin neurons under varying E2 exposure conditions in vitro. Preliminary results reveal ~150-170 proteins up-regulated by E2 exposure and ~200-220 proteins downregulated by E2 exposure in exosomes of both KTaR-1 and KTaV-3 Kisspeptin neurons. Estrogen-regulated Kiss-1 exosomal proteins include several candidates involved in bone remodeling (pentraxin, osteonectin, osteoclast-stimulating factor-1) and neuronal synaptic plasticity and signaling (annexins, semaphorins, connexins). Current work is exploring the effects of exposure of purified exosomes on morphology and gene expression in immortalized GnRH neurons and osteoblasts. While further study is required, initial results suggest that exosomes may represent additional cellular communication pathways utilized by Kisspeptin neurons to elicit changes in brain and bone.

Changing salinity in estuaries due to sea level rise and altered rainfall patterns, as a result of climate change, has the potential to influence the interactions of aquatic pollutants as well as to alter their toxicity. From a chemical property point of view, ionic concentration can increase the octanol–water partition coefficient and thus decrease the water solubility of a compound. Biologically, organism physiology and enzyme metabolism are also altered at different salinities with implications for drug metabolism and toxic effects. This highlights the need to understand the influence of salinity on pesticide toxicity when assessing risk to estuarine and marine fishes, particularly considering that climate change is predicted to alter salinity regimes globally and many risk assessments and regulatory decisions are made using freshwater studies. Therefore, we exposed the Inland Silverside (Menidia beryllina) at an early life stage to seven commonly used pesticides at two salinities relevant to estuarine waters (5 PSU and 15 PSU). Triadimefon was the only compound to show a statistically significant increase in toxicity at the 15 PSU LC50. However, all compounds showed a decrease in LC50 values at the higher salinity, and all but one showed a decrease in the LC10 value. Many organisms rely on estuaries as nurseries and increased toxicity at higher salinities may mean that organisms in critical life stages of development are at risk of experiencing adverse, toxic effects. The differences in toxicity demonstrated here have important implications for organisms living within estuarine and marine ecosystems in the Anthropocene as climate change alters estuarine salinity regimes globally.

Although errors can be a powerful impetus for learning, conventional pedagogy often emphasizes error-avoidance strategies that reward correct answers and disfavor mistakes. Error management training (EMT) takes an explicitly positive approach to errors, using them to create an active and self-directed learning environment. Using a surgical knot-tying model, we aimed to determine the efficacy of EMT among veterinary students with no prior surgical experience. We hypothesized that EMT would result in improved performance in unfamiliar scenarios (adaptive transfer) compared with an error-avoidance method. In this prospective double-blinded study, 42 students were equally divided between error avoidance training (EAT) and EMT groups. Performance in instrument- and hand-tied knots was evaluated for technique, time, number of attempts, and, when applicable, knot-leaking pressure. All participants demonstrated significant improvement between a pre-test and an analogous test 48 hours after training for all six outcomes (Wilcoxon matched pairs; two-tailed ps ≤ .013). An adaptive transfer test found no significant differences between EMT and EAT at 48 hours (ps ≥ .053). All participants demonstrated a significant performance decline in six of eight outcomes at 7 weeks post-training (ps ≤ .021). This decline was not significant for four of six EMT outcomes yet significant for five of six EAT outcomes. These data suggest that students trained in both EMT and EAT experience comparable gains in short-term performance, including adaptive transfer. Compared with EAT, EMT may help attenuate performance decline after a sustained period of quiescence. Educators may consider actively incorporating EMT into veterinary curricula.

Despite its fundamental importance, the educational mission of most schools of veterinary medicine receives far less recognition and support than the missions of research and discovery. This disparity is evident in promotion and tenure processes. Despite the frequent assertion that education is every college's core mission, there is a broad consensus that faculty are promoted primarily on the basis of meeting expectations relative to publications and grant funding. This expectation is evident in the promotion packets faculty are expected to produce and the criteria by which those packets are reviewed. Among the outcomes is increasing difficulty in hiring and retaining faculty, including young clinicians and basic scientists who are drawn to academic institutions because of the opportunity to teach. The Regional Teaching Academy (RTA) of the West Region Consortium of Colleges of Veterinary Medicine initiated an inter-institutional collaboration to address the most important obstacles to recognizing and rewarding teaching in its five member colleges. Working from the medical education literature, the RTA developed an Educator's Promotion Dossier, workshops to train promotion applicants, and an external review process. Initial use has shown that the reviews are efficient and complete. Administrators have expressed strong support for the product, a letter of external review that is returned to a promotion applicant's home institution. The overall result is an evidence-based, structured process by which teaching-intensive faculty can more fully document their achievements in teaching and educational leadership and a more rigorous external review process by which member colleges can assess quality, impact, and scholarly approach.

Abstract In female animals, ovarian estradiol (E2) can act as both a negative feedback inhibitor of GnRH secretion, as well as a positive feedback stimulator at the time of ovulation. Both of these E2-regulated mechanisms work via stimulation or repression of two distinct neuronal populations of Kisspeptin (KP)-synthesizing neurons. While it is clear that AVPV KP neurons increase kiss1 expression during the preovulatory surge on proestrus, subsequent secretory mechanisms required for potentiation of GnRH surge release remain unclear. Two KP-secreting cell lines, KTaV-3, which demonstrate increased kiss1 expression under high E2 exposure, and KTaR-1, which exhibit kiss1 suppression under low E2 exposure, were used to probe the presence of GnRH receptor (GnRHR) expression under different E2 exposure conditions. KTaV-3 and KtaR-1 cells were treated with a range of doses of E2 (5-100pM) and/or progesterone (20nM) for varying durations (4-96h), exposed to steroid hormones either constitutively or via modulating levels over time, approximating concentration changes found during the murine estrous cycle. Following RNA isolation, cDNAs were probed with primers for gnrhr. Preliminary results in KTaV-3 cells reveal the expression of gnrhr is induced only following elevated (50-100pM) E2 treatment for 18-24h. These same E2 exposure conditions were also found to increase expression of the homeobox protein dlx3, a transcription factor required for GnRHR expression in pituitary gonadotropes. In Arc-derived KTaR-1 cells, gnrhr expression was observed only following decreases in E2 concentration, while dlx3 remained constitutively elevated in this cell line. While reciprocal GnRH-Kisspeptin connections have not yet been observed in vivo, these observations suggest the potential for Kisspeptin neurons to respond to GnRH secretory changes under particular E2 exposure conditions, by modulating receptivity to GnRH at the level of the AVPV and/or Arcuate nuclei. We are continuing to explore the temporal parameters of this induction of GnRHR in KP cells, and if exposure of immortalized KP neurons to GnRH in vitro elicits expression and signaling changes in a time- and E2-dependent manner. Results will provide a more complete understanding of positive and negative feedback mechanisms required for normal neuroendocrine regulation of reproduction.

Abstract While the sex steroid hormone estrogen (E2) is essential for maximum reproductive fitness, circulating E2 is also involved in maintaining skeletal homeostasis in females. E2 in male and female animals has been shown to stimulate cancellous bone formation via activation of estrogen receptor alpha (ERα), and it was widely thought that this effect was mediated directly at the level of bone. A recent study, however, demonstrated a large increase in bone density in female mice in which ERα was deleted from specific neuroendocrine neurons in the arcuate nucleus of the hypothalamus, specifically those expressing kiss1, a population required for fertility and pubertal progression. Bone from transgenic Kiss1-cre X ERα floxed females showed higher osteoblast functioning, accompanied by increases in the expression of sp7 and runx2, positing the existence of a direct neural-bone regulatory axis that is altered by circulating E2 at the level of the brain (Herber et al., Nat Commun 2019). Our laboratory recently published a study demonstrating that GnRH and Kisspeptin, typically thought to act primarily within the neuroendocrine reproductive axis, are synthesized and secreted in an autocrine fashion by canine osteosarcoma cells in vitro, and that these neuropeptides can stimulate tumor cell proliferation (BMC Cancer 2019). Separately, our lab has also recently generated two immortalized Kiss1-expressing and –secreting cell lines, KTaR-1 (representative of female arcuate Kiss-1 neurons) and KTaV-3 cells (representative of female AVPV Kiss-1 neurons) (Endocrinology 2017). In the current study, we have combined these two in vitro models to explore if factors secreted by female ARC-derived KTaR-1 cells may affect multiple parameters of osteoblast function, including sp7 and runx2 expression (evaluated by qPCR), and ability to form bone matrix (evaluated by Alizarin Red assay). Preliminary results suggest that exposure canine osteosarcoma (COS) cells to conditioned media from KTaR-1 cells leads to increases in sp7 expression in an E2-dependent manner, such that 24h E2-deprivation of these neurons stimulates secretion of osteogenic factors. Additionally, media from both KTaR-1 and KTaV-3 cells stimulated Ca2+ production from cultured osteoblasts, as evaluated by Alizarin Red. We are continuing to explore these in vitro interactions using COS cells, as well as normal osteoblasts (cNOB) and immortalized cNOBs, and are evaluating media and exosomal proteomics to further characterize putative factors. While further study is required, these initial results suggest that our immortalized neuronal KP cell models may provide useful molecular tools to explore the regulation of this newly-proposed neural-bone axis.

Veterinary medical education is a relatively small community with limited numbers of institutions, people, and resources widely dispersed geographically. The problems faced, however, are large—and not very different from the problems faced by (human) medical education. As part of an effort to share resources and build a community of practice around common issues, five colleges in the westernmost region of the United States came together to form a regional inter-institutional consortium. This article describes the processes by which the consortium was formed and the initiation of its first collaborative endeavor, an inter-institutional medical/biomedical teaching academy (the Regional Teaching Academy, or RTA). We report outcomes, including the successful launch of three RTA initiatives, and the strategies that have been considered key to the academy’s success. These include strong support from the consortium deans, including an ongoing financial commitment, a dedicated part-time Executive Coordinator, regular face-to-face meetings that supplement virtual meetings, an organization-wide biennial conference, an effective organizational structure, and a core group of dedicated leaders and RTA Fellows. The western consortium and RTA share these processes, insights, and outcomes to provide a model upon which other colleges of veterinary medicine can build to further leverage inter-institutional collaboration.

BackgroundOsteosarcoma strikes hundreds of people each year, of both advanced and younger ages, and is often terminal. Like many tumor types, these bone tumors will frequently undergo a neuroendocrine transition, utilizing autocrine and/or paracrine hormones as growth factors and/or promoters of angiogenesis to facilitate progression and metastasis. While many of these factors and their actions on tumor growth are characterized, some tumor-derived neuropeptides remain unexplored.MethodsUsing validated canine osteosarcoma cell lines in vitro, as well as cells derived from spontaneous tumors in dogs, we explored the autocrine production of two neuropeptides typically found in the hypothalamus, and most closely associated with reproduction: gonadotropin-releasing hormone (GnRH) and kisspeptin (Kiss-1). We evaluated gene expression and protein secretion of these hormones using quantitative RT-PCR and a sensitive radioimmunoassay, and explored changes in cell proliferation determined by MTS cell viability assays.ResultsOur current studies reveal that several canine osteosarcoma cell lines (COS, POS, HMPOS, D17, C4) synthesize and secrete GnRH and express the GnRH receptor, while COS and POS also express kiss1 and its cognate receptor. We have further found that GnRH and kisspeptin, exogenously applied to these tumor cells, exert significant effects on both gene expression and proliferation. Of particular interest, kisspeptin exposure stimulated GnRH secretion from COS, similarly to the functional relationship observed within the neuroendocrine reproductive axis. Additionally, GnRH and kisspeptin treatment both increased COS proliferation, which additionally manifested in increased expression of the bone remodeling ligand rankl within these cells. These effects were blocked by treatment with a specific GnRH receptor inhibitor. Both neuropeptides were found to increase expression of the specific serotonin (5HT) receptor htr2a, the activation of which has previously been associated with cellular proliferation, suggesting that production of these factors by osteosarcoma cells may act to sensitize tumors to circulating 5HT of local and/or enteric origin.ConclusionsHere we report that kisspeptin and GnRH act as autocrine growth factors in canine osteosarcoma cells in vitro, modulating RANKL and serotonin receptor expression in a manner consistent with pro-proliferative effects. Pharmacological targeting of these hormones may represent new avenues of osteosarcoma treatment.

Abstract Breast cancer is a leading cause of cancer deaths among women, with an estimated 12.4% of American women diagnosed with this tumor type. Evidence suggests that exogenous disruptors can exert epigenetic changes leading to aberrant gene expression, putatively shifting the balance toward oncogenesis. While chemical pollutant exposure is well-studied, light exposure may exert diverse effects that are often overlooked. Mounting epidemiological evidence suggests that chronic circadian dysregulation is associated with increased breast cancer risk. Exposure to light at night (LAN) via ambient nighttime lighting, digital technology, shiftwork, and trans-meridian travel can alter temporal organization of cellular processes such as cell cycle regulation and tissue proliferation. Previous studies demonstrated that animals exposed to altered light cycles exhibited marked DNA methylation pattern changes, and aberrant DNA methylation is noted across multiple loci in the cancer genome. LAN exposure may thus contribute to the etiology of breast cancer via alteration of the epigenome and subsequent temporal disruption of typical gene expression patterns. We previously found (unpublished data) that mPer2::luc females exposed to 21 days of LAN (18:6 LD) exhibited profound circadian disruption particularly in mammary chain, in comparison to control-exposed (12:12 LD) mice, and that LAN exposure significantly decreased mammary ERα and ERβ expression. We are continuing to explore LAN-induced molecular alterations in the mammary via whole-genome bisulfite sequencing, to determine if inappropriate light exposure is an etiological risk factor of hormone-dependent mammary cancer. Post-pubertal female mice were exposed to LAN or control light cycles for 21d prior to mammary tissue harvest as described above for mPer2::luc knock-in mice. Genomic DNA samples were bisulfite-converted and sequenced, aligned to a bisulfite-converted genome, and analyzed to identify differential DNA methylation, using probes for regulatory regions (-500 to +2000bp), exons, introns, and CpG islands. LAN exposure induced changes in mean methylation of probes across 6.4% of regulatory regions (2044 of 32025), 5.5% of exons (1752 of 32025), 0.03% of introns (577 of 190791), and 1.9% of CpG islands (269 of 13840) individually analyzed and annotated with surrounding or downstream genes (adjusted p<0.05, abs. min. difference of means >5). Significant methylation changes resulting from LAN were noted in multiple genetic loci implicated in cancer, including directional methylation changes intermediate between control and 4T1 breast cancer models across various Hox genes, Hic1, Cdkn1c, and other genes associated with proliferation and oncogenesis, with commensurate changes in gene expression also observed. We are currently working to confirm relative expression of commensurate genes via qRT-PCR. Together, these results point to a potential mechanism by which LAN exposure may initiate cellular dysregulation within mammary tissue. Citation Format: Rebecca E. Veitch, Shay Bracha, Patrick Chappell. Circadian dysregulation via exposure to light at night alters DNA methylation of cancer-associated genes in murine mammary tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-237.

The decline of female reproductive function is an early phenotype of aging, occurring only midway through the lifespan. Yet, women's ability to delay pregnancy and preserve fertility is an important reproductive freedom. The number of women delaying pregnancy continues to rise in industrialized societies due to personal or socioeconomic circumstances, often resulting in subfertility or difficulty conceiving. There are few defined mechanisms associated with this etiology, and equally few effective therapies. To combat this problem, we used a novel emerging model, Nothobranchius guentheri, with a lifespan of only 9–12 months, that recapitulates the age‐associated spectrum of changes that adversely affect human fertility. We hypothesized that activating SirT1 would maintain female fecundity into late life. SirT1 is an NAD+ dependent histone deacetylase, whose activity is regulated by the nicotinamide to NAD+ salvage pathway, especially the rate‐limiting enzyme NAMPT. In order to increase SirT1 activation via NAMPT, we fed N. guentheri a diet containing the polyphenol resveratrol (RSV) at 300 μg RSV/g food, beginning at sexual maturity. Our previous work showed that RSV‐fed fish at 5 months of age had significantly increased ovarian NAMPT protein levels, and our current data confirms that this result continues into late life (9+ months of age). Furthermore, we found an age‐related decline in NAMPT protein that was overcome by RSV supplementation. For the first time, we show that SirT1 activity, which declines with age, is preserved in the ovaries of our RSV‐fed model. Female N. guentheri fed RSV into late life also showed increased embryo production compared to those on Control diet without RSV. These results suggest that activating SirT1 via increasing NAD+ may have a positive effect on female fertility, and that dietary interventions may become an effective therapy to combat reproductive senescence.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The objective was to determine estrogen's influence on control of a skeletal muscle through measurements of motorneuron excitability (H:M ratio) and presynaptic inhibition (PI). Estrogen serum concentrations were measured at menses and ovulation of female subjects and compared to male controls. Data were analyzed from 12 women and 13 men reporting no history of knee ligament injury. Women reported regular menstrual cycles and no hormone‐based contraceptive use for the previous year. Women were tested at menses (Time1) and ovulation (Time2). Men were tested twice, approximately 14 days apart. Analysis indicated no difference in the H:M ratio between the sexes at either time point. A significant difference for the sexes was detected in the magnitude of estrogen change (∆EST) between observations. At Time1, the male and female estrogen concentrations were not different; however, they were different at Time2, primarily due to the large rise observed in the women. A significant difference between the sexes was also seen in the magnitude of change for PI (∆PI) between observations. As with EST, the levels of PI between the sexes at Time1 were not different; however, a difference existed at Time 2. Estrogen interacts with GABA at several nervous system locations affecting inhibition of synaptic transmission. This is the first study to investigate changes in PI of a skeletal muscle between times of low and high estrogen. Improving the understanding of estrogen's influence on skeletal muscles may provide answers to why noncontact anterior cruciate ligament injuries of the knee occur more frequently in women.