Bridging conservation across the ex situ-in situ spectrum: Insights into the reproductive ecology of the threatened narrow-headed gartersnake (Thamnophis rufipunctatus).

Zoo-based (ex situ) conservation breeding programs provide invaluable opportunities to uncover enigmatic behaviors and traits of focal species under managed care, which can support research and conservation management efforts. A suite of factors and a limited range have yielded population declines in the threatened narrow-headed gartersnake (Thamnophis rufipunctatus). Better understanding its cryptic ecology and life history (e.g., reproductive ecology) offers conservation benefits. We analyzed data on courtship behavior, parity and litter size, offspring size, and neonatal growth from an ex situ T. rufipunctatus population at the Phoenix Zoo from 2009 to 2018. Courtship behavior and parturition phenology are likely linked with the North American monsoon season, yet the courtship window may be wider than realized. We document the first instances of interannual iteroparity and multigenerational rearing of successful breeders at the ex situ level. Litter sizes varied but were relative to maternal body mass, suggesting that fecundity may be driven by intrinsic condition (e.g., age and size) of breeding females. Mean offspring body masses were equivalent between sexes, and neonate growth trends were quadratic during their first 9 months. Sexual dimorphism became apparent around 4-5 months age. Much of these data are novel for T. rufipunctatus and provide insight into their reproductive ecology. Phenology of reproductive ecology and body size metrics can guide field surveillance, age estimations, and population ecology monitoring, as well as inform ex situ adaptive management practices. Strategies spanning the ex situ-in situ spectrum are applicable to other imperiled taxa to better inform conservation management decisions.

Adaptive management in a conservation breeding program: Mimicking habitat complexities facilitates reproductive success in narrow-headed gartersnakes (Thamnophis rufipunctatus).

Mimicking natural parameters and complexities in zoo conservation breeding programs can facilitate natural physiological and behavioral traits, which in turn can inform more effective species reintroduction efforts. To curtail population declines of threatened narrow-headed gartersnakes (Thamnophis rufipunctatus), the Arizona Center for Nature Conservation/Phoenix Zoo partnered with a multiagency conservation working group to develop an ex situ propagation-for-release program. Initially, Zoo staff followed common snake husbandry protocols of manually inducing brumation (i.e., winter dormancy). Copulation was observed during the first few years, but no births resulted. Also, some older individuals developed post-brumation health abnormalities, prompting a strategic reassessment. To facilitate propagation and improve health, Zoo staff applied ecological knowledge of T. rufipunctatus and an adaptive management strategy to implement key parameters for success: sociality, refugia, breeding and foraging behaviors, and natural brumation. Zoo staff developed a large multisnake enclosure that mimicked natural ecological and habitat complexities including a hibernaculum to stimulate natural brumation. Gartersnakes were left mostly unimpeded to conduct natural behaviors across seasons in the enriched environment. We referenced change in body mass after ten brumation periods as a proxy for health. Under natural brumation, gartersnakes did not lose body mass, and this shift resulted in fully ex situ parturition events-the first for this imperiled species. We highlight the efficacy of adaptive management and incorporation of natural parameters and environmental complexities into conservation breeding programs. These actions can improve the health and success of animals under managed care-processes applicable to a range of taxa targeted for conservation translocations.

Toll-like receptor 7: A novel neuroimmune target to reduce excessive alcohol consumption.

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns in foreign pathogens and intrinsic danger/damage signals from cells. TLR7 is a nucleic acid sensing endosomal TLR that is activated by single-stranded RNAs from microbes or by small noncoding RNAs that act as endogenous ligands. TLR7 signals through the MyD88 adaptor protein and activates the transcription factor interferon regulatory factor 7 (IRF7). TLR7 is found throughout the brain and is highly expressed in microglia, the main immune cells of the brain that have also been implicated in alcohol drinking in mice. Upregulation of TLR7 mRNA and protein has been identified in postmortem hippocampus and cortex from AUD subjects that correlated positively with lifetime consumption of alcohol. Similarly, Tlr7 and downstream signaling genes were upregulated in rat hippocampal and cortical slice cultures after chronic alcohol exposure and in these regions after chronic binge-like alcohol treatment in mice. In addition, repeated administration of the synthetic TLR7 agonists imiquimod (R837) or resiquimod (R848) increased voluntary alcohol drinking in different rodent models and produced sustained upregulation of IRF7 in the brain. These findings suggest that chronic TLR7 activation may drive excessive alcohol drinking. In the brain, this could occur through increased levels of endogenous TLR7 activators, like microRNAs and Y RNAs. This review explores chronic TLR7 activation as a pathway of dysregulated neuroimmune signaling in AUD and the endogenous small RNA ligands in the brain that could perpetuate innate immune responses and escalate alcohol drinking.

Impact of Serum Proteins on the Uptake and RNAi Activity of GalNAc-Conjugated siRNAs.

Serum protein interactions are evaluated during the drug development process since they determine the free drug concentration in blood and thereby can influence the drug's pharmacokinetic and pharmacodynamic properties. While the impact of serum proteins on the disposition of small molecules is well understood, it is not yet well characterized for a new modality, RNA interference therapeutics. When administered systemically, small interfering RNAs (siRNAs) conjugated to the N-acetylgalactosamine (GalNAc) ligand bind to proteins present in circulation. However, it is not known if these protein interactions may impact the GalNAc-conjugated siRNA uptake into hepatocytes mediated through the asialoglycoprotein receptor (ASGPR) and thereby influence the activity of GalNAc-conjugated siRNAs. In this study, we assess the impact of serum proteins on the uptake and activity of GalNAc-conjugated siRNAs in primary human hepatocytes. We found that a significant portion of the GalNAc-conjugated siRNAs is bound to serum proteins. However, ASGPR-mediated uptake and activity of GalNAc-conjugated siRNAs were minimally impacted by the presence of serum relative to their uptake and activity in the absence of serum. Therefore, in contrast to small molecules, serum proteins are expected to have minimal impact on pharmacokinetic and pharmacodynamic properties of GalNAc-conjugated siRNAs.