Land cover and space use influence coyote carnivory: evidence from stable-isotope analysis.

For many species, the relationship between space use and diet composition is complex, with individuals adopting varying space use strategies such as territoriality to facilitate resource acquisition. Coyotes (Canis latrans) exhibit two disparate types of space use; defending mutually exclusive territories (residents) or moving nomadically across landscapes (transients). Resident coyotes have increased access to familiar food resources, thus improved foraging opportunities to compensate for the energetic costs of defending territories. Conversely, transients do not defend territories and are able to redirect energetic costs of territorial defense towards extensive movements in search of mates and breeding opportunities. These differences in space use attributed to different behavioral strategies likely influence foraging and ultimately diet composition, but these relationships have not been well studied. We investigated diet composition of resident and transient coyotes in the southeastern United States by pairing individual space use patterns with analysis of stable carbon (δ13C) and nitrogen (δ15N) isotope values to assess diet. During 2016-2017, we monitored 41 coyotes (26 residents, 15 transients) with GPS radio-collars along the Savannah River area in the southeastern United States. We observed a canopy effect on δ13C values and little anthropogenic food in coyote diets, suggesting 13C enrichment is likely more influenced by reduced canopy cover than consumption of human foods. We also observed other land cover effects, such as agricultural cover and road density, on δ15N values as well as reduced space used by coyotes, suggesting that cover types and localized, resident-like space use can influence the degree of carnivory in coyotes. Finally, diets and niche space did not differ between resident and transient coyotes despite differences observed in the proportional contribution of potential food sources to their diets. Although our stable isotope mixing models detected differences between the diets of resident and transient coyotes, both relied mostly on mammalian prey (52.8%, SD = 15.9 for residents, 42.0%, SD = 15.6 for transients). Resident coyotes consumed more game birds (21.3%, SD = 11.6 vs 13.7%, SD = 8.8) and less fruit (10.5%, SD = 6.9 vs 21.3%, SD = 10.7) and insects (7.2%, SD = 4.7 vs 14.3%, SD = 8.5) than did transients. Our findings indicate that coyote populations fall on a feeding continuum of omnivory to carnivory in which variability in feeding strategies is influenced by land cover characteristics and space use behaviors.

Lgr5+ intestinal stem cells are required for organoid survival after genotoxic injury.

Progenitors and mature cells can maintain the intestinal epithelium by dedifferentiation and facultative intestinal stem cell (fISC) function when active ISCs (aISCs) are lost to damage. Here, we sought to model fISC activation in intestinal organoids with doxorubicin (DXR), a chemotherapeutic known to ablate Lgr5+ aISCs in vivo. We identified low and high doses of DXR compatible with long-term organoid survival. Similar fISC gene activation was observed between organoids treated with low vs high DXR, despite significantly decreased survival at the higher dose. aISCs exhibit dose-dependent loss after DXR but survive at doses compatible with organoid survival. We ablated residual aISCs after DXR using a Lgr52A-DTR allele and observed that aISC survival of the initial genotoxic insult is required for organoid survival following DXR. These results suggest that while typical fISC genes are activated by DXR injury in organoids, functional stemness remains dependent on the aISC pool. Our data establish a reproducible model of DXR injury in intestinal organoids and reveal differences in in vitro responses to an established in vivo damage modality.

Acoustic arrival predictions using oceanographic measurements and models in the Beaufort Sea.

Acoustic propagation in the Beaufort Sea is particularly sensitive to upper-ocean sound-speed structure due to the presence of a subsurface duct known as the Beaufort duct. Comparisons of acoustic predictions based on existing Arctic models with predictions based on in situ data collected by Seaglider vehicles in the summer of 2017 show differences in the strength, depth, and number of ducts, highlighting the importance of in situ data. These differences have a significant effect on the later, more intense portion of the acoustic time front referred to as reverse geometric dispersion, where lower-order modes arrive prior to the final cutoff.

Body Mindsets are Associated With Pain and Threat-Related Risk Factors for Pain in Survivors of Childhood Cancer.

Pain is a common consequence of childhood cancer. While most research has examined biomedical predictors of post-cancer pain, biopsychosocial conceptualisations such as the cancer threat interpretation (CTI) model hold promise for guiding comprehensive pain management strategies. Guided by the CTI model, this cross-sectional study evaluated correlates of post-cancer pain in childhood cancer survivors including threat-related risk factors (bodily threat monitoring, fear of cancer recurrence, help-seeking) and mindsets about the body. In the preceding three months, 21.8% of the survivors reported chronic pain (>3 months), and 14.3% experienced pain most days. Greater bodily threat monitoring, more fear of cancer recurrence, and more help-seeking were associated with more pain. There was heterogeneity in the mindsets that survivors of childhood cancer hold about their bodies. Holding the mindset that the 'body is an adversary' was associated with more pain, greater bodily threat monitoring, and more fear of cancer recurrence. Holding the mindset that the 'body is responsive' was associated with less bodily threat monitoring, while the mindset that the 'body is capable' was associated with greater help-seeking. A path model demonstrated a significant combined indirect effect of the 'body is an adversary' mindset on pain through bodily threat monitoring and fear of cancer recurrence. Overall, this study supported that a sub-group of childhood cancer survivors experience persistent and interfering pain and provided cross-sectional support for threat-related correlates for pain aligning with the CTI model. Body mindsets were associated with pain and threat-related correlates and may represent a novel target to support survivors with pain. PERSPECTIVE: This article presents associations of body mindsets, threat-related risk factors, and pain in survivors of childhood cancer (aged 11-25), guided by the Cancer Threat Interpretation model. The study indicates that body mindsets may be novel targets to embed in comprehensive post-cancer pain management approaches to support young survivors with pain.

Transforming Care for Older Adults Living with Complex Health Conditions in Ontario Post-Covid: Conference Proceedings and Recommendations.

The virtual conference 'Transforming Care: Supporting Older Adults Post-COVID in Ontario' was held in October 2021. It was organized by Specialized Geriatric Services (SGS) East and held over three half-days. The guiding themes included: The Need, The Innovation, and The Transformation. Over 500 participants heard from ~50 clinicians, researchers, administrators, older adults, care partners, and community partners. The pandemic uncovered and exacerbated existing issues and pushed us to explore new ways to support older adults living with complex health conditions. The following key priorities were identified: older adults and their care partners call for personalized care experiences, and a lifespan approach to care delivery; aging in the community remains the most common preference; an integrated community care system that supports aging at-home should be prioritized; care delivery by SGS interprofessional teams and specialists is paramount to providing comprehensive care; building health human resource capacity should be a system priority; and promising innovations should be scaled and spread. Evidence shows that we cannot return to status-quo; post-pandemic planning of both who we serve and how we serve needs to be anchored in system renewal, not just recovery. Renewal means integrating lessons learned during the pandemic into the redesign of our systems of care. Investments in innovative, upstream strategies that support home and community-based care, and target health promotion and prevention are necessary. The provincial and regional infrastructure of SGS has the expertise and capacity to assist Ontario Health Teams in responding to the evolving health and social needs of this population.

The bodily threat monitoring scale: Development and preliminary validation in adult and childhood cancer survivors.

OBJECTIVE: Bodily threat monitoring is a core clinical feature of Fear of cancer recurrence (FCR) and is targeted in psycho-oncology treatments, yet no comprehensive self-report measure exists. The aim of this study was the theory-informed development and initial validation of the Bodily Threat Monitoring Scale (BTMS). METHODS: Adult survivors of breast and gynaecological cancers (Study 1: N = 306, age = 37-81 years) and childhood cancer survivors (Study 2: N = 126, age = 10-25 years) completed the BTMS, designed to assess how individuals monitor for and interpret uncertain symptoms as indicating that something is wrong with their body. Participants completed measures to assess construct and criterion validity of the BTMS, and childhood cancer survivors (Study 2) completed the BTMS again 2 weeks later to assess test-retest reliability. RESULTS: The 19-item BTMS demonstrated excellent internal consistency across adult and childhood cancer samples (α = 0.90-0.96). Factor analyses indicated two subscales capturing 1. Monitoring of bodily sensations and 2. Threatening interpretations of bodily sensations. Two-week stability estimates were acceptable. For construct validity, the BTMS correlated with body vigilance and anxiety sensitivity. The BTMS also demonstrated criterion validity, yielding significant associations with FCR, intolerance of uncertainty, help-seeking behaviours, and quality of life. The BTMS was associated with FCR while controlling for body vigilance and anxiety sensitivity, indicating a unique contribution of this theory-informed measure. CONCLUSIONS: The BTMS shows evidence of sound psychometric properties and could be used to elucidate the role of bodily threat monitoring in the maintenance and management of FCR.

Maturation of small nucleolar RNAs: from production to function.

Small Nucleolar RNAs (snoRNAs) are an abundant group of non-coding RNAs with well-defined roles in ribosomal RNA processing, folding and chemical modification. Besides their classic roles in ribosome biogenesis, snoRNAs are also implicated in several other cellular activities including regulation of splicing, transcription, RNA editing, cellular trafficking, and miRNA-like functions. Mature snoRNAs must undergo a series of processing steps tightly regulated by transiently associating factors and coordinated with other cellular processes including transcription and splicing. In addition to their mature forms, snoRNAs can contribute to gene expression regulation through their derivatives and degradation products. Here, we review the current knowledge on mechanisms of snoRNA maturation, including the different pathways of processing, and the regulatory mechanisms that control snoRNA levels and complex assembly. We also discuss the significance of studying snoRNA maturation, highlight the gaps in the current knowledge and suggest directions for future research in this area.

International Survey of Pediatric Oncologists' Beliefs and Communication Practices Regarding Symptom Self-Monitoring by Childhood Cancer Survivors.

PURPOSE: Childhood cancer survivors report self-monitoring for and worrying about symptoms of disease recurrence and secondary cancers, although symptom-related worry is associated with poorer health-related quality of life. This survey captured pediatric oncologists' beliefs and communication practices regarding symptom self-monitoring for childhood cancer survivors. METHODS: Using a closed-loop snowball sampling technique, pediatric oncologists completed an online survey regarding the importance of symptom self-monitoring for off-therapy patients, the degree to which symptom self-monitoring was perceived to cause stress and worry, and communication practices. RESULTS: 196 pediatric oncologists (White [78%]; female [64%]; Mage = 47 years) from every continent except Antarctica participated. Oncologists believed it is important for off-therapy patients to self-monitor for symptoms of cancer recurrence (90%) and treatment late effects (94%), although some noted that recurrence (30%) and late effects (55%) are typically detected by routine surveillance before symptoms appear. Oncologists varied in their beliefs that off-therapy patients do (31%) or do not (31%) worry unnecessarily about symptoms of recurrence. Two thirds (62%) of oncologists reported often/always discussing with off-therapy patients which symptoms could indicate cancer recurrence, whereas fewer than half (43%) often/always discussed which symptoms were unlikely to indicate recurrence. Oncologists identified a need for education regarding how to communicate around symptom self-monitoring and the potential utility of a screening tool to identify those who worry excessively. CONCLUSION: Despite nearly universal belief that their off-therapy patients should self-monitor for symptoms of disease recurrence and late effects, a substantial proportion of pediatric oncologists do not counsel patients on symptom self-monitoring. Since nearly one-third believe that off-therapy patients worry unnecessarily about symptoms of recurrence, improving patient education regarding which symptoms are and are not medically concerning could decrease stress and improve health-related quality of life for pediatric cancer survivors.

Generation of anti-GD2 CAR macrophages from human pluripotent stem cells for cancer immunotherapies.

Macrophages armed with chimeric antigen receptors (CARs) provide a potent new option for treating solid tumors. However, genetic engineering and scalable production of somatic macrophages remains significant challenges. Here, we used CRISPR-Cas9 gene editing methods to integrate an anti-GD2 CAR into the AAVS1 locus of human pluripotent stem cells (hPSCs). We then established a serum- and feeder-free differentiation protocol for generating CAR macrophages (CAR-Ms) through arterial endothelial-to-hematopoietic transition (EHT). CAR-M produced by this method displayed a potent cytotoxic activity against GD2-expressing neuroblastoma and melanoma in vitro and neuroblastoma in vivo. This study provides a new platform for the efficient generation of off-the-shelf CAR-Ms for antitumor immunotherapy.

Functional Changes in the Adult Mouse Retina using an Alpha7 Nicotinic Acetylcholine Receptor Agonist after Blast Exposure.

Currently, there is a lack of treatments for retinal neurotrauma. To address this issue, this study uses an alpha7 nAChR agonist, PNU-282987, to determine it effects on functional activity in the retina shortly after a traumatic blast exposure. The objectives of this research include: (1) examination of the cellular and functional damage associated with ocular blast exposure, and (2) evaluation of structural and functional changes that occur post PNU-282987 treatment. Significant ocular blast damage was induced in adult mice after exposure to a single blast of 35 psi to the left eye. Blast-exposed transgenic mice expressing tdTomato Müller glia were treated daily with eyedrops containing PNU-282987 for 4 weeks following the blast exposure. Antibody staining studies in these transgenic mice was conducted to examine lineage tracing and electroretinograms (ERGs) were obtained to examine functional changes. Blast exposure caused a significant loss of cells in all retinal layers after 4 weeks. Immunohistochemical analysis demonstrated tdTomato-positive labeled photoreceptors and retinal ganglion cells in blast-exposed mice treated with PNU-282987. ERG recordings were taken from control animals, from blast-damaged animals and from animals exposed to blast followed by 4 weeks of PNU-282987 treatment. Scotopic ERG recordings from blast-exposed mice had significantly decreased amplitudes of a-wave, b-wave, oscillatory potentials and flicker frequencies, which were prevented after PNU-282987 treatment. In photopic experiments, the PhNR response was reduced significantly after blast exposure but the decrease was prevented after treatment with PNU-282987. These are the first experiments that demonstrate preservation of retinal function after blast exposure using an alpha7 nAChR agonist.

Agile development of a digital exposure treatment for youth with chronic musculoskeletal pain: protocol of a user-centred design approach and examination of feasibility and preliminary efficacy.

INTRODUCTION: Chronic pain affects a significant number of children and impacts multiple domains including social, emotional and behavioural functioning, and negatively impacts family functioning. Roughly 5% of youth with chronic pain experience moderate to severe pain-related disability, with pain-related fear and avoidance of activities being identified as substantial barriers to treatment engagement. Evidence supports targeted psychological and physical interventions to address these barriers (eg, graded-exposure treatment), but accessibility to intervention is undermined by a shortage of services outside of urban areas, high treatment-related costs, and long provider waitlists; highlighting the need to develop digitally delivered behavioural intervention, using agile and iterative study designs that support rapid development and timely dissemination. METHODS AND ANALYSIS: This study seeks to develop an effective and scalable intervention for youth with chronic pain and their caregivers. This paper presents a user-centred protocol for the development and refinement of a digital exposure treatment for youth and caregivers, as well as the study design to examine feasibility and preliminary efficacy of the treatment using single-case experimental design (SCED). Assessments include daily diaries, completed from baseline and daily throughout the intervention (~6 weeks), and at 3-month follow-up, as well as self-report measures completed at baseline, end of intervention and 3-month follow-up. Primary outcomes include treatment satisfaction, treatment expectancy, adherence to daily dairies and functional disability. Secondary outcomes are pain-related fear and avoidance of activities, pain catastrophising and pain acceptance. We will present descriptive and model-based inference analyses, based on SCED reporting guidelines. We will calculate effect sizes for each individual on each outcome. We will examine mean treatment expectancy, credibility and satisfaction scores, and patient drop-out percentage. ETHICS AND DISSEMINATION: This study is approved by the Institutional Review Board at Stanford University (protocol #53323). Findings will be actively disseminated through peer-reviewed journals, conference presentations and social media. TRIAL REGISTRATION NUMBER: NCT05079984.

Transcriptome Changes in Retinal Pigment Epithelium Post-PNU-282987 Treatment Associated with Adult Retinal Neurogenesis in Mice.

PNU-282987, a selective alpha7 nicotinic acetylcholine receptor agonist, has previously been shown to have both neurogenic and broad regenerative effects in the adult murine retina. The objective of this study was to assay the molecular mechanism by which PNU-282987 promotes the production of Muller-derived progenitor cells through signaling via the resident retinal pigment epithelium. These Muller-derived progenitor cells generate a myriad of differentiated neurons throughout the retina that have previously been characterized by morphology. Herein, we demonstrate that topical application of PNU-282987 stimulates production of functional neurons as measured by electroretinograms. Further, we examine the mechanism of how this phenomenon occurs through activation of this atypical receptor using a transcriptomic approach isolated retinal pigment epithelium activated by PNU-282987 and in whole retina. We provide evidence that PNU-282987 causes a bi-modal signaling event in which early activation primes the retina with an inflammatory response and developmental signaling cues, followed by an inhibition of gliotic mechanisms and a decrease in the immune response, ending with upregulation of genes associated with specific retinal neuron generation. Taken together, these data provide evidence that PNU-282987 activates the retinal pigment epithelium to signal to Muller glia to produce Muller-derived progenitor cells, which can differentiate into new, functional neurons in adult mice. These data not only increase our understanding of how adult mammalian retinal regeneration can occur, but also provide therapeutic promise for treating functional vision loss.

Enabling Healthy Aging to AVOID Frailty in Community Dwelling Older Canadians.

The Canadian population is aging. With aging, biological and social changes occur increasing the risk of developing chronic conditions and functional loss leading to frailty. Older adults living with frailty are more vulnerable to minor stressors, take longer to recover from illness, and have difficulty participating in daily activities. The Canadian Frailty Network's (CFN) mission is to improve the lives of older adults living with frailty. In September 2019, CFN launched the Activity & Exercise, Vaccination, Optimization of medications, Interaction & Socialization, and Diet & Nutrition (AVOID) Frailty public health campaign to promote assessing and reducing risk factors leading to the development of frailty. As part of the campaign, CFN held an Enabling Healthy Aging Symposium with 36 stakeholders from across Canada. Stakeholders identified individual and community-level opportunities and challenges for the enablement of healthy aging and frailty mitigation, as part of a focused consultative process. Stakeholders ranked the three most important challenges and opportunities at the individual and community levels for implementing AVOID Frailty recommendations. Concrete actions, further research areas, policy changes, and existing resources/programs to enhance the AVOID Frailty campaign were identified. The results will help inform future priorities and behaviour change strategies for healthy aging in Canada.

Smartphone-based Ecological Momentary Assessment to study "scanxiety" among Adolescent and Young Adult survivors of childhood cancer: A feasibility study.

OBJECTIVE: Scan-related anxiety ("scanxiety") refers to the fear, stress, and anxiety in anticipation of tests and scans in follow-up cancer care. This study assessed the feasibility of Ecological Momentary Assessment (EMA) for real-world, real-time capture of scanxiety using patients' personal smartphone. METHODS: Adolescent and Young Adult survivors of childhood cancer were prompted to complete EMA surveys on a smartphone app three times per day for 11 days (33 surveys total) around their routine surveillance scans. Participants provided structured feedback on the EMA protocol. RESULTS: Thirty out of 46 contacted survivors (65%) enrolled, exceeding the preregistered feasibility cutoff of 55%. The survey completion rate (83%) greatly exceeded the preregistered feasibility cutoff of 65%. Participants generally found the smartphone app easy and enjoyable to use and reported low levels of distress from answering surveys. Participants reported significantly more daily fear of cancer recurrence (FCR) and negative affect in the days before compared to the days after surveillance scans, aligning with the expected trajectory of scanxiety. Participants who reported greater FCR and scanxiety using comprehensive measures at baseline also reported significantly more daily FCR around their surveillance scans, indicating validity of EMA items. Bodily threat monitoring was prospectively and concurrently associated with daily FCR, thus warranting further investigation as a risk factor for scanxiety. CONCLUSIONS: Findings indicate the feasibility, acceptability, and validity of EMA as a research tool to capture the dynamics and potential risk factors for scanxiety.

Sex Influences Age-Related Changes in Natural Antibodies and CD5+ B-1 Cells.

Natural Abs are primarily produced by B-1 cells and are essential for protection against Streptococcus pneumoniae The incidence and mortality rate for pneumococcal infection increases dramatically after age 65, disproportionately affecting males in both human and murine systems. To date, there is a significant gap in our understanding of the relationship among sex, aging, natural IgM efficacy, and the natural IgM repertoire. Our investigation demonstrates that the protective capacity of serum IgM against pneumococcal infection is maintained in IgM obtained from aged female mice but absent in IgM from aged male mice. To understand this difference in protective capacity, we examined serum Ig, discovering that the protective change was not associated with shifts in levels of phosphorylcholine (PC)- or pneumococcal capsular polysaccharide serotype 3-specific IgM. Interestingly, we observed that aged females have an increase in the total number of CD5+ B-1 cells, higher serum IL-5 levels, and a larger percentage of aged female CD5+ B-1 cells that express CD86 as compared with aged males. Furthermore, single-cell IgM repertoire analysis from peritoneal PC+, splenic PC+, and bone marrow CD5+ B-1 cell subsets demonstrated greater diversity with age and a higher level of germline status in female mice than previously observed in studies of aged male mice. Aged female CD5+ B-1 cells also expressed higher levels of transcripts associated with cell activity and self-renewal, such as Nanog and Hmga2 Taken together, these data indicate that females maintain a more diverse and active CD5+ B-1 cell pool and natural IgM repertoire, which has implications for sex-related susceptibility to infection and disease.

Resident and transient coyotes exhibit differential patterns of movement behavior across heterogeneous landscapes in the southeastern United States.

Coyotes (Canis latrans) are a highly adaptable canid species whose behavioral plasticity has allowed them to persist in a wide array of habitats throughout North America. As generalists, coyotes can alter movement patterns and change territorial strategies between residency (high site fidelity) and transiency (low site fidelity) to maximize fitness. Uncertainty remains about resident and transient coyote movement patterns and habitat use because research has reached conflicting conclusions regarding patterns of habitat use by both groups. We quantified effects of habitat on resident and transient coyote movement behavior using first passage time (FPT) analysis, which assesses recursive movement along an individual's movement path to delineate where they exhibit area-restricted search (ARS) behaviors relative to habitat attributes. We quantified monthly movement rates for 171 coyotes (76 residents and 53 transients) and then used estimated FPT values in generalized linear mixed models to quantify monthly habitat use for resident and transient coyotes. Transients had greater movement rates than residents across all months except January. Resident FPT values were positively correlated with agricultural land cover during fall and winter, but negatively correlated with agriculture during spring. Resident FPT values were also negatively correlated with developed habitats during May-August, deciduous land cover during June-August, and wetlands during September-January except November. FPT values of transient coyotes were positively correlated with developed areas throughout much of the year and near wetlands during July-September. Transient FPT values were negatively correlated with agriculture during all months except June and July. High FPT values (ARS behavior) of residents and transients were generally correlated with greater densities of edge habitat. Although we observed high individual variation in space use, our study found substantive differences in habitat use between residents and transients, providing further evidence that complexity and plasticity of coyote habitat use is influenced by territorial strategy.

Modulation of microbiome diversity and cytokine expression is influenced in a sex-dependent manner during aging.

The microbiome and immune system have a unique interplay, which influences homeostasis within the organism. Both the microbiome and immune system play important roles in health and diseases of the aged including development of cancer, autoimmune disorders, and susceptibility to infection. Various groups have demonstrated divergent changes in the gut microbiota during aging, yet the compounding factor of biological sex within the context of aging remains incompletely understood, and little is known about the effect of housing location in the composition of gut microbiota in the context of both sex and age. To better understand the roles of sex, aging, and location in influencing the gut microbiome, we obtained normal healthy BALB/cByJ mice from a single source and aged male and female mice in two different geographical locations. The 16S rRNA was analyzed from fecal samples of these mice and cytokine levels were measured from serum.16S rRNA microbiome analysis indicated that both age and sex play a role in microbiome composition, whereas location plays a lesser role in the diversity present. Interestingly, microbiome changes occurred with alterations in serum expression of several different cytokines including IL-10 and IL-6, which were also both differentially regulated in context to sex and aging. We found both IL-10 and IL-6 play a role in the constitutive expression of pSTAT-3 in CD5+ B-1 cells, which are known to regulate the microbiome. Additionally, significant correlations were found between cytokine expression and significantly abundant microbes. Based on these results, we conclude aging mice undergo sex-associated alterations in the gut microbiome and have a distinct cytokine profile. Further, there is significant interplay between B-1 cells and the microbiome which is influenced by aging in a sex-dependent manner. Together, these results illustrate the complex interrelationship among sex, aging, immunity, housing location, and the gut microbiome.

Age-related changes in antigen-specific natural antibodies are influenced by sex.

Introduction: Natural antibody (NAb) derived from CD5+ B-1 cells maintains tissue homeostasis, controls inflammation, aids in establishing long-term protective responses against pathogens, and provides immediate protection from infection. CD5+ B-1 cell NAbs recognize evolutionarily fixed epitopes, such as phosphatidylcholine (PtC), found on bacteria and senescent red blood cells. Anti-PtC antibodies are essential in protection against bacterial sepsis. CD5+ B-1 cell-derived NAbs have a unique germline-like structure that lacks N-additions, a feature critical for providing protection against infection. Previously, we demonstrated the repertoire and germline status of PtC+CD5+ B-1 cell IgM obtained from male mice changes with age depending on the anatomical location of the B-1 cells. More recently, we demonstrated serum antibody from aged female mice maintains protection against pneumococcal infection, whereas serum antibody from male mice does not provide protection. Results: Here, we show that aged female mice have significantly more splenic PtC+CD5+ B-1 cells and more PtC specific serum IgM than aged male mice. Furthermore, we find both age and biological sex related repertoire differences when comparing B cell receptor (BCR) sequencing results of PtC+CD5+ B-1 cells. While BCR germline status of PtC+CD5+ B-1 cells from aged male and female mice is similar in the peritoneal cavity, it differs significantly in the spleen, where aged females retain germline configuration and aged males do not. Nucleic acid sensing toll-like receptors are critical in the maintenance of PtC+ B-1 cells; therefore, to begin to understand the mechanism of differences observed between the male and female PtC+CD5+ B-1 cell repertoire, we analyzed levels of cell-free nucleic acids and found increases in aged females. Conclusion: Our results suggest the antigenic milieu differs between aged males and females, leading to differential selection of antigen-specific B-1 cells over time. Further elucidation of how biological sex differences influence the maintenance of B-1 cells within the aging environment will be essential to understand sex and age-related disparities in the susceptibility to bacterial infection and will aid in the development of more effective vaccination and/or therapeutic strategies specific for males and females.

Stimulation of α7 nAChR leads to regeneration of damaged neurons in adult mammalian retinal disease models.

The adult mammal lacks the ability to regenerate neurons lost to retinal damage or disease in a meaningful capacity. However, previous studies from this laboratory have demonstrated that PNU-282987, an α7 nicotinic acetylcholine receptor agonist, elicits a robust neurogenic response in the adult murine retina. With eye drop application of PNU-282987, Müller glia cells re-enter the cell cycle and produce progenitor-like cells that can differentiate into various types of retinal neurons. In this study, we analyzed the regenerative capability of PNU-282987 in two retinal disease models and identified the source of newly regenerated neurons. Wild-type mice and mice with a transgenic Müller-glia lineage tracer were manipulated to mimic loss of retinal cells associated with glaucoma or photoreceptor degeneration. Following treatment with PNU-282987, the regenerative response of retinal neurons was quantified and characterized. After onset of photoreceptor degeneration, PNU-282987 was able to successfully regenerate both rod and cone photoreceptors. Quantification of this response demonstrated significant regeneration, restoring photoreceptors to near wild-type density. In mice that had glaucoma-like conditions induced, PNU-282987 treatment led to a significant increase in retinal ganglion cells. Retrograde labeling of optic nerve axon fibers demonstrated that newly regenerated axons projected into the optic nerve. Lineage tracing analysis demonstrated that these new neurons were derived from Müller glia. These results demonstrate that PNU-282987 can induce retinal regeneration in adult mice following onset of retinal damage. The ability of PNU-282987 to regenerate retinal neurons in a robust manner offers a new direction for developing novel and potentially transformative treatments to combat neurodegenerative disease.

Involvement of HB-EGF/Ascl1/Lin28a Genes in Dedifferentiation of Adult Mammalian Müller Glia.

Previous studies from this lab have determined that dedifferentiation of Müller glia occurs after eye drop application of an α7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, to the adult rodent eye. PNU-282987 acts on α7 nAChRs on retinal pigment epithelial cells to stimulate production of Müller-derived progenitor cells (MDPCs) and ultimately lead to neurogenesis. This current study was designed to test the hypothesis that the activation of genes involved in the HB-EGF/Ascl1/Lin28a signaling pathway in Müller glia leads to the genesis of MDPCs. RNA-seq was performed on a Müller glial cell line (rMC-1) following contact with supernatant collected from a retinal pigment epithelial (RPE) cell line treated with PNU-282987. Differentially regulated genes were compared with published literature of Müller glia dedifferentiation that occurs in lower vertebrate regeneration and early mammalian development. HB-EGF was significantly up-regulated by 8 h and expression increased through 12 h. By 48 h, up-regulation of Ascl1 and Lin28a was observed, two genes known to be rapidly induced in dedifferentiating zebrafish Müller glia. Up-regulation of other genes known to be involved in mammalian development and zebrafish regeneration were also observed, as well as down-regulation of some factors necessary for Müller glia cell identity. RNA-seq results were verified using qRT-PCR. Using immunocytochemistry, the presence of markers associated with MDCP identity, Otx2, Nestin, and Vsx2, were found to be expressed in the 48 h treatment group cultures. This study is novel in its demonstration that Müller glia in adult rodents can be induced into regenerative activity by stimulating genes involved in the HB-EGF/Ascl1/Lin28a pathway that leads to MDPCs after introducing conditioned media from PNU-282987 treated RPE. This study furthers our understanding of the mechanism by which Müller glia dedifferentiate in response to PNU-282987 in the adult mammalian retina.