United States

Background
Our group has shown that high density mesenchymal stromal cell (MSC) sheet implantation improves bone healing in a critical-sized bone defect model; however, these cells exhibit increased aging and cellular senescence in prolonged sheet culture, diminishing their therapeutic potential. Promisingly, rapamycin has emerged as a potential agent to rejuvenate aged MSCs. This study aimed to confirm rapamycin’s ability to prevent senescence and isolate specific pathways/genes responsible.

Methods
hPMSCs were seeded at high density (65,000 cell/cm2) and treated with either rapamycin (10 nM) or control vehicle (DMSO) for up to 7 days. Senescence and autophagy were assessed by senescence-associated 𝛽-Galactosidase (SA 𝛽-Gal) staining and DAPI nuclear staining, respectively. Additionally, cell culture media was extracted at different time points to assess senescence-associated cytokines IL-6 and IL-8 via ELISA. Finally, total RNA was extracted for differential gene analysis via GeneChip Clariom S Human Arrays.

Results 
Rapamycin significantly decreased SA-B Gal staining at all time points, with 67.7% and 72.9% less area when compared to control at day 4 and 7, respectively. Senescent-associated cytokine IL-6 levels were significantly reduced when compared to control, with 614.3 pg/ml and 286.6 pg/ml decreases at day 2 and 4 of treatment, respectively. Interestingly, IL-8 expression had increased significantly at all time points, with a 979.9 pg/ml difference at day 4 of treatment. Autophagy associated vacuoles-stained area had a 3.94-fold increase at day 4 of treatment compared to vehicle. Microarray gene analysis identified a total of 429 genes with a 2-fold or greater change in expression following treatment. FoxO, cellular senescence, and cell cycle pathways are some of many effected with significantly altered expression of FBXO32, PSAT1, CXCL8, and MYC, acting as potential players in rapamycin’s anti-aging effects.

Conclusion 
Rapamycin has proven effective in prolonging pMSC lifespan. The speculation and understanding of genes enacting this function may aid in isolating this specific role for usage in prolonging sheet cultures to enhance bioengineering applications. Numerous pathways identified in this study may explain rapamycin’s anti-aging functionality and lead to the creation of therapeutics specifically targeting relevant genes. The presence of enhanced IL-8 may prove beneficial in preserving stem cell functionality, specifically for wound healing applications. These findings prove rapamycin is a candidate for improving stem cell preparation and support previous studies indicating IL-8 may enhance stem cell viability, results useful in advancing stem cell therapy usage for bone remodeling/bioengineering.


2023 AMA Research Challenge

Rapamycin Prevents Senescence and Functional Decline of High-Density Placental Mesenchymal Stem Cells

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Background
Osteoporosis is one of the major causes of fractures in adults. As of 2004, it was estimated that approximately 10 million Americans >50 years have osteoporosis with annual cost of $25 billion. Osteoporosis can lead to many fragility fractures, especially hip fractures. Hip fractures are often associated with high levels of mortality. The cumulative mortality that is associated with one-year post hip fracture is between 20-40% with an additional risk of refracture. Although there are non-pharmacologic and pharmacologic treatment options available for osteoporosis, their use post hip fracture is low. Of the patients who present with fragility fractures, only 9-20% of the patients are treated for osteoporosis, increasing the chance of re-fracture in untreated patients.

Methods
A literature search was performed using PubMed and the following key words: “Osteoporosis” and “Hip fractures” in Title/Abstract. Initial search led to 3345 results. Studies on only humans were included. Literature after 2013 was included. Only Clinical Trials, Meta-Analyses, Randomized Controlled Trials, Reviews, Systematic Reviews were included. After an extensive two-tier review process, 14 articles are included in this review. 

Results
Fracture Liaison Service (FLS) and Orthogeriatric Service (OGS) are multidisciplinary team approaches that provide care for patients post-fragility fractures. FLS is shown to increase the rates of DEXA evaluations, improved prescription initiation rates for osteoporosis medications, and reduction in re-fracture rates. When comparing the effectiveness of FLS at tertiary hospitals, the hospital with FLS approximately 30% reduction in any refractures and 40% reduction in new major fractures over a three-year period. FLS is shown to be cost-effective as it prevented more fractures and increased quality-adjusted life years (QALYs). OGS is also shown to decrease length of stay at the hospital, lower risk of in-hospital mortality, and delirium. In addition, patients receiving OGS had improved mobility at 12 months post fracture when compared to usual orthopedic care. Implementation of FLS and OGS also shows decrease in mortality within 30 days and within one year in patients with hip fracture.

Conclusion
The introduction of FLS and OGS models changed the field of post fracture osteoporosis management. Initiation of pharmacological and nonpharmacological treatments post fracture has the potential to improve bone health, decrease re-fracture rates, decrease mortality and morbidity, and improve quality of life. The costs of follow up medical care have been shown to be reduced these services These models have been effective at providing the care that fragility fracture patients need.

The Importance of Fracture-Liaison Services and Orthogeriatric Services for Osteoporotic fractures

Background
Cartilage damage is challenging to treat due to its limited self-healing capacity caused by its avascular nature. However, 3D tissue engineering approaches that utilize biomaterials, additives, and cells offer a promising solution to regenerate functional cartilage. These innovative techniques have the potential to restore native cartilage properties, prevent the development of osteoarthritis, and restore joint function. In this study, we investigate a novel alginate-based 3D bioink composed of human articular cartilage for cartilage tissue engineering.

Methods
The bioink was developed by encapsulating C20A4 human articular chondrocytes in a mixture consisting of 20% medium viscosity sodium alginate (Alg), 5% polyvinyl alcohol (PVA), 5% Gum Arabic (GA), and 5% human articular cartilage. The human articular cartilage was obtained from cartilage shavings of distal femurs and ground into a powder for incorporation into the bioink. The bioink was printed into concentric discs using a BIO-X 3D Bioprinter, and the resulting 3D-biofabricated constructs were cross-linked with calcium chloride, rinsed, and cultured in chondrogenic media. Thirty-six total constructs were fabricated. Immediately after 3D printing, a portion of constructs were visualized with SEM (n=1) and underwent compression testing (n=5) to examine the microstructure and mechanical strength respectively. The remaining constructs were placed in culture for 1,7 or 14 days and subsequently analyzed for cellular viability (n=5) and gene expression (n=5) at each time point.

Results
SEM imaging revealed that the 3D-printed constructs exhibited a porous structure. The average elastic modulus of the constructs was 0.48 MPa. Post-printing, the cellular viability was 87.2% which decreased significantly to 76.4% at day 7 (p<0.001) but recovered to 85.9% at day 14 in culture. Furthermore, gene expression analysis showed a significant increase in transcriptional activity of chondrogenic genes Col2a1 (p=0.008) and Sox9 (p=0.021) at day 14 compared to day 1.

Conclusion
This study explored the use of an innovative alginate-based 3D bioink composed of human articular cartilage for cartilage tissue engineering. The bioink successfully formed porous 3D constructs that withstood the printing process. Additionally, the elastic modulus of these engineered constructs approached that of native cartilage (0.45-0.8MPa). Although cellular viability initially decreased, it gradually recovered over a two-week culture period. Moreover, the upregulation of chondrogenic genes indicates the potential of the bioink to promote cartilage regeneration. These findings suggest this bioink holds promise as an effective approach for cartilage tissue engineering, offering possibilities for future therapeutic interventions in cartilage repair and regeneration.

Novel 3D Extrusion Bioink Containing Processed Human Articular Cartilage for Application in Cartilage Tissue Engineering

Cerebrospinal fluid (CSF) homeostasis in the brain relies on a delicate balance in CSF production, circulation, and excretion. When this balance is compromised, patients develop a spectrum of disorders, including hydrocephalus and neurodegenerative diseases. Emerging studies suggest that the lymphatic system, referred to as the glymphatic system in the brain, may have a previously unknown role in CSF homeostasis. Interestingly, it is unknown whether the glymphatic system has any role in embryonic CSF regulation. Frog Xenopus has been widely used to study fundamental neurodevelopmental processes and is a unique model to investigate CSF dynamics. In Xenopus, unlike any other model system, whole brain CSF circulation can be visualized in real-time and manipulated easily, providing unprecedented insight into CSF hemostasis. In this work, we asked whether frogs have a glymphatic system and its role in CSF regulation. Using the frog Xenopus model system, we show that the glymphatic system is essential in CSF drainage during embryonic brain development. We used post-fertilization day 4-Xenopus tadpoles, where we injected conjugated ovalbumin into the brain ventricle and simultaneously into the tadpole's heart. Then we used confocal imaging to visualize CSF circulation and systemic cardiac circulation in parallel. We showed that there was no overlap between the two circulations. However, when we disrupted lymphatic formation, we showed that CSF excretion was disrupted, suggesting that Xenopus CSF excretion relies on the glymphatic system rather than cardiac circulation. In further studies, we will look at how CSF excretion is conserved among species and relate to our patients with hydrocephalus, Alzheimer’s, and other neurodegenerative diseases where abnormal CSF excretion is critical for disease progression.

The glymphatic system in Xenopus tropicalis – a model system for Congenital Hydrocephalus

Background
Migraines represent one of the leading causes of disability worldwide. While the pathophysiology of migraine pain is complex, the sensitization of the trigeminovascular pathway is implicated. Osteopathic manipulative treatment (OMT) has been used clinically to supplement pharmacologic treatment for patients suffering from migraines, but wide-spread adoption of these treatments has been limited by a lack of physiological and mechanistic evidence supporting its application. We aim to determine if OMT is an effective method of treating the pain associated with an acute migraine, abortively or prophylactically, and investigate its potential mechanisms of action.

Methods
Rats were randomly divided into treatment groups and assessed by behavioral testing and immunohistochemical (IHC) staining. Migraines were induced by a two-hit method. Animals were primed with Complete Freund’s Adjuvant (CFA) via injection and were exposed to umbellulone, a human migraine trigger, via inhalation on day 6. The presence of cutaneous allodynia, indicating a sensitized pain response, at the periorbital and hindpaw areas was determined using von Frey filaments. Subjects receiving OMT were given 2 osteopathic treatments for 1 minute each either before inhalation (prophylactic) or after inhalation of umbellulone (abortive). For the IHC studies, subjects were sacrificed and brain tissue was collected. The trigeminal nucleus caudalis (TNC) and the trigeminal ganglion (TG) were analyzed to determine the activation of the second-order sensory neurons by measuring expression of calcitonin gene-related peptide (CGRP), a neuropeptide implicated in migraine pathophysiology, and c-fos, an immediate early response gene. The experimenter was blinded for the treatment groups.

Results
Our behavioral testing showed that CFA + umbellulone induced significant allodynia. OMT employed as an abortive measure partially blocked the development of cutaneous allodynia. Multiple prophylactic episodes of OMT prevented the development of cutaneous allodynia. IHC analysis showed a significant increase of c-fos expression in TNC samples from subjects treated with CFA + umbellulone, which was blocked by prophylactic OMT. The expression of CGRP positive neurons in the TG samples increased significantly in subjects treated with CFA + umbellulone, which was normalized by prophylactic OMT.

Conclusion
Two osteopathic treatments demonstrated effectiveness in mitigating migraine pain as both a prophylactic and abortive measure. These findings provide insight into the efficacy of OMT for migraines and support its potential as a non-pharmacological approach for migraine management. Future studies will assess other forms of OMT as well as treatment directed to other regions associated with the trigeminovascular pathway.

OMT alleviated migraine-like pain via blockade of trigeminal activation

Background
Ex-vivo perfusion (EVP) is an emerging technology used to limit irreversible tissue damage and reduce failure in organ transplantation. Assessment of the viability of vascularized-composite allografts (VCA) during EVP is challenging because of lack of organ-specific viability parameters.
Infrared thermography (IRT) provides a non-invasive method of measuring skin temperature. We hypothesize that skin temperature changes are correlated with edema formation and weight gain during perfusion and can be used to assess VCA viability.
Methods
Eleven superior epigastric artery perforator flaps were procured from Yorkshire pigs and preserved by EVP utilizing a normothermic colloid perfusate with a hemoglobin-based oxygenated carrier (HBOC)-201 (HbO2 Therapeutics, Souderton, PA) for 12 and 24 hours. IRT images were recorded hourly (Fluke TiS Thermal Imager, Everett, WA) and mean skin temperature and percent temperature changes were analyzed (SmartView3.4 Software, Everett, WA). Skin biopsies were collected every 6 hours and analyzed after H&E staining. Indocyanine Green (ICG) angiography (Stryker SPY Elite System, Munich, Germany) was performed before pedicle division and at perfusion endpoint to assess peripheral perfusion. Median pixel brightness (MPB) was measured (ImageJ, Bethesda, MD) and normalized to account for variations in recording conditions. Flap weight and temperature changes from baseline were analyzed with paired T-tests. Correlation of flap temperature changes and MPB were with endpoint weight change (edema formation) was assessed using Pearson correlation tests.
Results
Flap weight did not change significantly from baseline to either 12 hours of EVP (TP0=188+/-5 g, TP12=198+/-5 g, p=0.6763) or to 24 hours (TP24=195+/-5 g, p=0.6412). Median percent flap weight change from baseline was determined: 12hr EVP -1.12% [-4.54-0.93%] versus 24hr EVP 2.0% [0-3.01%]. There was no significant difference between flap weight at 12 hours versus 24 hours (p=0.905). Normalized MPB at endpoint was not significantly different after 12hr EVP (0.47 [0.38-0.57]) compared to 24 hr EVP: 0.82 [0.7-094] (p=0.09524). Skin temperature was not significantly different at 12 hours (28.09°C, [27.56-29.1]) versus 24 hours (28.1°C, [26.9-29]) (p=0.4923). H&E staining of flaps showed preserved skin architecture compared to baseline. Skin temperature change significantly correlated with percent weight change (r=-0.7399, p=0.009) and and with MPB( r= -0.8335, p=0.019).
Conclusion
Flap skin temperature during EVP correlates with flap edema. This finding suggests that a decline in temperature could serve as a non-invasive real time indicator of flap injury during EVP.

Infrared Thermography as a Viability Measure During  Ex Vivo Preservation of Vascularized Composite Allografts

Background:
Psychiatric disorders, including anxiety, depression, schizophrenia, and substance use disorders, impose a significant burden on global public health. Conventional treatments targeting dopamine receptors often have limitations in terms of efficacy and tolerability. Therefore, there is an urgent need to explore and discover new active molecules that can effectively modulate dopamine receptors with improved therapeutic properties. Cannabidiol (CBD) and CBD-like molecules have emerged as potential candidates due to their diverse pharmacological effects and favorable safety profiles.


Methods:
A comprehensive review of existing literature, encompassing Computational Chemical Approach (in silico), was conducted to elucidate the mechanisms of action and biological effects of CBD on dopamine receptors. for this goal, an in silico approximation was carried out. Basically, Several CBD and CBD-like were docked against Dopamine receptors and compared with dopamine molecules. In order to estimate the dopamine receptor-ligands stability, molecular dynamics simulation was applied. Then molecular analyses were employed to explore the impact of CBD and its analogues on dopamine receptor signaling and endocannabinoid system regulation and to suggest new potential ligands.

Results:
The Findings from literature indicate that CBD and CBD- Like molecules modulates dopamine receptor activity and neurotransmission, suggesting potential therapeutic effects on psychiatric disorders associated with dopamine dysregulation. Additionally, CBD and CBD- like molecules interacts with the endocannabinoid system, specifically targeting cannabinoid receptors and influencing endocannabinoid signaling pathways involved in mood regulation, stress response, and neuroinflammation.The in silico results allow to suggest new molecules with dopamine receptor activity potential. 

Conclusion:

This comprehensive study highlights the therapeutic potential of CBD and CBD- Like molecules on dopamine receptors. The modulation of dopamine receptor activity and signaling pathways may contribute to a beneficial effect. The investigation by using computational chemical approach (in silico) enhances our understanding of the binding modes and interactions between these compounds and dopamine receptors. Continued research in this area may lead to the discovery of new active molecules with enhanced therapeutic efficacy and improved treatment strategies for individuals with psychiatric disorders


Exploring the Action Mechanisms of Cannabidiol and Cannabidiol-Like Molecules on Dopamine Receptors: A Computational Chemical Approach.

Background: 
With more than half of U.S. adults reporting alcohol consumption the last 30 days and nearly 6% reporting heavy drinking, alcohol imposes a significant socioeconomic burden. While there are a host of current medications approved to treat alcoholism, they have limited effectiveness and lack support from both providers and patients. Nevertheless, new medications are slow in coming. Semaglutide, a medication originally developed for obese type 2 diabetics, may offer a distinctly new approach. 
Methods: 
A survey to assess the impact of semaglutide on alcohol consumption and desire was designed and distributed in semaglutide-user Facebook and Reddit groups and frequencies and proportions were calculated.
Results: 
Of those who had reported drinking before starting semaglutide, 21% of respondents (N= 121) ceased drinking altogether, and 88·4% of respondents (N=538) reported a reduction in their desire to consume alcohol after commencing their medication. Respondents also shared further personal accounts of decreased occurrences of compulsive or "addictive" behaviors beyond a reduction in the desire to consume alcohol.
Conclusions: 
Considering the growing number of reports detailing experiences such as these, combined with our survey results indicating a reduction in both frequency and quantity of alcohol consumption, we strongly recommend conducting scientifically rigorous investigations and clinical trials to explore the potential of semaglutide for addressing conditions associated with alcohol use. 


Semaglutide: A Promising Treatment for Alcohol Use Disorder

Pneumococcal pneumonia caused by Streptococcus pneumoniae (Spn) remains one of the main causes of morbidity and mortality worldwide in children and adults and is a leading pathogen of meningitis and sepsis in children. Laboratory tests to diagnose pneumococcal disease (PD) can be time consuming. Real-time PCR methods can deliver results within 3-4 hours, but with sample processing and DNA extraction, final results may take eight hours. The goal of this study was to evaluate the sensitivity and specificity of the antibodies to identify Spn strains.
We utilized nasopharyngeal (NP) specimens in which Spn+ (N=19) were identified by lytA-based qPCR, isolated by culture, and serotype obtained by Quellung reactions. To confirm specificity, lytA-negative NPs [Spn- (N=3)] were analyzed. NPs were DNA extracted, and serotype-specific qPCR reactions obtained bacterial load. An aliquot of the NP specimens was stained with serotype-specific antibodies labeled with Alexa-488 or type-specific Anti-S4. Stained NP and clinical specimens were mounted on slides and observed using fluorescence microscope.
Serotype-specific qPCR assays quantified bacterial load in Spn+ NP specimens ranging from 1.37x108 to 6.71x105 cfu/ml. The microbiota was observed in Spn+ and Spn- specimens. In most Spn+ specimens (N=19) the anti-Spn antibodies detected individual cells, diplococci and/or pneumococcal chains. Semi-quantitative detection by fluorescence microscopy (ie, +, ++, +++, or ++++) correlated with bacterial load obtained by qPCR. Identification of Spn, in Spn+ specimens, was completed within thirty minutes. Spn signal via fluorescence was not observed in nasopharyngeal homogenates from uninfected specimens (Spn-). 
Of the clinical specimens (N=23) obtained from either culture swab or endotracheal tube, fluorescence using the Alexa-488 antibody only was observed within thirty minutes in 17 samples total. Spn fluorescence was not observed in six samples that were suspended in a charcoal medium.
The goal of this study was to evaluate the sensitivity and specificity of antibodies to identify Spn strains. We evaluated fluorescent antibodies using in vitro studies with isolated strains and in vitro cultured lung cells infected with Spn to assess samples including nasopharyngeal swabs from healthy children, lung specimens Spn-infected mice, and clinical specimens from patients with PD. Identification with Spn was highly specific and sensitive, and detection using fluorescence microscopy occurred in thirty minutes. This study validates a conceptual model that can identify, within minutes, Spn strains in clinical specimens using fluorescence microscopy. Rapid and accurate identification of respiratory pathogens has the potential to save lives and improve antibiotic stewardship. 


Rapid, specific, and highly sensitive detection of S. pneumoniae directly in clinical specimens using a fluorescence-based method

Background
Bladder Cancer is the 6th most common and 8th most deadly cancer in the United States. It is pathologically staged as pTa (non-invasive), pT1 (invasion of lamina propria), or pT2 (muscle invasive disease). Treatment for pT1 disease is transurethral resection of the bladder tumor (TURBT), while treatment for pT2 disease is a radical cystectomy. Given the distinct treatments and implications on quality of life, differentiating between the two disease states is highly advantageous. However, it is challenging to determine if T1 will progress to T2 disease. As such, a prognostic test, ideally from urine samples, in the high-risk patient could aid clinical decision making.

Methods
We have collected over 800 urine samples from patients with bladder cancer dating back to 2013. These specimens include samples from throughout the clinical course and are matched to each patient's demographics and clinical history. The urine samples are fixed in methanol, and these methanolic extracts are traditionally discarded. We developed a method to use these extracts to run liquid chronography–mass spectroscopy and profile the sample’s metabolome. We have termed this process CytabolomicsⒸ. Ultimately, we aim to discover urine biomarkers to aid in predicting whether T1 disease is likely to progress.

Results
There are a total of 264 patients in the cohort. The patients were followed for a median of 7.6 years. There are 72 patients staged pTa, 85 staged pT1, and 74 staged pT2. The remainder of the patients are staged pTis, pT3, or pT4. The wide distribution of patients with pT1 and pT2 will allow for ample comparison of urine samples from each stage of disease.
As of the writing of this abstract, we have prospectively collected sample metabolomic data from 20 bladder cancer patients’ urines as a test run. This is encouraging for the future of the study because it demonstrates CytabolomicsⒸ is a suitable method for studying urine samples. It is also an exciting new development in the study of preserved tissue samples, as it allows for additional data to be collected and analyzed from what is usually discarded.

Conclusion
Bladder cancer is a common cancer in the United States, yet there is a gap in knowledge about how to identify stable versus progressive disease. The demographic data from the cohort of bladder cancer patients as well as the novel method used to study the corresponding urine samples provides a promising opportunity in furthering our knowledge regarding this disease.

Employing a Novel Research Method To Investigate Urine Biomarkers  

Background: Nocturia, or frequent night-time urination, significantly impacts patients’ sleep, quality of life, and health. Both age and biological sex contribute to nocturia. The water-regulatory hormone arginine vasopressin (AVP) controls urine production. AVP binds to arginine vasopressin receptor 2 (AVPR), a G-protein-coupled receptor, in various organs, including the kidney and bladder. Desmopressin, an AVPR2-specific agonist, is a mainstay treatment for nocturia, but its mechanism of action in the bladder is not fully understood. A better understanding of vasopressin signaling in the bladder is crucial for refining treatment options. The fruit bat Carollia perspicillata have CNS anatomy more closely resembling humans and primates and can be kept in captivity for well over ten years, making it an attractive translational model to study aging, including nocturia. We aim (1) to identify commercial antibodies recognizing AVPRs in a novel animal model for nocturia, the fruit bat C. perspicillata (2) to develop an assay to precisely quantify arginine vasopressin receptor levels in bat bladder.

Methods: Total protein was isolated from mouse and bat kidney, liver, and bladder by homogenization in RIPA buffer with a homemade bead mill and centrifugation. 10 µg protein each, as measured by BCA assay, was electrophoresed with size standards in gels and electroblotted onto membranes. AVP1aR and AVP2R proteins were detected with commercial 1° antibodies followed by fluorescent 2° antibodies, while total protein was detected with Fast Green. Fluorescence was measured by scanning (Odyssey M, Li-Cor) and quantitated with Fiji/ImageJ software.
The mouse bladder was dissected, pinned flat and fixed overnight in a 4% w/v formaldehyde buffer. The sample was permeabilized with a blocking solution for a few hours. The same sample was then incubated with 1° commercial antibody AVP1aR for 12hrs and washed in a solution. Next the sample was incubated with 2° immunofluorescent antibody and washed again. The tissue sample was mounted and visualized with confocal microscope.

Results: AVPR2 were detected in the kidney and liver of both mouse and bat at the same expected apparent molecular masses of ~44 kDa. In the bladder of both species, the predominant apparent molecular masses were ~80 kDa, indicating dimerization, a common phenomenon with G-protein-coupled receptors. Relative protein levels were similar in the kidney and liver.
AVPR1A proteins were detected throughout the detrusor muscle of the mouse using confocal microscopy at 20x magnification.

Discussions/Conclusions: Commercial antibodies against human and rodent AVPRs recognize the orthologous proteins from the fruit bat C. perspicillata. The sizes of AVPR1A and AVPR2 proteins observed for bats match those for mice and those reported in the literature, indicating the specificity of the antibodies. AVPR1A and AVPR2 are expressed in bat kidney and bladder, the organs responsible for urine production, and are known in humans to be involved in nocturia. The fruit bat, C. perspicillata, is thus a tractable model for studies of vasopressin signaling in nocturia. Our next step will be to determine the cellular location of AVPRs and to measure their levels in the kidney and bladder of young and old bats.

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