United States

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.

AMA Research Challenge 2023

Vasopressin Receptors in Bat: A Novel Animal Model for Nocturia

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Background
Parkinson’s disease (PD) involves the accumulation of alpha-synuclein (αS) in Lewy bodies in the substantia nigra of the brain. αS consists of an N-terminal 100-residue membrane-binding region and a 40-residue C-terminal tail. Molecular progression of PD throughout the brain is thought to occur as αS forms toxic aggregate species that are released from neurons and subsequently internalized by surrounding neurons to further seed aggregation of endogenous protein. Although the mechanism by which neurons internalize extracellular αS is unclear, one protein that is of interest as a putative receptor for αS is the presynaptic protein neurexin 1-beta (N1β). Previous work has shown that N1β drives cellular internalization of αS in a manner dependent on αS N-terminal acetylation (NTA); N-terminally acetylated αS (αSacetyl), the primary form of the protein in vivo, exhibits increased alpha-helical structure at its N-terminus compared to unmodified αS (αSunmod). Thus, αS appears to interact with N1β on neuronal membranes in an NTA-dependent manner, leading to neuronal uptake of extracellular αS and contributing to its intercellular propagation in PD. The objective of this study is to characterize αSacetyl-N1β binding with residue-specific resolution using nuclear magnetic resonance (NMR) spectroscopy. We hypothesized that this interaction primarily involves the N-terminal 10 residues of αS due to their known alpha-helical propensity with NTA. Mapping the αSacetyl-N1β interaction will offer insight into the specific regions of αS involved in its recognition and neuronal internalization, as well as inform potential therapeutic targets for inhibition of internalization and slowing of PD progression.

Methods
Established bacterial transformation, growth, and purification methods were used to generate αS samples. Heteronuclear single quantum coherence (HSQC) NMR studies were performed to generate chemical shift peaks corresponding to the local chemical environments of each residue for 15N-labeled αSunmod and αSacetyl either in the presence or absence of N1β. Changes in αS NMR peak position and/or intensity in the presence of N1β demonstrate involvement of the corresponding αS residues in αS-N1β binding.

Results
HSQC NMR studies demonstrated reduced NMR peak intensity in the N-terminal 100 residues of αSacetyl in the presence of N1β, with a more pronounced reduction in the first 11 residues.

Conclusion
αS interacts with N1β most strongly at its first 11 residues in an NTA-dependent manner, consistent with our hypothesis. However, αSacetyl also interacts with N1β across its 100-residue membrane-binding domain, suggesting that this entire region contributes to N1β-mediated αSacetyl internalization.

Structural characterization of the interaction between alpha-synuclein and neurexin-1 beta in the context of pathologic alpha-synuclein propagation in Parkinson's Disease

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**Abstract:**



Intracellular Signaling Pathways of TLR2/1 and TLR2/6 Initiated Reactive Oxygen Species Production in Neutrophils

SARS-CoV-2 is a contagious virus that causes COVID-19, a disease that has affected millions of people worldwide and claimed over six million lives. The pandemic has unveiled an urgent need for new antivirals to control emerging infectious diseases and potential future pandemics. Classic antivirals are currently designed to directly interfere with pathogens. However, antivirals are often insufficient to rapidly clear infections in the absence of an effective immune response. Immunotherapy could complement the use of antivirals; however, its application to infectious diseases remains largely unexplored. 
Adenosine analogs are antiviral drugs which resemble the structure of adenosine, a metabolite that derives from the breakdown of ATP and can suppress the immune response through activation of the Adenosine A2A Receptor (A2AR) in immune cells. Adenosine is overproduced in some infectious diseases, like COVID-19, where it restricts protective antiviral immune responses. Previous work from our lab showed that adenosine-mediated activation of A2AR causes suppression of CD8+ T-cells effector functions in COVID-19 mouse models. Importantly, our group showed that adenosine analogs could restore antiviral T-cell responses in COVID-19 models, through blocking the A2AR. Therefore, we propose that adenosine analogs that interact with A2AR may be endowed with dual – immunomodulatory and intrinsic - antiviral functions.
In this study, we used a drug discovery technique, Molecular Docking, to investigate possible adenosine analogs that could be used as antiviral and immunomodulatory dual agents. We screened adenosine analogs, known or predicted to inhibit SARS-CoV-2 replication, to test if they may bind to A2AR. Eight adenosine analogs were chosen and docked to the A2AR. Out of the ten analogs, three - forodesine, riboprine, and 8-chloroadenosine - were predicted by the docking modeling to bind with high affinity to the A2AR. We then tested whether forodesine could block A2AR activation. To investigate this, we treated CD8+ T cells from mouse spleen and lymph nodes with forodesine or CGS-21680 (CGS), an A2AR agonist, or a combination of the two. Then, we measured the proliferation of T-cells using flow cytometry. We found that forodesine treatment restored T-cell proliferation from the A2AR agonist CGS in CD8+ T-cells, suggesting that forodesine blocks A2AR. Our results indicate that adenosine analogs, like forodesine, may be endowed with immunomodulatory properties through A2AR, in addition to their intrinsic antiviral properties. In conclusion, adenosine analogs, like forodesine, could be used as novel dual – immunomodulatory and intrinsic – antiviral drugs. These compounds could represent game-changing therapies, not only to control COVID-19, but also other emerging viral diseases and future pandemics.


Adenosine analogs have a dual immunomodulatory and antiviral function and are potential therapeutic agents against SARS-CoV-2

Background: Exosomes are membrane-bound nanovesicles known to be secreted by various cells, including pancreatic ductal adenocarcinoma cells (PDAC). In the context of pancreatic cancer, exosomes are thought to play important roles in cellular communication during metastasis, structuring the tumor microenvironment, and paraneoplastic disorders. Paraneoplastic disorders are clinical symptoms based on an altered immune response or aberrant signaling associated with occult malignancy without metastatic dissemination. Currently, the paraneoplastic disorder most associated with pancreatic cancer includes neuropathic pain, but investigations into brain involvement have yet to be done despite recent evidence of PDAC-induced neuroinflammation underlying cachexia in PDAC models. Using genetically engineered mouse models where pancreatic precancerous (PanIN) and cancerous (PDAC) lesions express mCherry tagged exosomes, we have observed the interesting phenomenon of pancreatic cancer derived exosomes coalescing amongst neurons. From these findings, we hypothesize that pancreatic cancer exosomes from PanIN and PDAC lesions contribute to paraneoplastic neurological syndrome. 

Methods: To investigate this hypothesis, we have used a genetically engineered mouse model of spontaneous pancreatic ductal adenocarcinoma (PDA) where only PDA cells express cytoplasmic YFP, and exosomes (which express CD9) derived from these cells are tagged with mCherry. To answer the question of which neuronal populations have positive mCherry-CD9 signaling we performed immunofluorescence with NeuN, Calbindin, Map2, GAD65, and DCX. If we observed certain populations that appear to have a strong exosome coalescence, we selected those for further quantification. We quantified the percentage of neuronal cells with exosome coalescence with mCherry-CD9 signaling between normal, pre-disease (before PanIN lesion development), PanIN, and PDAC. We counted 30 cells within selected regions of each mouse with 2 mice per group and performed statistical analysis to observe potential differences between the mean of each group.

Results: Interestingly, we saw widespread exosome coalescence amongst different neuronal populations and brain regions. Purkinje and hippocampal neurons had an increased density of mCherry-CD9 tagged exosomes thus were selected for quantification. Preliminary results displayed a significant difference between the mean of normal (p<0.0025), pre disease(p<0.0006), and PanIN (p<0.0020) groups versus PDAC. 

Conclusions: The current results demonstrate that pancreas-derived exosomes not only enter the brain but appear to coalesce amongst Purkinje and Hippocampal neurons. In PDAC staged mice, a higher percentage of neurons with positive mCherry-CD9 exosomes coalescence. Future work is focused on elucidating the functional consequences of exosome travels amongst neurons to possibly understand the paraneoplastic neurological effects of pancreatic exosomes in patients.

Early-stage pancreatic cancer associated exosomes contribute to paraneoplastic neurological syndrome

Background
The ability to reduce ice growth and inhibit ice recrystallization has become a key factor in cryopreserving biological specimens, such as cells and tissues, for cell-based therapeutics like vaccines. However, current cryopreservation protocols can result in loss of cell viability due to cellular damage related to the freezing and thawing process. The use of cryoprotective agents has been effective in increasing post-thaw recoveries and functionality in appropriate models. Nevertheless, many cryoprotectant compounds demonstrate toxicity to cells, preventing their clinical application. Betaine is an organic molecule which has the potential to function as a cryoprotectant due to its physiological role in the body as an osmolyte. Therefore, the goal of this preliminary study was to observe the ability of betaine to inhibit ice recrystallization and to assess its cryopreservation properties. 

Methods
Ice recrystallization was assessed through a splat cooling assay. The betaine sample was dissolved in phosphate buffered saline at concentrations of 0.5, 2, 10, 15, 18, 20, 25, 50 mM. 10 𝜇 L of dissolved betaine was dropped from a height of 2m onto a -80 °C aluminum block forming an ice wafer which was transferred to a Peltier unit at -6.4°C for 5 minutes. The wafer was placed under a microscope and the ice crystals were photographed. Mean ice crystal size was obtained and compared to the negative control; phosphate buffered saline. A dose response curve representing the ice recrystallization inhibition activity was obtained. 

The cytotoxicity of betaine was assessed through a resazurin assay with HEPG2 liver cells. Cytotoxicity of Betaine was assessed at 0.5, 2, 10, 15, 18, 20, 25, and 50 mM. 1%- Triton-X was used as a negative control. 

Results
Dose response curves obtained from splat cooling assays indicated that betaine could reduce ice crystal growth at concentrations above 200mg/mL. However, it was unable to completely inhibit ice recrystallization. The half maximal inhibitory concentration (IC50) calculated from the dore response curves was found to be between 119.9mg/mL and 226.4mg/mL, which is not comparable to cryo-additives used in current cryoprotective protocols. In vitro cytotoxicity studies performed indicated biocompatibility up to 10mg/mL.

Conclusion
Our quantitative assessment suggests that the cryoprotective properties of betaine are not sufficient for its use as a cryoprotectant alone. Future directions include assessing the post-thaw viability of betaine and its cryoprotective abilities when combined with another cryoprotectant-like compound. 


A quantitative assessment of the cryopreservation properties of betaine

ATPase Inhibitory Factor 1 (IF1) is a nuclear-encoded ATP synthase-interacting mitochondrial protein. IF1 is known to be upregulated during periods of elevated stress, such as during myocardial infarctions or cardiac hypertrophy. Previously conducted lab research has demonstrated that mice with the absence of the protein IF1 were protected against pathological cardiac hypertrophy. To further investigate the extent of such findings, an experiment was designed to investigate the role of the IF1 protein in cardiotoxic conditions induced by Doxorubicin (DOX), a widely used anti-cancer drug. DOX is known to induce cardiotoxicity through the generation of reactive oxygen species, increased levels of lipid peroxidation, and mitochondrial dysfunction. Doxorubicin-induced cardiomyopathy is associated with a low prognosis and can serve to be fatal in certain populations. We hypothesized that mice with the absence of IF1 will demonstrate more preserved cardiac functions in the presence of DOX. 

To test the hypothesis, DOX was intraperitoneally injected into two separate groups of mice: mice for which the IF1 protein was knocked out (IF1-KO) and wildtype mice normally expressing the IF1 protein (IF1-WT). The drug was dissolved in a solvent consisting of dimethyl sulfoxide (DMSO), PEG-300, Tween-80, and distilled water in pre-designated proportions. Injections were performed on three separate days with 6 mg/kg of DOX administered on each injection day. Injections were conducted on Day 0, Day 2, and Day 4. Control mice were injected simply with solvent. Subsequently, transthoracic echocardiograms were performed using a VEVO F2 system at one-week intervals for three weeks ie. Day 7, Day 14, and Day 21. Long-axis M-Mode images were obtained to analyze systolic parameters: ejection fraction, fractional shortening, left ventricular mass, as well as left ventricle anterior and posterior wall thickness. Body mass was also measured initially and after the three-week interval. Findings demonstrated that in the presence of DOX, ejection fraction and fractional shortening, the two parameters of cardiac contraction, significantly decreased in IF1-WT mice; whereas no significant decrease was seen in IF1-KO mice. Furthermore, left ventricular anterior wall thickness was found to be significantly reduced in both IF1-WT and IF1-KO mice, suggesting that DOX may lead to a decrease in heart size. These results demonstrate that the absence of IF1 may provide protection against the cardiotoxic effects of DOX, thus supporting the hypothesis. Further studies could investigate the role of increased doses of DOX on cardiac physiology as well as the effects of increased time intervals through the performance of transthoracic echocardiograms after five to seven weeks. Future experiments using other pathophysiological and biochemical methodologies will gain further insights into the mechanisms of the protective effects of eliminating IF1. Experimental findings may yield new insights into the development of interventions to mitigate adverse effects of this drug in cancer patients under chemotherapy treatments.


Analysis of the Role of ATPase Inhibitory Factor 1 (IF1) in Doxorubicin-Induced Cardiotoxicity

Background: The larynx is important in breathing, swallowing, and speech production. The closure and opening of the space between the vocal folds (“glottis”), allows the larynx to fulfill its functions. The larynx is a symmetric organ. To move all parts of the larynx appropriately and achieve glottal sufficiency, symmetry is beneficial. Previous studies have reported on various forms of shape asymmetry and their consequence for function. Asymmetry in how the larynx is positioned within the neck has not been investigated before but could play a critical role in the development of shape asymmetry and thereby influence larynx functions. 
Methods: We investigated a stratified sample of 210 human subjects (105 females, 105 males) for positional asymmetry. Positional asymmetry was defined as the angle between the mid-sagittal organ plane and the mid-sagittal body plane. The larger the angle between the two, the greater the positional asymmetry. Positional asymmetry was measured in all subjects. With the help of regression models, the relationships between positional asymmetry and body size, larynx organ size, and larynx shape were explored. 
Results: Positional asymmetry ranged between 0 and 18°. It was larger in males (4.71°±3.05; mean±stdev) than in females (3.49°±3.11) (t-test, T105,105 = -2.85; p < 0.01). Larynx midsagittal plane was deflected either to the left or the right side. In females, the distribution was almost even (51.43%) but it was biased towards the right side in males (62.86%). Positional asymmetry was associated with larynx overall shape (females: R2= 0.05, P= 0.017; males: R2= 0.09, P= 0.002), and shape asymmetry (female: R2 = 0.059; p< 0.01, male: R2= 0.21; p<0.001), with larynx size in females (females: R2= 0.042, P= 0.036; males: R2= 0.003, P= 0.609), with age in females (females: R2 = 0.467; p= 0.027; males: R2 = 0.0232; p= 0.12), but not with body height (males: R2= 0.0082; P= 0.357 ; females: R2= 0.0061; P= 0.43) or with BMI (Body Mass Index) (female: R2 = 0.018; p= 0.169, males: R2= 0; p= 0.97). 
Conclusions: Our data highlight the value and challenges of investigating human phenotypic variation. Positional asymmetry of the larynx was associated with the shape and size variation of the vocal organ as well as with a person’s age.

Assessing Positional Asymmetry of the Larynx

The role of the triglyceride-glucose index (TyG) in obesity and its progression to Type 2 diabetes

Various growth factors have been shown to affect the proliferation, migration, and adhesion of keratinocytes in the face of wound healing. The expression of wound repair genes is suspected to be regulated by circadian rhythm. How wound repair functions over the course of a circadian rhythm has yet to be fully described. Moreover, much is unknown about the impact of bacteria on the circadian expression of wound healing genes. Here, we intend to use periodontal disease as a model to describe the effect of a septic environment on expression of wound healing genes by epithelial cells. Furthermore, we aim to demonstrate that stimulation of epithelial cells with pathogenic bacteria alters the circadian patterns of wound repair gene expression. In the experiment, OKF4 cells were seeded, and artificial wounds were created in each well. The cells were challenged with pathogenic oral bacteria, and cell lysates were collected at T=0, T=8, and T=22 hours. PCR quantified gene expression levels. Microscopically, increased cell spreading was observed after T=22 hours in the wounded cells. Impaired cell spreading was observed in the wells challenged with pathogenic bacteria. The expression of EGF and FGF-7 appeared to follow a circadian rhythm. The presence of pathogenic bacteria downregulated the expression of genes involved in keratinocyte migration. These findings support the hypothesis that genes involved in wound healing follow a circadian rhythm and are altered in the face of infection. Continued research into this topic can lead to the development of therapies targeted towards restoring the expression of genes that aid in wound repair complicated by a septic environment or overlying infection. Further elucidating the circadian pattern of expression of these genes can also result in optimization of the timing that therapies are administered to maximize their therapeutic effect thereby improving patient outcomes. 


Altered Wound Healing Response after Challenge with Pathogenic Bacterium

Background: Neuroinflammation has been implicated in the development and progression of numerous neurodegenerative disorders and studies suggest systemic inflammation may precede central inflammation. Chronic low-grade systemic inflammation is a hallmark of metabolic disorders like Type II diabetes mellitus (T2DM), which is a risk factor for cognitive dysfunction. In fact, researchers have proposed the term “Type III diabetes” for Alzheimer’s disease due to the shared molecular and cellular mechanisms among diabetes and cognitive decline. For both of these pathologies, diet has been recognized as a modifiable risk factor, yet the specific impact of diet on neuroinflammation remains poorly understood. Current literature suggests that a high intake of saturated fats and processed foods may promote neuroinflammation and worsen neurodegeneration, while a ketogenic diet has been linked to cognitive improvement in dementia via regulation of neuroinflammatory pathways. This ongoing work seeks to uncover how diet composition influences neuroinflammation, specifically how diet modulates the activity of the NLRP3-inflammasome.

Methods: To address this question, we utilized a novel biosensor mouse developed in our laboratory that allows us to measure caspase-1/NLRP3-inflammasome activity in vivo. Due to the strong association of dementia and T2DM, and the established abnormalities in blood glucose levels in T2DM, this study focuses on high glycemic index (HGD) and ketogenic (KD) diets. These diets will allow us to determine whether rapid spikes in blood sugar seen with the HGD increase NLRP3-inflammasome neuroinflammation, potentially contributing to neurodegeneration, and if the extremely low carbohydrate load in the KD has the opposite effect.

Results: Long term administration of a KD promotes glucose intolerance but does not increase neuroinflammation, which a HGD increases NLRP3-inflammasome-dependent neuroinflammation without altering glucose regulation in wild-type male mice.

Conclusion: We found that the ketogenic diet impairs glucose metabolism, but does not lead to increased neuroinflammation, while the HGD showed the opposite phenotype. Future experiments will assess inflammatory cytokines systemically and centrally, determine levels of pro-inflammatory transcripts and cleaved caspase-1 protein levels in the brain, and evaluate behavior deficits to probe at the mechanism behind these unexpected results.


Next from AMA Research Challenge 2023

Structural characterization of the interaction between alpha-synuclein and neurexin-1 beta in the context of pathologic alpha-synuclein propagation in Parkinson's Disease

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