United States

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.

2023 AMA Research Challenge

OMT alleviated migraine-like pain via blockade of trigeminal activation

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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.

Vasopressin Receptors in Bat: A Novel Animal Model for Nocturia

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
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. 


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OMT alleviated migraine-like pain via blockade of trigeminal activation

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

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OMT alleviated migraine-like pain via blockade of trigeminal activation

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

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