United States

Background: Sinoatrial node (SAN) cells, the natural pacemakers of the heart, determine the rate and rhythm of the heart’s electrical conduction system. Damage to these cells can lead to conditions such as arrhythmias and Sick Sinus Syndrome. These conditions are commonly treated with electronic pacemaker implants which can lead to risks of complications, such as infection. A more biologically compatible therapeutic alternative that could reduce the risk of complications could eventually include cell transplantation with SAN cells derived from human pluripotent stem cells (hPSCs). However, a protocol for the generation of SAN cells from hPSCs in vitro has not been well-established, although there are well-established protocols for the generation of other cardiomyocytes, such as ventricular and atrial cells. Our study aims to generate SAN-like cells in vitro by using the hPSC model.

Methods: To generate the SAN-like cells, we combined aspects of protocols for the generation of ventricular and atrial cells from hPSCs with a newer, published, successful protocol by Protze et al. proposed for generating SAN-like cells from hPSCs. By incorporating these protocols, SAN-like cells were generated by selective induction of cardiac progenitors and controlled maturation of the SAN lineage by mimicking their in vivo development. The cells were then characterized via light and fluorescent microscopy, flow cytometry, and immunofluorescence, and viability was assessed via light microscopy and flow cytometry.

Results: We successfully generated SAN-like cells as demonstrated by their faster beating pattern on light and fluorescent microscopy and a lack of NKX2-5 expression on immunofluorescence, two characteristics that distinguish SAN cells from other cardiomyocytes. However, flow cytometry demonstrated a small proportion of live cells and light microscopy observations demonstrated increased cellular debris and cell death morphology of the SAN-like cells, suggesting that viability of these cells was greatly reduced compared to other cardiomyocytes generated in vitro.

Conclusion: The successful generation of SAN-like cells from hPSCs provides a promising avenue in the development of novel biologic therapies that could improve outcomes in the most susceptible populations for rhythm disorders. Future studies need to address the issue of reduced viability of these cells compared to other in vitro generated cardiomyocytes.

AMA Research Challenge 2022

Cardiology - In vitro generation of sinoatrial node-like cardiomyocytes derived from human pluripotent stem cells - Cardiology

Welcome to the largest national, multi-specialty research event **featuring research from 750 medical students, residents and fellows, and international medical graduates.**


#### Event Details
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At the conclusion of this event, the AMA will announce the top five finalists who will advance to the AMA Research Challenge finals, present their research to an elite panel of judges and compete to win a $10,000 grand prize sponsored by Laurel Road.
 

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Ricardo Correa, MD, EdD
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Charles Lopresto, DO

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Welcome to the largest national, multi-specialty research event featuring research from 750 medical students, residents and fellows, and international medical graduates.


poster

In this study we define a novel role for the SDF-1/CXCR4 axis in the hibernation and treatment of myocardial tissue in diabetic cardiomyopathy (DCM).
Wild-type and cardiac CXCR4 null (CM-CXCR4 null) mice were randomly assigned to low-fat diet (LFD) or high-fat diet (HFD) for 30 weeks. Serial echocardiography was used to assess cardiac function. SDF-1 plasmid (80 ug) was injected into the LV wall of half group of the HFD mice (27 weeks). SDF-1, CXCR4, SIRT1, SIRT3, and SIRT6 mRNA levels in the hearts were quantified by qPCR at different times.
Compared to age-matched LFD mice, 27-week-old HFD mice showed a 33% decrease in Ejection Fraction (EF). Direct myocardial injection of hSDF-1 plasmid led to a 15% and 30% improvement in EF 1 and 3 weeks later, respectively, compared to control. To determine if CM-CXCR4 expression induces CM hibernation as a mechanism of DCM, we quantified cardiac function in HFD CM-CXCR4 null mice. We observed an early loss of cardiac function in HFD CM-CXCR4 null mice compared to HFD control mice. Interestingly, SIRT1 and SIRT6 were statistically decreased in HFD mice, suggesting that SIRT signaling may be involved in blunted SDF-1 signaling in DCM.
Our data demonstrated that the SDF-1/CXCR4 axis has an important role in DCM. Our data suggests that cardiac myocyte over-expression of CXCR4 is involved in cardiac myocyte hibernation. These data suggest that modulation of SDF-1/CXCR4 expression will delay or alleviate DCM.

Cardiology - Stromal Cell-Derived Factor 1 (SDF-1) and its Receptor CXCR4 Improve Diabetic Cardiomyopathy (DCM) - Cardiology

Dermatology - Association of Mental Health Disorders and Bacterial Cultures in Hidradenitis Suppurativa - Dermatology

Nonmelanoma skin cancer (NMSC), the most common form of cancer worldwide, is caused by chronic exposure to ultraviolet radiation (UV). UV generates reactive oxygen species, which lead to mutations and inflammation that enhance the oncogenic transformation of skin cells. Because current sunscreens have limited efficacy at protecting from UV and preventing skin cancer, there is a need for alternative preventive approaches. To this end, Pro-NP, a nanoparticle formulation consisting of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), was used for sustained delivery of these enzymes. We hypothesized that topical application of Pro-NP would safely deliver SOD and CAT to skin cells and suppress UV-induced DNA damage and skin carcinogenesis. To test this, Pro-NP or vehicle-treated mice were assessed for biomarkers of UV-induced skin cancer after short-term and long-term irradiation, enabling us to determine the photoprotective effects of Pro-NP in vivo. Pro-NP decreased UV-induced DNA damage in male and female mice, yet only protected female mice from UV-induced cancer formation. Interestingly, Pro-NP reduced UV-induced proliferation and inflammation to a greater extent in female skin. Taken together, these findings demonstrate that topical application of antioxidant regimens such as Pro-NP may protect from DNA damage and inflammation following UV exposure and suppress skin cancer formation.

Dermatology - Nanoparticle-mediated Delivery of Antioxidant Enzymesprotects From Uvr Damage and Skin Tumorigenesis - Dermatology

Dermatology - Representation of Skin of Color Images in Dermatological Artificial Intelligence Databases - Dermatology

Abstract Title:
Exploring subcutaneous encapsulated islet cell transplantation as a treatment for Type 1 diabetes.
Background
According to the JDRF, over 1.6 million persons living in the United States live with Type 1 Diabetes (T1D) as of 2020. Current protocols utilize the liver as a transplant site in patients undergoing islet cell transplantation as a treatment for Type 1 diabetes (T1D). However, the need for an alternative and more accessible site is evident with only 50% of patients maintaining insulin independence one year after transplantation. In this study, we set out to evaluate whether transplantation of non- and (PVPON/TA/CTLA-4-Ig)-encapsulated islets can restore normoglycemia to streptozotocin (STZ)-treated diabetic NOD.SCID mice when transplanted into a prevascularized subcutaneous space.
Methods
We took seven mice that were previously implanted with a nylon catheter subcutaneously in order to induce a foreign body response and increase vascularization. After four weeks, these mice were then administered a dose of 190 mg/kg of STZ in order to induce diabetes. All of the mice were confirmed to be diabetic by a blood glucose value of more than 300 mg/dL. Three mice were transplanted with 500 non-encapsulated islets while the remaining four were transplanted with 500 islets that were encapsulated with a poly (N-vinylpyrrolidone (PVPON)/Tannic acid (TA)/CTLA-4-Ig nanolayer coating. Blood glucose values were then taken from the tail vein of each mouse to assess whether normoglycemia could be restored.
Results
Normoglycemia was restored in five out of the seven mice (71.4%) one day after transplantation. Of the two mice (28.6%) in which normoglycemia was not restored, one mouse (14.3%) had an adverse reaction to the STZ treatment and did not survive 24 hours post transplantation. The other mouse (14.3%) was not engrafted with islet cells and remained diabetic.

Conclusion
Transplantation into a prevascularized subcutaneous pocket using both non-encapsulated and encapsulated islet cells was found to restore normoglycemia in NOD.SCID mice. This therapy for T1D presents an alternative option to the current protocol and should be further explored in future studies. Future studies may make use of other techniques such as encapsulation with PD-L1 (CD274) in order to further diminish the T-cell response and facilitate islet cell survival.


Endocrinology - Exploring Subcutaneous Encapsulated Islet Cell Transplantation as a Treatment for Type 1 Diabetes - Endocrinology

Background: The adipocyte-derived hormone leptin is a key mediator in homeostatic appetite regulation and satiety via the arcuate nucleus (ARC) of the hypothalamus. Interestingly, serotonin (5-HT) produced in the dorsal raphe nucleus (DRN) modulates anorectic effects of its own in the ARC, yet the underlying mechanism through which 5-HT acts in tandem with leptin remains inconclusive. Our lab has identified leptin-sensitive serotonergic neurons in the DRN that project to the ARC, and has demonstrated that activation of this circuit significantly decreases food intake. Through the unique co-expression of leptin receptors and 5-HT within these neurons, the current study aimed to elucidate the role of 5-HT in leptin’s regulation of food intake between the DRN and ARC.

Methods: To examine the role of 5-HT on leptin’s action in the DRN, adult male Sprague Dawley rats underwent stereotaxic surgery for guide cannula implantation in the DRN. After recovery, rats received 100 μg of intra-raphe p-chlorophenylalanine (PCPA), an inhibitor of 5-HT synthesis, each day for four days. On the fourth day, an additional injection of leptin was administered in the DRN (5 μg/rat) and food intake was measured over a 24-hour time course. To examine the role of 5-HT on leptin’s hypothalamic action, a subsequent experiment was conducted by implanting an additional cannula into the ARC for the administration of leptin on the fourth day of treatment. Post-mortem, DRN tissue sections were immunolabeled to confirm 5-HT depletion and visualize leptin-sensitive serotonergic neurons.

Results: 24-hour food intake analyses across both experiments revealed only vehicle-treated rats administered leptin exhibited a significant decrease in food intake compared to controls, an effect not observed in PCPA-treated rats. These results suggest depletion of 5-HT within the DRN attenuated leptin’s ability to decrease food intake in both the DRN and ARC. 5-HT immunolabeling confirmed PCPA significantly decreased 5-HT in the DRN of all treated rats and displayed co-localization of 5-HT and phosphorylated-STAT3 (a marker for leptin receptor activation) in DRN neurons.

Conclusion: Depleting DRN-derived 5-HT diminishes leptin’s action on hypothalamic and DRN leptin receptors, suggesting 5-HT is a regulator of leptin-mediated food intake control within this novel neuronal circuit.

Endocrinology - Serotonin, Leptin, and a Novel Neuronal Circuit: Rewiring Central Appetite Regulation As We Know It - Endocrinology

TITLE: EFFECTS OF ADENOSINE ON LIPID ACCUMULATION IN A HUMAN HEPATOMA CELL CULTURE MODEL WHEN CHALLENGED WITH EXCESS FATTY ACID


Authors: Michael Brown B.S., School of Medicine, New York Medical College MS2
Dr. Robin DaSilva, PhD, Department of Food Science and Human Nutrition, University of Florida
Background:

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disorder in western populations and is rapidly becoming one of the most common indications for orthotopic liver transplantation in the world. Fully understanding lipid metabolism in the liver is necessary for further understanding the progression of NAFLD to non-alcoholic steatohepatitis (NASH) and in finding an alternative treatment to weight loss for those diagnosed with NAFLD.

Design/Methods:

HepG2 cells treated with oleic acid (OA) were used as a model of liver cell lipid accumulation.
Methionine, choline and purines are important metabolites and nutrients that are dysregulated in
NAFLD, so we used methionine and choline deficiency (MCD) and excess purines in cultured
HepG2 cells to assess the influence of these conditions.

Cell Culture
Quantitative real-time polymerase chain reaction
Western Blot
TG Assay

Statistics:
Analysis for statistics and all figures were made using the software Prism (GraphPad). A one
way-ANOVA was used to test for statistical significance. A P-value less than 0.05 was taken as
a significant difference.

Results:
MCD HepG2 cells had fewer total triglycerides and had higher expression of carnitine palmitoyltransferase 1 a (CPT-1a) and PPARa, genes involved in b-oxidation, as compared to control cells. MCD cells treated with extracellular adenosine or inosine with OA lowered phosphorylated-acetyl-CoA carboxylase (p-ACC). Adenosine was found to affect lipid metabolism through its role in activating AMP-activated protein kinase (AMPK) in the AMPK-ACC-CPT 1 pathway.
Conclusions:
HepG2 cells under a methionine and choline-deficient condition increased fatty acid oxidation and prevented cells from lipid-loading after treatment with exogenous fatty acids. The addition of excess purine molecules exacerbated lipid-loading in HepG2 cells in all conditions by decreasing fatty acid oxidation and potentially by increasing fatty acid synthesis. Impaired adenosine production in the MCD condition may reduce the inhibition of fatty acid synthesis and result in enhanced lipid oxidation.


Effects of Adenosine on Lipid Accumulation in a Human Hepatoma Cell Culture Model When Challenged with Excess Fatty Acid

With the increasing prevalence of type 2 diabetes and non-alcoholic fatty liver disease (NAFLD), there is still an unmet need to better treat hyperglycemia and hyperlipidemia. Isthmin (Ism1) is a recently identified adipokine that promotes glucose uptake in adipocytes while suppressing lipid production in hepatocytes. The mechanism by which Ism1 controls lipid metabolism is still unknown but the data show that Ism1 treatment induces a signaling pathway that counteracts lipid production, which could be directly related to its function in reversing hepatic steatosis. Different mechanisms are known to be responsible for the development of hepatosteatosis, including enhanced de novo lipogenesis, impaired fatty acid oxidation or impaired lipid transport. The hypothesis is that Ism1 signaling affects one or several of these mechanisms to control lipid metabolism and reduce hepatosteatosis. The objective of this research was to identify the different lipid species that are controlled by Ism1 treatment or are altered in livers from mice lacking the Ism1 gene, to better understand which of the lipid pathways are regulated by Ism1. To do so, we used a NAFLD mouse model in which mice were pharmacologically treated with Ism1 or in which Ism1 was genetically knocked out. We quantified the liver lipid content by mass spectrometry. We observed that Ism1 treatment and Ism1 loss of function impacted the quantity of many lipids in NAFLD mice liver including diglycerides, triglycerides, and fatty acids, consistent with a major role for Ism1 in regulating de novo lipogenesis. Although Ism1’s mechanism of action needs to be identified, these results suggest that Ism1 may have a therapeutic potential to treat NAFLD.

Isthmin Suppresses Global Hepatic Lipid Synthesis as Revealed by Untargeted Lipidomics of Murine Livers

Sex Specific Differences in Regulation of Estrogen Receptor 𝛣 Signaling in Ulcerative Colitis using Latent Variable Meta-Modeling

Background
Patients with inflammatory bowel disease (IBD) are more susceptible to mental health problems than the general population. The prevalence of psychiatric diagnoses in patients with IBD is lacking in India.
Methods
We ascertained the presence of anxiety, depression, or perceived stress disorder among Indian patients with IBD [Ulcerative colitis (UC) or Crohn's disease(CD)] using the Hospital Anxiety and Depression Scale and Perceived Stress Scale. Patients with prior anxiety, depression, or post-traumatic stress disorder were excluded to create the study cohort.
Results
A total of 318 patients with IBD (mean age 40.13±12.06 years, 168 (52.82%) males) were asked to fill out the questionnaire. Most of the enrolled patients had UC (n=255, 80.2%). The prevalence of anxiety and depression was 14.15% and 12.57%, respectively. According to the perceived stress scale, 58.2%, 36.8%, and 5% of patients had low, moderate, and high stress, respectively. Upon subgroup analysis, the prevalence of anxiety and depression were 12.94% and 12.15% in patients with UC and 19.04% and 14.28% in patients with CD, respectively. Patients with CD had a higher perception of stress as compared to patients with UC.
Conclusion
The prevalence of anxiety, depression, and perceived stress is high among Indian patients with IBD.

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