United States

Background: At present, damage to nervous tissue is difficult to treat, often irreversible, and furthermore nerve growth to new sites is very limited. I hypothesize that teeth have a special capability to induce nerves and that we can isolate tissues and factors that could be used in a clinical setting to completely regenerate nerves. In human clinical studies where developing third molar teeth have been transplanted to donor sites, the teeth have continued to grow but more astoundingly have gained the ability for nociception in response to cold stimuli. This finding implies that developing teeth transplanted to a bony defect induce de-novo nerve growth at the recipient site. In this working study I utilize the rat model to demonstrate de-novo nerve growth by transplanting whole teeth to iatrogenic bony-defects created in autologous tibia and performing histological and biochemical analysis. Demonstrating innervation in transplanted teeth of post-natal rats would indicate that dental tissues are indeed capable of nerve growth and would greatly enhance our understanding of nerve induction.

Methods: Post-natal rats (Sprague-Dawley) aged 28-30 days were anesthetized and intact tooth germs with fully developed crowns undergoing root development were isolated. A bony defect was then created in the ipsilateral anterior tibia and the extracted tooth was transplanted to the defect. The wound was closed and hemostasis established. Hematoxylin and Eosin staining, and fluorescent immunohistochemistry were performed for nerve specific markers including anti-acetylated tubulin III antibodies. 

Results: Histology reveals a tooth well integrated into the tibia and a significant reduction in inflammation and bony remodeling, with dental specific enamel and dentin present in the leg. Moreover, the transplanted tooth is vascularized and early immunohistochemistry indicates evidence of innervation of transplanted teeth in line with the hypothesis that teeth have nerve inductive capacity postnatally.

Conclusion: Teeth transplanted autologously to tibia can integrate into the leg, survive as independent organs with their own blood supply, and early histology indicates innervation of the transplanted tooth. These data are applicable to patients’ needing re-innervation of eye, spine, extremity, teeth and many other organs. The implications are far-reaching and may lead to novel nerve regeneration therapeutics.




AMA Research Challenge 2022

De-Novo Innnervation of a Transplant Organ

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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Ricardo Correa, MD, EdD
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Jessica Li, MD/PhD Candidate
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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

Background: Obesity, metabolic syndrome, and type 2 diabetes have dramatically grown in prevalence over the last decade. Weight loss and lifestyle modifications are often prescribed, as adherence to a resistance exercise training program reduces adiposity and improves glucose sensitivity. Despite the popularity of high protein diets for weight loss and muscle building, prospective and retrospective population-based studies as well as randomized clinical trials have shown that reduced protein intake is associated with better metabolic health in humans, the majority of whom are sedentary. Similar results are observed in sedentary rodents, where reduced protein intake improves metabolic health and body composition. We hypothesized that a unifying explanation of these results is that exercise is protective against high dietary protein intake. Here, we investigate how dietary protein intake interacts with resistance exercise training in mice to determine outcomes related to metabolic health. 

Methods: Male mice were assigned a group with either a high (36%) or low (7%) protein diet with or without a previously validated 12-week progressive overload resistance training program. Food consumption, body composition, and fitness were measured along with metabolic phenotyping outcomes. 

Results: Similar to previous studies, the low protein diets improved body composition in male mice when compared to high protein. However, high protein-fed sedentary animals accumulated significantly more fat mass than high protein-fed exercising mice. Low protein consumption significantly improves blood sugar control compared to high protein diets independently of resistance exercise, though resistance exercise itself may also promote glucose tolerance. Increasing dietary protein intake leads to more rapid strength and lean muscle gain in response to resistance exercise training. However, exercising low protein-fed mice eventually gained similar strength to high protein-fed exercised mice. 

Conclusions: Our results suggest that while low protein diets maximize metabolic health for sedentary mice, resistance exercise and muscle growth may be protective against some, but not all, negative metabolic effects of high protein diets and lead to greater strength and muscle mass. Future studies will be needed to determine if resistance or other forms of exercise are protective against the negative metabolic effects of high protein diets in humans, and to examine if resistance exercise allows high-protein-fed animals to live long, healthy lifespans.

Dietary protein restriction promotes leanness while enabling strength gain in mice

Background: Gestational diabetes causes placental lipid accumulation and essential long-chain polyunsaturated fatty acid (LCPUFA) deficiency in infants which signals poorer outcomes for both mother and baby. Physiologic LCPUFA biomagnification in the fetus suggests placental fatty acid transport occurs by both passive and active mechanisms. This study’s objective was to determine whether differential fatty acid uptake in human trophoblasts is mitochondrial-mediated.
Methods: Using MitoTracker fluorescent tagging of mitochondria and 4,4-difluoro-3a,4a-diaza-s-indacene, BODIPY, a fluorophore attached to fatty acids of variable lengths, we tracked live-cell uptake in BeWo and isolated cytotrophoblasts (CTB) from consenting mothers. Experiments were repeated in the presence of oligomycin (OM), which inhibits ATP-synthase, 2-deoxyglucose (2dG) which competitively inhibits glycolysis, etomoxir (Eto) which inhibits fatty acid oxidation, and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) which uncouples respiration. Statistical analysis was conducted using Student’s T-test per drug or 2-way ANOVA to determine fatty acid-specific, drug-based, and interaction effects. Significance was set at p<0.05.
Results: Live-cell imaging shows rapid uptake of both BODIPY-C12, which mimics a 16-carbon saturated fatty acid like palmitate, and BODIPY-C16, which mimics a 20-carbon LCPUFA occurs in BeWo and CTB. The time to peak uptake was longer for BODIPY-C16 than BODIPY-C12 (p<0.0001). Intracellularly, both fatty acids colocalize with mitochondria. Interestingly, both BODIPY-C12 and C16 stimulate visible cytoplasmic projections that facilitate peripheral uptake, followed by perinuclear accumulation. Treatment with 1uM OM, 50 mM 2dG, or 150 uM Eto significantly decreased ATP levels in BeWo while 0.3 uM FCCP did not (p=0.017, 0.002, 0.012, 0.458 respectively; n=3). ATP-inhibitors, slowed the rate of BODIPY-C16 uptake (p<0.0001 with 2dG and Eto; n=4). Conversely, 2dG and Eto increased the rate of BODIPY-C12 uptake (p=0.007, p=0.012 respectively; n=4). Primary cytotrophoblasts follow a similar trend for fatty acid uptake and response to ATP-inhibitors, taking up the BODIPY-C16 slower than BODIPY-12 (p<0.05 with OM and Eto; untreated control includes average of 4 patients while treatment was replicates from a single patient).
Conclusion: Differential fatty acid uptake in human trophoblasts is ATP-dependent which may explain placental lipotoxicity and LCPUFA deficiency associated with poorer pregnancy outcomes in GDM.

Differential fatty acid uptake in human trophoblast cells is ATP-dependent

Abstract Title: 
Dual role of CASP8AP2/FLASH in regulating EMT plasticity

Background
Metastasis and chemoresistance remain major challenges and the leading cause of cancer-associated deaths in most tumors. During metastasis cells undergo epithelial-to-mesenchymal transition (EMT), a process characterized by loss of epithelial cell markers, increased invasive potential and chemoresistance. EMT plasticity characterized by a seemingly back-and-forth transition between an epithelial and a mesenchymal phenotype plays a fundamental role in cancer progression. EMT occurs through broad transcriptome reprogramming mediated by key regulators, the EMT transcription factors (EMT-TFs). We identified FLASH (or CASP8AP2) as a critical novel EMT-TFs regulator. FLASH regulates a diverse set of cellular processes including apoptosis, histone mRNA processing and cell cycle S-phase progression. Here we show that FLASH is critical for EMT plasticity in multiple cancer cell lines due to its dual role in regulating EMT-TFs. We uncover that FLASH acts a repressor of SNAIL, but an activator of ZEB1, two critical EMT-TFs. Importantly, FLASH is highly expressed in tumors associated with early and aggressive metastasis such as small cell lung carcinoma. 

Methods
To address the role of FLASH in EMT we employ whole-genome NGS analysis, gene expression quantification by Q-PCR and computational EMT scoring using sets of established gene lists that are expressed in multiple cancers and have been validated as markers of the EMT phenotype.

Results
Our investigation reveals that: (1) the role of FLASH in EMT is independent of its role in cell cycle progression; (2) loss of FLASH in cancer cells drives a hybrid EMT phenotype with high epithelial scores; (3) FLASH expression inversely correlates with multiple EMT markers in cell lines from the Cancer Cell Line Encyclopedia (CCLE) dataset, owing to its dual role in EMT plasticity. 

Conclusion
The function of FLASH as a master regulator of pathways involved in tumor progression is novel. Here we show that FLASH regulates both epithelial and mesenchymal markers, thus controls EMT plasticity independent of its function in cell-cycle progression. Because FLASH function is conserved in a number of cancer cell lines including cervical, breast, pancreatic, prostate and lung cancer, FLASH expression modulation in cancer cells may play a critical role in EMT-driven metastasis and subsequent therapeutic resistance.


Dual role of CASP8AP2/FLASH in regulating EMT plasticity

Potential Use of Peripheral Nerve Grafts to Treat CNS Diseases

Autism spectrum disorder (ASD) is a developmental disorder associated with prenatal exposure to valproic acid (VPA) in humans, and prenatal exposure to VPA is a validated animal model of ASD. Animals exposed to VPA in utero have smaller bodies, and lower body and brain weights. We believe that this is because of impaired caloric intake due to impaired oropharyngeal function. 
 
Hypothesis: We hypothesize that prenatal VPA exposure results in hypoplasia of oropharyngeal components and fewer brainstem lower motor neurons (LMN). Specifically, we hypothesize that VPA exposure will lead to smaller muscles of mastication, poorly developed hard palates and mandibles, smaller skulls, and reduced esophageal muscle thickness compared to control animals. We also hypothesize that VPA exposure will result in fewer brainstem LMNs and surviving neurons will be smaller and abnormally arranged, resulting in an imbalance in the number and distribution of excitatory and inhibitory inputs on brainstem LMNs.
 
Results: Repeated prenatal VPA exposure on E10 and E12 results in smaller muscle fibers in the tongue and masseter muscles at P0. Brainstems from P28 rats showed LMN neuronal cell bodies in the trigeminal, facial, hypoglossal nuclei, and nucleus ambiguous to be significantly smaller than those in control animals. The cell body shape was also significantly different in the facial and hypoglossal nuclei. There was an imbalance of synaptic puncta and excitatory/inhibitory ratios in the trigeminal motor and hypoglossal nuclei, and the nucleus ambiguous. It was also found that VPA exposure had no significant impact on the skull mass or morphology, or the morphology of the esophagus.

Conclusion: We have shown that prenatal exposure to VPA impacts the orofacial innervation which leads to possible oropharyngeal muscular weakness and dysfunction. The excitatory and inhibitory imbalance may impede the ability to feed through weaker neuromuscular signals, overexcitation leading to spasticity, or denervation through excitotoxicity in the LMNs.


Repeated In Utero Valproic Acid Exposure on Oropharyngeal Dysfunction in Autism Spectrum Disorder Rat Models

Background 
Galectins are soluble proteins found intracellularly and extracellularly. They are carbohydrate binding proteins that play a role in adhesion, signaling, immune regulation, apoptosis, and other cellular processes. It has been established that galectin expression is altered in cancer pathogenesis, and that galectins are potential therapeutic targets for cancer treatment. However, little research has established the relationship between serum concentration of galectins and breast cancer subtypes. Breast cancer has a high incidence, and treatment is often specific to histological or molecular subtype. The current method for breast cancer subtype identification is an invasive biopsy. Our study analyzes the differences in galectin concentrations in breast cancer patients of different subtypes to establish potential biomarkers for less invasive cancer identification or targets for treatment. 

Methods 
Enzyme-linked Immunosorbent Assay (ELISA) was used to establish the concentrations of galectin -1, -3, -7, and -9 in 100 breast cancer patient serum samples. The galectin concentrations were compared by histological subtype, stage, and other patient characteristics. Statistical analysis of the results was performed using a Student’s T-test and the Wilcoxon Method. 

Results 
Samples from patients with invasive lobular carcinoma had significantly higher concentrations (mean, 13.04 ng/mL) of galectin-3 than invasive ductal carcinoma samples (mean, 9.32 ng/mL; p-value,: 0.0108). We also found that concentrations of galectin-1 increased by stage from breast cancer stages I to III, with a significant difference found between samples from patients with stage I breast cancer (mean, 19.02 ng/mL) and both stage II (mean, 24.15 ng/mL; p-value,: 0.0127) and stage III (mean, 28.37 ng/mL; p-value,: 0.0026) breast cancer. 

Conclusion 
Our results point to a potential use of serum galectin-3 concentrations to distinguish between lobular carcinoma versus ductal carcinoma and could offer a non-invasive diagnostic tool. Based on our results, research could be performed to further establish the relationship between galectin-3 and other breast cancer histological subtypes, as well as molecular subtypes.

Serum Galectin-3 Concentrations Differentiate Between Lobular and Ductal Carcinomas in Breast Cancer Patients

Background: Wound healing is a complex balance of cascading events orchestrated by dynamic autocrine/paracrine signaling of heterogenous factors. An imbalanced composition of bioactive compounds results in a cycle of sustained, inappropriate cellular signaling, and progression towards chronic wounds. The diverse and complex nature of chronic wounds has resulted in a lack of development of focused and effective therapeutic interventions. Therefore, there remains a critical need for a system that can generate customizable therapies for chronic wounds that can aid in reestablishing proper balance in wound healing signaling. Recent investigations have demonstrated that the diverse milieu of secreted factors from Adipose-derived Mesenchymal Stem Cells (ASCs) could offer a unique approach to treating chronic wounds that circumvents the limitations of other autologous cell therapies. Objective: Investigate the ability to tailor ASC biologics with “wound stimuli” priming.

Methods: A novel 3D-printed tissue-mimetic hydrogel system was utilized as a customizable in vitro bioreactor system to generate ASC-derived acellular biologics. ASCs were exposed to a variety of different native “wound stimuli”, including exposure to collagen peptides and fibrin(ogen). The “wound stimuli” were assessed for their ability to modulate the secretory composition of the ASC secretome and to enhance the wound healing activity of keratinocytes, fibroblasts, and endothelial cells.

Results: Our novel 3D system enhanced the regenerative activity of ASCs. Proteomic evaluation demonstrated ~15% increase in protein secretion, including a number of pro-wound healing growth factors and immunomodulatory compounds. Similarly, ASCs exhibited enhanced production of antioxidants (~25%) and EV/Exosomes (~4-5x). Treatment of keratinocytes, fibroblasts, and endothelial cells with the ASC-derived biologics resulted in enhanced wound healing activity, including increases in metabolic, proliferative, and migratory activity. Furthermore, introduction of “wound stimuli” to the 3D system resulted in modulation of the ASC secretome composition and further enhancement of its wound healing capacity. 

Conclusion: Together, this data demonstrates the potential of a novel 3D system to enhance the regenerative capacity of ASCs and permit the priming of ASCs to tailor secreted biologics. Thus, these studies provide insight towards advancing the development of tailored acellular biologics for future regenerative therapeutics for a variety of soft tissue application, including wound healing.

Tailoring Stem Cell Biologics to Enhance Regenerative Wound Healing

Objective:
The main objective of this article is to study the efficacy and safety of vedolizumab in the management of moderate to severe Ulcerative Colitis (UC) patients.
Methods:
We used Preferred Reporting Items for Systematic Reviews and Meta-Analyses (
guidelines, conducted a literature search in PubMed, Google Scholar, and Science Direct, and nine studies were included in the systematic review.
Results:
At week six, vedolizumab showed a significant clinical response At week 52 vedolizumab showed significant mucosal healing and clinical remission The
most commonly associated adverse effects are nasopharyngitis, oropharyngeal infection, and gastrointestinal infection.
Conclusions:
Most of the included studies showed a significant clinical response and clinical remission of the disease Additionally, vedolizumab is well tolerated with
fewer side effects and is safe to use in UC patients.

Vedolizumab Therapy For Management OF Moderate to Severe Ulcerative Colitis

Background: Alzheimer's Disease (AD), related diseases (ADRD), and Multiple Cognitive Impairments (MCI) portend cognitive decline in the elderly, causing significant time course-dependent deficits in language, memory, comprehension, attention, judgment, and reasoning. There is a relative paucity of literature that characterizes sociodemographic implications of these diseases and even fewer systematic reviews that assess the disease burden. The Cleveland Alzheimer’s Disease Research Center (CADRC) is a collaborative effort to establish a National Institute on Aging (NIA) funded Alzheimer’s disease research center; our aim is to determine if people with AD and MCI due to AD who live in rural communities demonstrate greater utilization of healthcare resources compared to those living in non-rural settings.

Methods: Traditional methods of large-scale systematic review are laborious, inefficient, and circuitous for first-pass filtering. Alternatively, our utilization of the machine learning-based method known as Rayyan QCRI allows the first pass researcher to quickly screen with predefined inclusion and exclusion criteria. Repeated screening reinforces the neural network to prefer literature similar to those that have been included, deduplicates imported publications, and provides quality of life features that traditional methods cannot offer.

Results: Of 2239 publications selected for review, 107 were marked as potential “Include,” 290 were marked as “Maybe,” and the remaining 1802 as “Exclude” in a span of 30 hours of in-software time. “Include” and “Maybe” articles were passed into second-pass analysis for data extraction with machine-learning ratings in mind. In comparison, 35 publications were reviewed by traditional methods, yielding 2 potential “Include”, and 5 “Maybe” in a span of 10 hours. 

Conclusion: Rayyan is an effective method for first-pass systematic review and has distinct qualities that make it a preferential alternative to traditional methods.

A Modern Procedure for Systematic Review: Comparing Classic Methodology to a Machine Learning–Based Technique

Background

Shared Decision-Making (SDM) is a collaborative process where patients and clinicians work together to determine a treatment plan, balancing evidence-based medicine with patient values. Little is known about how SDM is documented by clinicians in the Electronic Medical Record (EMR). Understanding how patient preferences are reflected in clinical documentation is important as clinicians strive to make care more patient-centered. This study seeks to evaluate whether clinical documentation is reflective of verbal communication in pediatric clinical encounters.

Methods

This Mixed Methods Sequential Exploratory Analysis included 109 pediatric surgical consultations. For each encounter, the transcript of the verbal clinician-family communication was descriptively evaluated for elements of SDM: discussion of risks/benefits/alternatives, parental preferences, and expression of parental concern. Next, the corresponding EMR documentation was evaluated for these elements. Qualitative methods were used to describe similarities and differences of these elements in both types of communication. A basic descriptive quantitative analysis was conducted to further describe the similarities/differences between these two types of communication describing the same clinical encounter.

Results

In the discussion of risks/benefits/alternatives, the EMR documentation for 48% of encounters used a standard dot phrase, which often differed from what was discussed verbally. Common discrepancies also included a parental preference being documented in the EMR when no preference was verbally expressed by the parent, occurring in 12% of consultations. The opposite was also seen: in 14% of consultations, a parental preference was expressed but was not documented. Overall, expression of parental concern was infrequent, occurring in 30% of the consultations. Of the 34 parental concerns that were expressed, only 21% were documented. 

Conclusion

In conclusion, there are substantial discrepancies between what is verbally communicated in pediatric surgical encounters and what is subsequently documented in EMR documentation. As clinicians evolve in delivering patient-centered care, understanding how patient perspectives are reflected in medical documentation could significantly impact clinical practice, informing interventions to make documentation more inclusive of patient values. This research is particularly significant as patient care is becoming more transparent with increased patient access to their EMR, emphasizing the importance of understanding whether SDM is accurately reflected in clinical documentation.


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Dietary protein restriction promotes leanness while enabling strength gain in mice

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