2021
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Item Development and in vitro characterization of gemcitabine loaded nanoparticles for pancreatic cancer therapy(2021) Pham, Jennifer; Ranjan, Amalendu; Vishwanatha, JamboorPurpose: Pancreatic Ductal Adenocarcinoma (PDAC) is the 4th leading cause of cancer deaths in the United States and the most common type of pancreatic malignancy (90%). With a poor five- year survival rate of only 5-8%, complete surgical resection remains the only curative treatment. However, most patients are diagnosed at a later stage where chemotherapy and radiotherapy are the only options. Gemcitabine is the FDA-approved treatment for PDAC but the current therapy leads to more severe side effects due to the instability of gemcitabine in the blood stream and its poor membrane permeability. Nanoparticles are effective in cancer therapy because they allow modifications that make for a more effective delivery method which also reduces the toxicity to normal tissue. Methods: In this proposed study, we aim to formulate, optimize and evaluate the in vitro effectiveness of gemcitabine loaded nanoparticles in PDAC cell lines in order to improve the effectiveness of current chemotherapy treatments for pancreatic ductal adenocarcinoma. Results: We found that out of the three types of nanoplatforms used for encapsulating gemcitabine (GEM-NPs): polymeric, liposomal and a hybrid of the polymeric and the liposomal forms, the liposomal nanoparticles were the most effective in the encapsulation of gemcitabine according to the physicochemical properties, such as average particle size, zeta potential, encapsulation efficiency or drug loading, etc., and in vitro functional evaluation in PDAC cell lines. Conclusion: This study suggests that the use of liposomal nanoparticles is the most beneficial in the encapsulation and delivery of gemcitabine.Item Glial cell line-derived neurotrophic factor and Parkinson's disease: Is its family receptor the missing link?(2021) Kasanga, Ella; McElroy, Christopher; Shifflet, Marla; Salvatore, MichaelPurpose: Glial cell line-derived neurotrophic factor (GDNF) improved motor function in Parkinson's disease (PD) preclinical and Phase I trials whereas mixed results were generated for Phase II trials. This disparity in outcome may be attributed to several factors including disease severity at treatment onset. Although GDNF is delivered to the striatum, increasing reports suggest that its recovery in the substantia nigra (SN) is vital for motor recovery, a process which may be compromised with PD progression. GDNF delivery in aged rats, wherein bradykinesia is evident, induces expression of its family receptor, GFR-ɑ1, in the SN. Indeed, replenishment of GFR-ɑ1, which is decreased in the SN in aged models, improves motor function, suggesting that GFR-ɑ1 may be critical for motor benefits. Using a hemi-Parkinson's model wherein bradykinesia is also evident, we evaluated for changes in GFR-ɑ1 expression with lesion progression. Method: 3-month old male Sprague-Dawley rats were euthanized on days 7 and 28 post-lesion induction for neurochemical assays. Results: A lesion-associated decline in GFR-ɑ1 protein expression was observed in the striatum at both time points. Also, there was a more significant reduction in the 28-day versus 7-day cohort denoting a progressive loss of GFR-ɑ1. Conclusion: These results suggest that GFR-ɑ1 in the striatum declines with nigrostriatal dysfunction progression and thus, may be a limiting factor in GDNF efficacy. Maintaining GFR-ɑ1 expression in the striatum may therefore be a link in the restorative capacity of GDNF.Item Exoskeleton & Gait Speed in Patients with Spinal Cord Injury(2021) Walls, Paige; Diu, Pam; Salem, Yasser; Liu, HowePurpose: Robot assisted gait training (RAGT) is an intervention used to improve gait in individuals with lower extremity motor dysfunction. The purpose of this review is to evaluate both prior and recent literature on the effectiveness of exoskeleton use as an intervention for gait speed in individuals with spinal cord injury. Methods: Electronic databases used in the search included PubMed, CINAHL, PEDro, & Scopus. Two reviewers searched all results from the databases and categorically organized relevant articles based on inclusion & exclusion criteria. Studies were included if they involved exoskeleton as an intervention for gait speed and were conducted on adults diagnosed with spinal cord injury. Results: 37 final articles were reviewed. The sample sizes ranged from 1 to 170 participants. Participant ages ranged from 18 to 81 years old. Duration of treatment ranged from a single session to 16 weeks, frequency ranging from 1-2 sessions to 5 per week. Length of sessions ranged from 20 to 90 minutes. The primary outcome measure for gait speed used in most studies was the 10MWT. Others included the 6MWT, 2MWT, TUG, treadmill speed, and motion capture analysis. Conclusion: The evidence suggests that the use of robotic assistance may effectively improve gait speed in individuals with spinal cord injury. However, evidence is inconclusive as to whether RAGT is more effective in improving gait speed when compared to other interventions. Studies report that robotic assisted interventions are safe & feasible interventions without adverse events or exacerbation of symptoms for patients of this population.Item Assessing Fall Events in Geriatric Cancer Patients who are Prescribed an Opioid and/or Benzodiazepine(2021) Bhachawat, Neal; Rasu, Rafia; Agbor, WalterIRBexempt#2020-013. Purpose: Opioids & benzodiazepines are commonly used in cancer pain treatment however their sedating effects increase a patient's fall risk. BEERS criteria was established to reduce adverse events related to medication use in elderly population. Falls are a leading cause of death in the geriatric population and seniors with cancer confer an estimated 20%increased risk. Objectives:(1)identify the demographics of cancer patients age65+ who experienced a fall,(2)determine fall event trends based on patient-specific factors & medications (3)determine if BEERS criteria was followed. Methods: A cross-sectional study analyzing fall outcomes in cancer patients, age 65+, with analgesic medications used to manage acute/chronic pain. The population data was be compiled from the CDC National Ambulatory Medical Care Survey(NAMCS).Diagnosis was based on ICD9/10 and medication codes. Database findings based on sample of office visits. Results: In the NAMCS database between 2006–2017 was 276,166,738 (weighted freq.) cancer patient visits, age 65+ with 83.16%experiencing a fall. 194,560,411 were taking Benzodiazepine only.31,941,74 5were taking Opioids and 68% were prescribed a benzodiazepine as adjunct therapy. Fall incidence: Opioid group 84.6% (p=.03); Benzodiazepine alone 97.1% (p< .001); Benzodiazepine + opioid 93.2% (p< .001). Of the 83.16% of patients who experienced a fall, majority were white and female; 54% were age 75+; Types of cancers: prostate (12.3%), breast (7.3%), lung (5.2%), colorectal (4.5%) and others (70.7%). Fall incident peaked during 2012-2014 with 41.9% of falls occurring and declined to 8.3% during 2015-2017 (p< 0.001). Conclusion: The fall rates experienced by geriatric cancer patients taking opioids and/or benzodiazepines is far greater than the national average for the general geriatric population: 25%suffering a fall. In 2015 BEERS criteria and FDA advised against concurrent use of benzodiazepines + opioids. Our results indicate clinicians followed these changed guidelines, resulting in a decrease in fall events during data period 2015-2017 and illustrating the crucial role BEERS criteria plays in patient safety.Item Smooth Muscle Contraction Is Regulated by Chloride Channels: Functional Evidence for TMEM16A in Porcine Coronary Arteries(2021) Dick, Gregory; Tune, JohnathanPurpose: Contraction of coronary smooth muscle is influenced by ion channels controlling membrane potential (Em) and Ca2+ influx. A great deal of attention has been focused on K+ channels, as their opening makes Em more negative, reduces Ca2+, and causes relaxation. We investigated ion channels whose opening would depolarize Em, increase Ca2+, and promote contraction. A candidate for study is TMEM16A, a Ca2+-activated Cl- channel expressed in a variety of smooth muscles. We tested the hypothesis that drugs which influence TMEM16A would alter contraction. We predicted that contraction would be enhanced by a TMEM16A activator (Eact), whereas it would be attenuated by a TMEM16A inhibitor (T16Ainh-A01). Methods: We used isometric tension recording methods on epicardial coronary artery segments from domestic swine. Contractions to K+ were recorded before and after treatment with 5 µM Eact or 5 µM T16Ainh-01. Extracellular K+ was varied by adding K-gluconate, rather than KCl, to keep Cl- constant. Results: K+ contracted rings with an EC50 of 19.1 ± 0.6 mM and a maximum of 11.8 ± 1.4 g. Drug vehicle had no effect on EC50 or maximum. Eact shifted contraction to the left (17.8 ± 0.9 mM; P < 0.05) but did not affect the maximum (105 ± 3% of control). T16Ainh-A01 shifted contraction to the right (20.4 ± 0.6 mM; P < 0.05) but did not affect the maximum (90 ± 1% of control). Conclusions: These data suggest that TMEM16A is expressed in porcine coronary arteries and influences electromechanical coupling.Item Characterization of adipose tissue and adipose-tissue derived stem cells in Lipedema(2021) Al-Ghadban, Sara; Bunnell, BrucePurpose: Lipedema is a painful adipose tissue (AT) disorder that occurs almost exclusively in women, with onset manifesting at puberty or at times of hormonal change. This disorder is characterized by a symmetrical increase of fat deposition in the legs and the arms, sparing the hands and the feet. The goal of this study was to determine the histology of the skin and fat of lipedema tissue and characterize the adipose tissue-derived stem cells (ASCs) obtained from the stromal vascular fraction (SVF) of thigh AT of non-lipedema and lipedema patients. Methods: Histological sections from AT were stained with H&E. Adipocyte area was quantified using ImageJ software. Markers for macrophages (CD68) was investigated by immunohistochemistry. ASCs were characterized by the expression of stemness markers and their multi-differentiation potential. Flow cytometry, RT-PCR and immunofluorescence assays were performed. Results: The data show hypertrophic adipocytes, increased numbers of macrophages and blood vessels in thigh tissue of women with lipedema compared to non-lipedema patients. Additionally, at the cellular level, an increase in the adipogenic differentiation potential with no change in the expression of mesenchymal markers (CD73, CD90 and CD105) or extracellular markers (collagen, fibronectin and laminin) was detected in lipedema ASCs compared to non-lipedema ASCs. Conclusion: Infiltration of immune cells, increase in adipocyte size and adipogenesis stimulates angiogenesis and fibrosis in lipedema AT. Defining the structure and the components of AT will provide insights into the pathophysiology of lipedema and will help researchers develop potential treatment for the disease.Item Involvement of the c-Jun N-terminus kinase (JNK) pathway in Endothelin (ET-1) mediated neurodegeneration of retinal ganglion cells(2021) Kodati, Bindu; Stankowska, Dorota; Krishnamoorthy, Vignesh; Krishnamoorthy, RaghuPURPOSE: Endothelins contribute to neurodegeneration in glaucoma, however, the mechanisms are not completely understood. The goal of this study was to determine if JNK2 plays a causative role in endothelin-1 (ET-1)-mediated loss of RGCs in mice. METHODS: JNK2-/- and wild type (C57BL6) mice (n=4) were intravitreally injected in one eye with 2 nmoles of ET-1, while the contralateral eye was injected with 2 µl of vehicle. The mice were euthanized at 2 h and 24 h post-injection. Retinal sections from the JNK2-/- and wild type (C57BL6) mice were used for immunohistochemical analysis of the phosphorylation of JNK substrate, c-Jun. In a separate set of experiments, JNK2 -/- and wild type mice (n=6) were intravitreally injected with either 2 nmoles of ET-1 or vehicle, and euthanized 7 days post-injection. RGC counts and axonal degeneration were assessed. RESULTS: Intravitreal ET-1 administration produced a significant increase in immunostaining for phospho c-Jun in wild type mice which was appreciably lower in the JNK2 -/- mice. Following ET-1 administration, a significant (p< 0.05) 26% loss of RGCs was found in wild type mice, while JNK2-/- mice showed no significant (p=0.36) loss of RGCs. A significant decrease in the axonal counts and an increase in the collapsed axons was found in ET-1 injected eyes in wild type mice. CONCLUSION: JNK2 appears to play a major role in ET-1 mediated loss of RGCs in mice.Item AT1R sniffer cells detects spontaneous and evoke release of AngII in the AP-NTS pathway(2021) Gusson Shimoura Almeida Lima, Caroline; Paundralingga, Obed; Farmer, George; Cunningham, JosephAlthough angiontesin II (AngII) has multiple actions in the brain, the existence of a brain RAS is still controversial. Our previous studies have used angiotensin sensitive sniffer cells to test whether angiotensin peptides are released from subfornical organ projections to the median preoptic nucleus. In these studies, we examined another pathway involving the area postrema (AP) and nucleus of tractus solitarius (NTS). The AP is angiotensin sensitive and projects to the NTS, so the purpose of this study was to test for the release of angiotensin peptides in the NTS after stimulation of AP. Sniffer cells were produced by transfecting Chinese Hamster Ovary cells with commercially available plasmids for the angiotensin 1a receptor (Origene Tech.) and R-GECO (Addgene #32462). These sniffer cells are sensitive to AngII and III but not angiotensin 1-7, bradykinin, or neurotransmitters such as glutamate or acetylcholine. Sniffer cells were placed on coronal brainstem slices containing both AP and NTS from adult male Sprague – Dawley rats. Changes in fluorescent intensity of sniffer cells in the NTS was determined following electric stimulation of the AP (100Hz, 10ms, 1mA). Electrical stimulation increased fluorescence intensity 134 ± 11%, n=13 of sniffer cells on the NTS with a mean response latency of 4 ± 0.7sec, n=13. Some cells demonstrated spontaneous changes in fluorescence intensity 2±0.1, n=28 that were not observed in cells located outside of the NTS. The results indicate that sniffer cells placed on the NTS demonstrated evidence of spontaneous and evoked release of angiotensin peptides.Item Self-Management Program for Brain Health(2021) Jose, Roslin; Kannan, Srijaa; Ross, Sarah; Quiceno, Mary; Severance, JenniferPurpose: While developing dementia may be unavoidable, various factors contribute to its onset including lifestyle choices. The purpose of this study is to implement a program supporting lifestyle changes to improve brain health and cognitive functioning with hopes of preventing or delaying dementia. Methods: Health coaching, education and targeted assessments with feedback will be used to provide a personalized approach to addressing brain health. Inclusion criteria: ≥ 18 years old without dementia or uncontrolled psychiatric illness. Participants can choose to receive health coaching through a digital tool or in-person. They will complete assessments for each of the pillars of Brain Health: Diet, Exercise, Social Engagement, Cognitive Activity, Sleep, Mindfulness & Outlook, and General Health. Demographics, motivation to change and cognitive status will also be assessed. Health coaching will last 3 months with assessments for the pillars given 3 months before starting, at the start, at the end, and 3 months after completion. We will evaluate scores for each assessment at various points in time for improvement or other trends. Results: This program is in the beginning stages of implementation. Conclusion: This self-management program intends to promote lifestyle changes that will reduce the risk of developing dementia. If individuals make improvements in the seven pillars, they can expect optimization of cognitive functioning and risk reduction for developing dementia. Clinical implications include the development of an evidence-based program for addressing risk factors for dementia with potential for preventing or delaying its onset.Item Synthesis and Bioactivity of Nitric Oxide Donor and Antioxidant Drug Hybrid(2021) Khowaja, Sanober; Nguyen, Maria; Weston, Courtney; Acharya, SuchismitaPurpose: In ischemic stroke or peripheral artery disease, there is a blockage of the arteries that results in increased free radicals and cell death. Our goal was to synthesize a hybrid compound SA-9-01 and assess its NO releasing and reactive oxygen species (ROS) scavenging ability using chemical assays. We hypothesize that the hybrid compound will prevent cell death by improving blood circulation and neutralizing ROS. Methods: SA-9-01 was synthesized using a synthetic procedure similar to a previously designed analog SA-2 and structure was verified by 1HNMR. The NO releasing activity was determined using the Griess assay by measuring the total nitrite formation. The xanthine oxidase assay was used to measure the ROS scavenging activity. Results: Compound SA-9 (25 mM) released NO at concentrations between 1.35-1.40 µM at t=90 mins sufficient to provide therapeutic activity, while a known NO donor SIN-1 released between 2-15 µM at same concentration and time point. From the xanthine oxidase assay, the scavenging ratio for SA-9-01 (250 µM) was about 20-25% at t=100 mins and comparable to previously described compound SA-2 and Baicalein (the positive control). Conclusion: Compound SA-9-01 was synthesized successfully with the correct chemical structure and was found to be a tautomer of the first batch SA-9. The Griess assay demonstrated that SA-9 releases physiological level of NO. SA-9-01 also demonstrated ROS scavenging activity. The testing of SA-9-01 in cells is under progress.Item Racial Differences in Treatment and Outcomes of Chronic Low Back Pain(2021) Timmer, Benjamin; Licciardone, John C.Purpose: Chronic low back pain (CLBP) is the leading cause of patient visits to primary care physicians in the United States. Black patients with chronic pain are at higher risk of progressing to disability due to lack of access to healthcare, yet limited studies have addressed the epidemiology of CLBP treatment and outcomes in the Black population. Methods: This cross-sectional study utilized the Pain Registry for Epidemiologic, Clinical, and Interventional Studies and Innovation (PRECISION) to study racial differences in selected patient-reported treatment and clinical outcomes, including a numerical rating scale (NRS) for pain, Roland-Morris Disability Questionnaire, Pain Sensitivity Questionnaire, and Pain Catastrophizing Scale. Results: A total of 848 patients with CLBP participated, including 184 (22%) who were Black. The percentage (95% CI) of Black patients who used non-pharmacological treatments was 70%(63%-77%) vs. 87%(84%-89%) of White patients (p=< 0.001). Black patients were also less likely to use nonsteroidal anti-inflammatory drugs for pain, 72%(65%-78%) vs 86%(83%-89%) (p=< 0.001). However, opioid use was not different between races. Black patients experienced greater pain than White patients based on reported mean (95% CI) NRS values of 7.2(6.9-7.4) vs. 5.8(5.7-5.9) (p=< 0.001). Additionally, Black patients experienced greater disability, pain sensitivity, and pain catastrophizing than White patients (p=< 0.001 for each contrast). Conclusions: These findings suggest that racial health disparities exist among patients with CLBP. Greater efforts to increase awareness of and access to non-pharmacological treatments for Black patients with CLBP may improve their outcomes.Item CMS MIPS Quality Measures Are Blind to High-Need High-Cost Homebound Older Adults Cared for in Home-Based Medical Care(2021) Bhatnagar, ShivaniPurpose: Multiple studies find home-based medical care a solution for high-need, high-cost homebound older adults. However, home-based medical care needs proper representation among the Merit-based Incentive Payment System (MIPS) and Center for Medicare and Medicaid Services (CMS)-specified quality measures to be financially viable in modern value-based care. Unfortunately, many useful measures for this population are unusable due to inadequate home medical visit codes in these measures' denominators. The objective of this study is to determine what proportion of the CMS 2019 MIPS Quality Measures that are potentially appropriate for patients receiving home-based medical care have home visit codes in their denominators. Methods: All 257 CMS 2019 MIPS Quality Measures were reviewed for applicability to homebound older adults cared for by home-based medical care providers. Measure details were reviewed in public documents from five CMS websites across 2017-2019. Results: Of the 257 CMS 2019 MIPS Quality Measures, 179 were inapplicable for this population, most commonly because they applied to specialist physicians outside of geriatric or palliative providers. Of the 78 measures that were deemed potentially appropriate, only 37 (47%) had home visit codes. Conclusions: The majority of CMS MIPS Quality Measures that are potentially appropriate for the care of homebound older adults, who receive home-based medical care, are unusable by home-based medical care practitioners for MIPS reporting. This represents a barrier for home-based medical providers in delivering effective care to this high-need, high-cost vulnerable patient population.Item The Effect of Sex on GABAA Receptor Activation in Vasopressin Neurons from the Supraoptic Nucleus(2021) Brock, Courtney; Bachelor, Martha E.; Little, Joel; Farmer, George; Balapattabi, Kirthikaa; Cunningham, JosephPurpose: Arginine Vasopressin (AVP) is important in maintaining proper fluid balance and plasma osmolality. Disruption in its regulation occurs in patients with chronic heart failure (CHF) and liver failure, which leads to poorer patient outcomes. AVP neurons from the supraoptic nucleus (SON) receive input from GABA, yet it is unknown what effects GABAA receptor activation has on these neurons under pathophysiological conditions or whether the effects are sex-specific. What is known is that under pathophysiological conditions, AVP neurons are unaffected by negative feedback which leads to excessive AVP release. Understanding the role of the GABAA receptor in these conditions is important. Here, we investigate whether activation of the GABAA receptor leads to sex dependent effects. Methods: Adult, intact, Sprague Dawley rats were anesthetized and bilaterally injected with the AAV2-0VP1-ClophensorN virus directly into the SON. After a two-week recovery, the animals were sacrificed and the brains were rapidly removed. Cells from the SON were dissociated and incubated for two hours. After incubation, recordings were taken using ratiometric live cell imaging. Selected neurons were sequentially excited at 445nm and 556nm and then emission data was collected between 500-550nm and 580-653nm respectively. After 40 cycles of 3-second recordings, muscimol (100nM), a GABAA receptor agonist was transiently applied to the cells. Results: In both males and females, application of muscimol resulted in chloride influx, which implies neuronal inhibition. Conclusion: Under normal physiological conditions, GABAA receptor activation does not show sex specific effects in neurons from the SON.Item Review: A 15-Year-Old Boy with Abdominal Pain, Hematochezia, and Anemia(2021) Nguyen, Ly; Modi, Saman; Huang, CliftonBackground: Intussusception is the telescoping of part of the intestine into a more distal segment. It can cause abdominal pain and hematochezia. Intussusception typically presents between 6 and 36 months of age, however, 3-4% of cases can occur in children over 10 years of age and are usually associated with a pathologic lead point. Case information: A 15-year-old male presented to the emergency department with recurrent abdominal pain, diarrhea, and hematochezia. The painless rectal bleeding was first noted when the patient was 9 years old. At that time, abdominal and colonoscopy exams and stool cultures were normal. Laboratory studies were consistent with iron deficiency and β-thalassemia (low Hb, Hct, MCV, elevated TIBC, low ferritin, low serum iron, elevated HbA2), but were otherwise unremarkable. The abdominal pain began 8 weeks prior to admission. The patient has a history significant for iron-deficiency anemia, β-thalassemia minor, and allergic rhinitis with nasal polyps, and a family history of Peutz-Jeghers syndrome with nasal polyposis in two siblings. Prior to admission, an abdominal and pelvic CT scan revealed an ileocolonic intussusception without a distinct mass. A diagnostic procedure and surgical resection revealed a resolved intussusception with a dysplastic polyp as the lead point. No evidence of carcinoma was noted. Conclusions: This case highlights a unique presentation of intussusception with a pathologic lead point in an atypical age group.Item The Influence of Ecogeographic Variation in Human Nasal Morphology on Thermal Conditioning of Inspired Air(2021) Thai, Elizabeth; Amaranayaka, Hasintha; Patil, Sandeep; Yokley, Todd; Dennis, Brian; Maddux, Scott D.Most air conditioning of inspired air occurs in the nasal passages and is largely dependent on external environments. Studies show strong associations between climate and ecogeographic patterning of human nasal morphology. Individuals indigenous to cold-dry environments exhibit relatively longer/taller/narrower nasal passages than individuals from hot-humid climates, and these morphologies are assumed to reflect functional differences. To test these associations between nasal morphology and air-conditioning function, we assessed cranial CT scans of 2 individuals"—one of European ancestry (EA) and one of West African ancestry (WA). 3D models of nasal passages were created using 3D Slicer software and were artificially dilated in-silico to simulate fully decongested nasal passages prior to collecting morphometric measurements, mucosal surface area (SA), and airway volume (AV). 3D models of each individual were then employed in Computational Fluid Dynamics (CFD) simulations, via ANSYS fluent software, to assess differences in intranasal airflow heat and moisture transfer. Ambient air conditions were set at -5°C, 35% relative humidity. As expected, the EA individual exhibited longer/taller/narrower nasal passages compared to the WA individual. The EA individual exhibited higher mucosal SA and lower AV resulting in a higher surface-area-to-volume (SA/V) ratio compared to the WA individual. Our CFD simulations also followed theoretical predictions. The higher SA/V ratio of the EA individual resulted in increased heat transfer compared to the WA individual. The results of our study provide support for assertions that ecogeographic variation in human nasal passages reflects climate-mediated evolutionary demands for intranasal air-conditioning.Item Optimization of CRISPR-Cas9 via the synergy of MD simulation and machine learning(2021) Liu, Jin; Wang, Duen-Shian; Liang, IvyCRISPR-Cas9, a promising gene-editing tool, sheds light on gene therapy. The normal DNA cleavage of CRISPR-Cas9 is programmed by a guide RNA (gRNA) template. However, recent studies showed that Cas9 cleavage occurs even without guidance from the gRNA in the presence of Mn2+ ions, implying the issue of off-target effect of Cas9. Here, we report a mechanism of this RNA-independent off-target cleavage (RI-cleavage) elucidated by molecular dynamic (MD) simulations. We further used machine learning algorithms developed by our lab to facilitate the design of novel Cas9 variants to reduce such RI-cleavage. In this study, we revealed the possible mechanism of RI-cleavage and further engineered Cas9 to reduce RI-cleavage via the power of machine learning. Our research serves as an excellent example showing the potential in the synergy of MD simulation and machine learning to optimize CRISPR-Cas9.Item Impact of Sleep Quality on Cardiovascular Responses to Simulated Hemorrhage in Humans(2021) Hua, Vincent; Barnes, Haley J.; Rosenberg, Alexander; Anderson, Garen K.; Luu, My-Loan; Rickards, CarolinePoor sleep quality may limit cardiovascular responsiveness to physiological stress. We hypothesized that subjects with poor sleep quality would be less tolerant to simulated hemorrhage, which would be associated with lower arterial pressure and cerebral blood flow, and higher heart rates compared to subjects with good sleep quality. Hemorrhage was simulated in 20 human subjects with lower body negative pressure (LBNP). Sleep quality was classified as POOR in 5 subjects (Global Pittsburgh Sleep Quality Index (PSQI) score ≥5), and GOOD in 15 subjects (Global PSQI score < 5). Markers of cardiovascular function were measured continuously throughout the LBNP protocol. Sleep quality had no effect on LBNP tolerance (POOR: 1453±223 s vs. GOOD: 1535±88 s; P=0.34), and there were no differences in the magnitude of central hypovolemia at presyncope (%Δ stroke volume, POOR: -53±8 % vs. GOOD: -49±4 %; P=0.32). However, there were differences in the magnitude of hypotension (%Δ mean arterial pressure, POOR: -18±3 % vs. GOOD: -22±2 %; P=0.08), cerebral blood flow reduction (%Δ MCAv, POOR: -19±6 % vs. GOOD: -28±2 %; P=0.03), and reflex tachycardia (% Δ heart rate, POOR: 103±30 % vs. GOOD: 72±9 %; P=0.09). There was a moderate association between sleep quality and the magnitude of MCAv reduction at presyncope (r=0.53; P=0.02). Sleep quality did not affect tolerance to simulated hemorrhage in healthy human subjects. While there were differences in hemodynamic responses, this may be related to premature termination of the protocol due to early onset of subjective presyncopal symptoms.Item Evaluating the anti-leukemic effect of clotam and copper-clotam using CCRF-CEM cell lines(2021) Patel, Krishna; Siraj, Sohail; Basha, Riyaz; Sankpal, UmeshPurpose: Acute lymphoblastic leukemia (ALL) is the most common type of cancer in children younger than 5 years. Patients with ALL have bone marrow that produces immature white blood cells, which are unable to effectively fight infections. NSAIDs are common pain reliving agents that act through COX inhibition, which stops the production of prostaglandins. Clotam (Tolfenamic Acid/TA) is an NSAID that has anti-tumor proliferative effects. It works through targeting specificity protein (Sp) transcription factors that assist cancer cells in inhibiting apoptosis. Our objective is to test TA and copper-TA (Cu-TA), a derivative of TA, to induce an anti-leukemic response. Methods: The T-cell ALL cell line CCRF-CEM was obtained from the American Type Culture Collection (Manassas, VA) and cells were cultured as per the supplier's instructions. A cell viability assay was performed in which cells were plated in a 96-well plate and treated with increasing concentrations of TA and Cu-TA. After 48-hours, the cells were lysed, and the amount of ATP in the cells was measured using luminescence. Using this data, IC50 values were calculated. Results: The IC50 values showed both TA and Cu-TA had anti-cancer proliferative effects. Cu-TA was 15 times more potent than TA in its ability to kill CCRF-CEM cells. Conclusion: Our results demonstrate that Cu-TA is more effective than TA for killing CCRF-CEM cells. This study suggests better implications of Cu-TA in ALL therapy, if further tested using pre-clinical models.Item An agent-based model for simulating viral infections(2021) Dobrovolny, Hana; Fain, BaylorAs we have seen in the past year, new viruses can spread rapidly and cause outbreaks that need a quick response from researchers to develop or re-purpose treatments. While experiments and clinical studies form the basis of this response, the data generated by these studies can be further leveraged through the use of mathematical models. Properly calibrated and validated mathematical models can make predictions about scenarios that are difficult to test experimentally, but are also faster and cheaper when testing possible treatment regimens. We have developed a realistic agent-based model of viral infections that runs on graphical processing units (GPUs), so it runs fast enough to simulate typical in vitro viral studies in a few hours. We present here testing and validation of the model for influenza infections and show that it can be calibrated to simulate different viral infections.Item Ternary Pseudo-Triphasic Phase Diagram for Lipid Formulation(2021) Dulie Kom Nzia, Jeanne; Dong, XiaoweiPurpose: Lipid-based formulations are frequently studied, they improve oral bioavailability of water-insoluble drugs and constitute 40% of new drugs. A ternary pseudo-phase diagram(TPPD) is a thesaurus tool used to define specific physical compositions of a thermodynamically stable solution. The aim of this study was to construct TPPD of lipid-based formulations and identify optimum composition for drug delivery. Method: The aqueous titration experimental method was used. A lipid and surfactant components were mixed in a fixed ratio. No cosurfactant was used. The mixture was heated at 45°C. Water was used as a titer and was added in increment of 5%-10% to up to 95% of the total mixture. After equilibrium, the mixture was observed for characterization. Data were gathered on a table, showing each increment of water, the amount of each component, and the percentage of each component in the mixture. Data collected were used to build TPPD through excel. Result: Comparing one set of 3 phase diagrams(PDs), the PD(Miglyol-812, TPGS, Water) presents a larger area of clear solution(CS), a very small area of microemulsion, and a small area of emulsion. The PD(Miglyol-812, Tween 80, Water) presents a smaller area of CS, a small area of microemulsion, and a larger area of emulsion. The PD(Miglyol-812, K El, Water) presents a small area of CS, a small area of microemulsion, and a large area of emulsion. Conclusion: All PD built present stable area of emulsion and microemulsion that can be reconstituted for optimum delivery.