bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of Curcumin and its Analogs as Adenosine Receptor Agonists for Treating Chronic Pain
Authors: Hamilton Luke J. , Pattabiraman Mahesh , Chandra Surabhi
Published: April 2020

Chronic pain debilitates millions of people throughout the world. From the mid‐1990s until the present, the principle pharmacologic treatment for chronic pain has been long‐term administration of opioid medications. However, between 1999 and 2007, the number of deaths related to opioid drug overdose rose throughout the world and more than tripled in the USA, causing concern. In response, studies have been initiated to discover new analgesics that carry a lower risk of addiction and overdose‐related death. All four adenosine receptor (AR) subtypes (A1AR, A2AAR, A2BAR, A3AR) have been investigated as targets for analgesic drugs, but concerns about cardiovascular side effects are impeding translation to the clinic. Drugs that are selective for A3AR offer a solution, because they inhibit pain without cardiovascular effects. Our aim is to clarify the structural characteristics of molecules that make them more selective for A3AR over other ARs. To generate ARs for testing, three human embryonic kidney cell lines are being grown, expressing A2A, A2B, and A3ARs respectively. We have selected curcumin as our test molecule because it has documented analgesic properties and because its binding affinity for ARs has not previously been described. We use photochemistry to alter curcumin, changing it from trans‐trans to cis‐trans. Both forms of curcumin are then evaluated for their binding affinity (Ki) for each of the AR subtypes using fluorescent ligand competitive binding assays. Results thus far show that isomerized curcumin binds to ARs more strongly than unmodified curcumin. Further binding assays will reveal whether isomerized curcumin is selective for A3AR over other ARs. Further testing with cAMP ELISAs will reveal whether binding of isomerized curcumin is associated with receptor activation. Activation of A3AR will result in depressed cAMP levels because A3AR is coupled to Gαi. Activation of A2AAR and A2BAR will result in elevated cAMP levels, because both A2 ARs are coupled to Gαs. In summary, the structure activity relationships discovered by this study will help future pharmacologists to design molecules that target A3ARs for the alleviation of chronic pain.

Support or Funding Information

We thank the Great Plains IDeA CTR program (NIGMS # 1U54GM115458‐01) and the American Chemical Society Petroleum Research Fund (# 54862‐UR4) for their funding support.
Functional Implications of Tyrosine Hydroxylase Immunoreactivity in Rat Brain
Authors: Ayles Brittney , Rahmani Sana , Kiriakedis Satomi , Li Hui-Yun
Published: April 2020

Hypothalamo‐pituitary‐adrenal axis is involved in the stress regulation. Understanding the cellular mechanisms involved in this system is critical in studying stress response. Tyrosine hydroxylase (TH), the rate‐limiting enzyme of catecholamine synthesis, has been suggested to play a pivotal role in regulating the neuronal functions under stress. Previous findings showed the brainstem ascending TH‐immunoreactive fibers participate in the cellular activation of paraventricular nucleus of the hypothalamus in response to neurogenic stress. Current study aimed to study the role of TH in regulating brain areas which are involved in the neural circuits in stress response. Immunocytochemical staining of TH confirmed the TH‐immunoreactive positive neurons are found in the locus coeruleus, ventral lateral medulla, nucleus of the solitary tract and zona incerta; wihile TH‐ir fibers are widely found in the forebrain region including the striatum, paraventricular nucleus of hypothalamus, and amygdala. These are areas rich in neurons bearing dopamine receptor, corticotropin‐releasing factor and oxytocin. Using double immunohistochemical staining, we are able to demonstrate the functional significance of tyrosine hydroxylase inputs to these different populations of neurons.

Support or Funding Information

We are grateful to Oregon Tech for supporting undergraduate research as well as Dr. Paul Sawchenko for his generosity in donating rat brain tissues.
Airway Relaxation Mechanisms and Structural Basis of Osthole to Improve Lung Function in Asthma
Authors: Tu Yaping , Wang Sheng , Xie Yan , Huo Yan-Wu , Li Yan , Abel Peter W. , Jiang Haihong , Zou Xiaohan , Jiao Hai-Zhan , Kuang Xiaolin , Wolff Dennis W. , Huang You-Guo , Casale Thomas B. , Wei TaoTao , Cao Zhengyu
Published: April 2020

Background and objective

Overuse of β2‐adrenoceptor agonist bronchodilators causes receptor desensitization, resulting in decreased efficacy and an increased risk of death in asthma patients. This is a critical unmet problem in asthma management, and novel drugs are urgently needed. Here, we report that osthole, a coumarin derived from a traditional Chinese medicine, induces complete relaxation of preconstricted airways in mouse precision‐cut lung slices, irrespective of β2‐adrenoceptor desensitization.

Methods

Mouse precision‐cut lung slices as an in vitro model were used to analyze airway relaxation. The cAMP level in human airway smooth muscle (ASM) or HEK293 cells was determined by phosphorylation of vasodilator‐stimulated phosphoprotein using Western blot or cAMP dependent reporter luciferase assay. The secretion of prostaglandin E2 (PGE2) in ASM cells or lung slices was measured by ELISA assay. The effect of osthole on airway hyperresponsiveness (AHR) was measured in both house dust mite (HDM)‐sensitized mice and naïve regulator of G‐protein signaling 2 (RGS2) KO mice. The binding sites of osthole with PDE4D5 catalytic domain were analyzed by crystallization and X‐ray diffraction.

Results

Osthole administered in vivo ameliorates AHR, a hallmark of asthma, in murine asthma models. Mechanistic studies revealed that osthole inhibits phosphodiesterase 4D (PDE4D) activity to amplify endogenous PGE2 signaling in ASM cells, which eventually triggers cAMP/protein kinase A‐dependent relaxation of airways. The crystal structure of the PDE4D complexed with osthole reveals that osthole binds to the catalytic site to prevent cAMP binding and hydrolysis. Together, our studies elucidate a specific molecular target and mechanism by which osthole induces airway relaxation.

Conclusion

Identification of osthole binding sites on PDE4D will guide further development of novel therapeutics for β2‐adrenoceptor agonist‐resistant asthma.

Support or Funding Information

This work was supported in part by the National Institutes of Health (R01HL116849) and Nebraska State LB595 Research Program, USA; the National Laboratory of Biomacromolecules and the Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences (CAS); the CAS/SAFEA International Partnership Program for Creative Research Teams; the National Key R&D Program of China (2017YFA0205501); the National Natural Science Foundation of China (21777192 and 31500623)
Role of novel receptor GPR171 in chemotherapy‐induced neuropathic pain
Authors: Edwards Talyor M. , Ram Akila , McCarty Ashley , Bobeck Erin N.
Published: April 2020

First‐line chemotherapies against solid tumors are highly efficacious in reducing the tumor burden, but have many adverse side‐effects including nerve damage, leading to chronic pain. Non‐addictive, efficacious pain relievers are an area of active interest, and we propose a novel target to address this pressing issue. GPR171 is a G‐Protein Coupled Receptor that was recently deorphanized and was identified to be expressed in the brain in regions that regulate reward, anxiety, and pain. Within the pain circuit, it was shown previously that systemic administration of the GPR171 agonist enhances morphine antinociception in acute pain tests. Preliminary data from our lab has shown that GPR171 activation can also alleviate persistent inflammatory pain. However, the role of this receptor has not been investigated in other chronic pain models. Given these findings in acute and inflammatory pain, we hypothesize that GPR171 can reduce neuropathic pain. To test this hypothesis, we investigate the role of GPR171 in chronic neuropathic pain. We tested the efficacy of a GPR171 agonist in a chemotherapy‐induced neuropathy mouse model. Neuropathic pain was induced by injecting paclitaxel (16 mg/kg) followed by assessment of mechanical pain thresholds using Von Frey filaments. Our results indicate that the GPR171 agonist can relieve neuropathic pain. In addition, using immunofluorescence we observed that mice with neuropathic pain show a decrease in GPR171 receptors within a key brain region involved in pain modulation, the periaqueductal gray (PAG). Overall, this study proposes that GPR171 may be a novel target for the treatment of neuropathic pain.
Role of WNK1/OSR1 in maintaining endothelial tight junction integrity
Authors: Jaykumar Ankita , Plumber Sakina , Stippec Steve , Earnest Svetlana , Cobb Melanie
Published: April 2020

Deletion of the WNK1 (with no lysine kinase‐1) gene in mice causes embryonic lethality due to failed vascular development. This phenotype can be rescued by endothelial‐specific expression of WNK1 or a constitutively active form of its substrate OSR1 (Oxidative Stress Responsive‐1). Additionally, depletion of WNK1/OSR1 decreased in vitro angiogenesis and cell migration in endothelial cells. This indicates an essential function of WNK1/OSR1 in angiogenesis. However, the underlying mechanism by which WNK1/OSR1 mediates this process is unclear. WNK1 is known to regulate TGF‐β/Smad signaling which induces epithelial‐mesenchymal transition (EMT), a process utilized in cell migration and angiogenic sprouting during physiological and pathophysiological vascular remodeling. OSR1 is reported to form a complex with the TGF‐βII receptor at tight junctions which mediates dissolution of tight junction upon activation by TGF‐β. Therefore, we hypothesize that WNK1/OSR1 axis is necessary for TGF‐β/Smad‐induced disruption of cell‐cell adhesion, thereby promoting cell migration and angiogenesis. We found that WNK1 pharmacological inhibition decreased the expression of TGF‐β‐induced EMT mediators such as the receptor tyrosine kinase Axl which is known to be important for maintaining tight junction integrity in HDMECs (Human Dermal Microvascular Endothelial Cells) (Normalized densitometry values; WNKi‐ Axl: 0.2 ± 0.05 vs. siControl: 1, n=3) and in primary HUVECs (Human Umbilical Vein Endothelial Cells) (normalized densitometry values; WNK1i: 0.4 ± 0.1 vs. DMSO: 1, n=3, *p<0.05). We also determined that OSR1 and Occludin, a tight junction protein are co‐localized and that inhibition of WNK1 using a small molecule WNK1 inhibitor, decreased their co‐localization and tight junction breakdown during TGF‐β signaling in primary HUVECs (Pearson’s coefficient; Control; 0.08 ± 0.2 vs. TGF‐β: 0.61 ± 0.15 vs. TGF‐β+WNK1i: 0.12 ± 0.25, n=2, 2 replicates per experiment). Finally, we observed that bands corresponding to phosphorylated form of Occludin on a Western blot were enhanced in WNK inhibited samples compared to control (normalized densitometry values; TGF‐β+WNKi: 1 vs. TGF‐β: 0.21 ± 0.07, n=2). Since Occludin is known to be dephosphorylated during tight junction breakdown, the presence of the phosphorylated form of Occludin with WNK inhibition may indicate the disruption of cellular processes leading to TGF‐β‐dependent tight junction breakdown. These data suggest WNK1‐dependent function of OSR1 in cell migration and angiogenesis through regulating tight junction integrity. Combined, these data indicate the importance of WNK1 function in endothelial cell migration and angiogenesis. Understanding how WNK1 regulates angiogenesis and cell migration is immediately relevant during development, fibrosis as well as cancer.

Support or Funding Information

American Heart Association postdoctoral fellowship to Ankita Jaykumar and NIH funding R01 HL147661 to Melanie H. Cobb
HYPE‐mediated AMPylation as a novel therapeutic target for neurodegeneration
Authors: Camara Ali
Published: April 2020

A major hallmark of Parkinson’s disease (PD) is the deposition of the intrinsically disordered protein α‐synuclein (αSyn) into intracellular inclusions termed Lewy bodies. HYPE—the sole human representative of a conserved family of adenylyltransferase enzymes—has been shown to covalently modify (AMPylate) αSyn in vitro. Remarkably, HYPE‐mediated AMPylation ameliorates many of the neurotoxic phenotypes of αSyn implicated in the progression of PD, such as αSyn fibrillation and membrane permeability. These potentially cytoprotective phenomena conferred by HYPE’s adenylyltransferase activity make it an attractive therapeutic target. Unfortunately, wild‐type HYPE is intrinsically inhibited, showing only basal AMPylation levels relative to a constitutively active mutant (E234G‐HYPE). To this end, we set out to screen both FDA‐approved and proprietary small‐molecule compound libraries towards the identification of novel manipulators of HYPE AMPylation. Employing fluorescence polarization (FP) of a labelled ATP analogue (ATP‐FAM) on a 384‐well microplate platform, we’ve developed a robust, high‐throughput assay suitable for monitoring changes in AMPylation. First‐pass selection of our combined ~10,000 compound libraries yielded promising hit percentages: 0.3 for activators of WT HYPE, and 1.2 for inhibitors of E234G‐HYPE. Challenging neuronal cell culture models of PD with these hits provides molecular insights into αSyn‐induced neurotoxicity, and paves the path for novel therapeutic strategies in combating PD.

Support or Funding Information

This project was made possible with support from Grant # 11000704 (S. Mattoo, PI), Grant # UL1TR002529 (A. Shekhar, PI), and Grant # TL1TR002531 (T. Hurley, PI), from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.
Effects of ECG Summarization Methods on the Characterization of Drug‐Induced QT Prolongation – a Demonstration of Need for Standard Methods of ECG Beat Summarization6.3.6
Authors: Ether Nicholas , Bailie Marc , Leishman Derek , Lauver Adam
Published: April 2020

A vital aspect of drug‐development is the pre‐clinical screening of drug‐induced cardiotoxicity. The effect a drug has on the ECG QT‐interval (QTi) is an especially vital screening due to the positive relationship between QT prolongation and arrhythmic events, such as Torsade de Pointes. Consequently, the accurate recording, measurement, and calculation of QTi and heart rate corrected QTi is necessary for cardiotoxicity studies. However, many aspects of these processes are subjective, and techniques vary greatly between researchers and across studies. Examples of subjective aspects of QT prolongation (QTp) characterization include, but are not limited to: the method of heart rate correction for QT (QTc), the number of data points considered, the number of ECG beats or amount of time averaged per data point, and the specific time points evaluated. While the effects of different QTc methods has previously been explored in the literature, the efficiency and accuracy of various ECG summarization methods still warrants further refinement. We evaluated the effects of different methods in order to begin identifying potential industry standards that can minimize the number of data points needed without compromising model prediction. In order to demonstrate the effect different summarization methods have on the identification of drug‐induced QTp, we used Ponemah software to analyze previously collected ECG telemetry data from non‐human primates. The subjects were administered vehicle, three doses of dofetilide, vehicle again, and three doses of quinidine, with a week between each dose. Data was summarized using various methods, including 1, 15, 30, 60 and 120 minute continuous averages as well as 1‐minute averages taken at 1‐hour intervals. We then quantified the effect size of the doses on QTi using restricted maximum likelihood (REML) estimation. Using these estimates, differences in results between the summarization methods were then compared. After finding dramatic differences, we performed power analyses on each summarization method to identify the most accurate and efficient methods. Results suggest that characterization of the dose response and power varied depending on the ECG summarization method used. Using continuous interval averages was more consistent and had greater power than selecting a 1‐minute average at various intervals. Further improvement is achieved by taking pharmacokinetics into account when selecting time intervals. These results confirm the need for careful consideration of ECG summarization methods during study design in order to avoid missing vital data or introducing researcher bias. It also provides evidence to support the recommendation for the introduction of a uniform industry standard for analyses of cardiovascular data. 6.3.6

Support or Funding Information

This work was funded in part by a research grant from Eli Lily and Co, Indianapolis, IN
CARING PROFESSIONAL HEALTH: A POWERFUL OF YOGA
Authors: Rossi Lia Mara , Galdeano Denival Soares , Marques L. S. , Barros Mirna Duarte
Published: April 2020

INTRODUCTION

Health professionals and medical science students have high levels of stress, anxiety and performance‐related musculoskeletal disorders. Long workloads and strenuous shifts, feelings of empathy for the suffering of patients and families, demands for cleanliness and hospital conduct, and the need for many hours of study cause mood swings and emotional instability, diminish immune system function and increase psychosomatic diseases in students and health professionals.

The goal of this study was to evaluate the effects for health professionals and students that participated in YogaSanta Program, an opened and free program of Yoga.

MATERIAL and METHODS

The World Health Organization Quality of Life assessment (WHOQOL‐BREF) was sent by e‐mail for health professional as well as for Medicine, Nursing e Speech Therapist students from Santa Casa de São Paulo School of Medical Sciences(São Paulo – Brazil). Who have shown WHOOQL‐BREF score under 50% were invited to participate of “YogaSanta Program”. YogaSanta aims “to take care of people who care for people” and offer free Yoga sessions for all. We selected 22 adults that practice one‐hour yoga session twice a week for 3 months consecutively. The sessions included Pranayama (breathing exercises), Asanas (postures), Yoganidra (relaxation) and Dhyana (meditation). All participants completed Visual Analogue Scale (VAS) for assessing stress‐level in two different moments: (VAS after = VAS‐A) immediately after Yoga class and (VAS before = VAS‐B) just before Yoga class. VAS varies from zero to 10 where “zero” corresponds to “no stress” and “10” corresponds to the “worst stress level”. VAS‐B and VAS‐A were compared through the t‐paired test and the results are shown in this study.

RESULTS

From 22 participants, 6 occupied administrative functions the hospital, 13 were students and two were faculty members. The mean ± standard deviation (SD) of participants age was 32.3 ± 12.2 yrs (Range: 22 – 60 yrs). The mean ± SD of VAS‐B and VAS‐A were compared (EVA‐B 3.34 ± 1.4 vs. VAS‐A 1.89 ± 1.4) and showed a low correlation = 0.574 with p<0.001.

DISCUSSION and CONCLUSION

Some previous articles suggest that physiological benefits like muscle strength, flexibility, blood circulation and oxygen uptake, hormone functions and the relaxation induced by meditation helps to stabilize the autonomic nervous system and this help yoga practitioners become more resilient to stressful conditions and reduce a variety of important risk factors for different diseases, especially cardio‐respiratory disorders.

Based on this and the results presented above, we believe that the YogaSanta program is an efficient option for producing improvements in psychological well‐being and decreasing mood disorders in health professionals and health students, thus contributing to improving performance and quality of life and help to stress reduction.
Amelioration of metaflammation induced in rats by exogenous hydrogen sulfide: Focus on mesenteric adipocyte oxidative stress
Authors: Zayachkivska Oksana , Revenko Oleg , Bula Nazar , Savytska Maryana , Yaschenko Antonina
Published: April 2020

Metaflammation (metabolic inflammation) is low‐grade chronic inflammatory status linking to nutrient excess. Visceral adipocytes in mesenterium (AM) related to high fructose diet (HFD) are sources for metaflammation process, seriously threatening human health. Novel pathogenesis of AM is still unclear and there are no specific effective drugs for treatment of AM. Recently hydrogen sulfide (H2S) demonstrated potent vasodilatation actions which is promised for AM that have reduced vascularization Therefore, the aim of this study was designed to investigate the effects and underlying mechanisms of H2S on the prevention and treatment of experimental rats with AM during HFD.

Methods

The study was done on rats with standard diet (SD, control group) and 28 days HFD, and acute water immersion restraint stress (WRS) was induced for evaluation AM adaptive response and testing therapeutic potential of exogenous H2S donor NaHS (100 μmol/kg/day) and the hybrid H2S‐Aspirin (ATB 340, H2S‐ASA, 17,5 mg/kg/day) vs conventional aspirin (ASA, 10 mg/kg/day) on AM were evaluated by damage index by electronic microscopic study, and glucose level, activity of Cystathionine gamma‐lyase (CSE), Cystathionine beta‐synthase (CBS), Thiosulfate‐dithiol sulfurtransferase (TST) and Carbonyl group (CG) level in blood were examined biochemically (Institutional Ethical Committee Approval: 23/04/2018 № 4).

Results

In HSD group with NaHS treatment protected the endothelial and sub‐endothelial structures in mesenterium destructions were absent, the nucleus of endothelial cells it was observed an uneven condensation of chromatin. Effect of hybrid H2S‐aspirin characterized as a protective on AM against HFD and WRS‐induced injury, exhibited a downward trend in the inflammatory infiltration, lowering the level CG, which may indicate a reduction in oxidative stress under the influence of hybrid H2S‐aspirin. ATB‐340 showed an increase CSE, CBS and TST activities vs ASA pretreatment (p<0.05). The use of aspirin and NaHS has shown an increase on 67% CSE and CBS activities in HSD rats vs saline group.

Conclusion

These findings suggested that exogenous H2S prevented and ameliorated AM metflammation via reduction of oxidative damage, and provided a theoretical basis for effective tool for prevention of HFD‐related AM against long‐term postprandial hyperglycemia.

Acknowledgment

Authors would like to thank Prof. JL Wallace for ATB 340 providing and his expert suggestions.
Upregulation of Renal ENaC Activity in Spontaneously Hypertensive Rats Is Independent of Aldosterone
Authors: Buncha Vadym , Cherezova Alena , Mamenko Mykola
Published: April 2020

Spontaneously hypertensive rat (SHR) has been widely used as a model to interrogate the contribution of the kidney to the pathogenesis of essential hypertension in humans. Elevation of blood pressure in SHR has been associated with altered renal function, since predisposition to hypertension can be transferred with a transplanted kidney into a normotensive Wistar‐Kyoto (WKY) strain. Renal defects result in excessive water and sodium retention leading to increased circulating volume and blood pressure. Abnormally elevated Na+ reabsorption in SHR is associated with the overactive renin‐angiotensin‐aldosterone system (RAAS) and with the increased abundance of the renal epithelial Na+ channel (ENaC) responsible for final regulation of sodium reuptake in the kidney and pivotal for long‐term blood pressure control. The existing data on regulation of ENaC by RAAS components in SHR is inconsistent and molecular determinants of excessive ENaC activation remain largely obscure. In the present study, we tested the efficacy of aldosterone antagonism to inhibit ENaC activity, decrease renal Na+ reabsorption and reduce blood pressure in SHR of both sexes. Where necessary, age‐matched (12–15 week‐old) WKY rats were used as controls. Consistent with previously published data, enzyme immunoassay analysis revealed elevated circulating aldosterone levels and semi‐quantitative immunoblotting – higher expression of renal ENaC protein in SHR when compared to WKY controls. Patch‐clamp experiments in split‐opened collecting ducts showed that ENaC activity was moderately increased in SHR when compared to WKY. Systemic administration of aldosterone antagonist, spironolactone (30 mg/kgBW·day) did not affect renal ENaC activity or protein abundance in SHR. Aldosterone antagonism did not alter the expression of sodium‐chloride co‐transporter (NCC) in the kidneys of SHR. Tail‐cuff plethysmography showed that treatment with spironolactone at doses 30–200 mg/kgBW·day for 2 weeks failed to decrease blood pressure in SHR and did not result in any detectable changes in urinary sodium excretion. We did not observe any sex‐specific differences pertaining to ENaC activity or responsiveness to spironolactone in SHR males and females. Thus, we conclude that aldosterone is not a primary driver of excessive ENaC‐dependent renal Na+ reabsorption in SHR.
Food Availability Regulates Circadian Blood Pressure Rhythms in Rats
Authors: Rhoads Megan K. , Jin Chunhua , Pollock David M.
Published: April 2020

Blood pressure (BP) changes throughout the day and follows a circadian rhythm that is believed to be controlled by so‐called clock genes. Loss of BP reductions during the sleep period are associated with increased risk of cardiovascular and renal disease. Recent human studies suggest that timing of food intake can affect BP. Therefore, the aim of our study was to determine if food availability affects blood pressure rhythms in the absence of light cues in rats with functional and dysfunctional molecular clocks. Individually housed Bmal1KO rats (n=4) and littermate controls (n=6) were implanted with DSI HD‐S10 telemetry transmitters at 10 weeks of age. Data were collected for 2 minutes every 10 minutes at 1000 samples/second. Animals were given ad lib access to food and water in a 12:12 light/dark (LD) cycle for 2 weeks to establish normal BP rhythms. After 2 weeks, animals were placed in constant darkness (DD) with ad lib food and water for 4 weeks. Food availability was then restricted to 12 hours per day (CT 14.5 to CT 2.5, CT=clock time) under DD conditions (DD‐TRF, TRF=time restricted feeding) for 1 week. Cosinor analysis was performed to determine period, amplitude and phase of activity and systolic blood pressure (SBP) rhythms. In LD conditions, period, amplitude, and phase of SBP and activity were similar between Bmal1KO and control rats. After 4 weeks of DD, control rats had SBP rhythms that peaked at CT 19.9±0.3 hours while Bmal1KO rats had SBP rhythms that peaked at CT 12.0±1.8 hours (p<0.05) indicating a significant change in acrophase. SBP amplitude and period were similar in both groups under DD conditions. For locomotor activity, period was shortened in Bmal1KO rats in DD compared to control rats (control: 24.1±0.01 hours vs. Bmal1‐KO: 23.8±0.08 hours). In DD‐TRF, control rats have SBP rhythms that peak at CT 20.69± 0.44 hours, when food was available. However, SBP rhythms of Bmal1 rats peaked at CT 13.94±3.4 hours. SBP amplitude, period, and activity period were similar between groups during DD‐TRF. These results suggest that food availability and the molecular clock contribute to the timing of systolic blood pressure rhythms in rats.
Transitioning from Traditional Fill‐In to Multiple Choice Question (MCQ) Format for Anatomy Practical Examinations: Should We?
Authors: Werle Laura , Stokoe Lily , Pravetz Matthew , Márquez Samuel
Published: April 2020

United States medical schools are moving to multiple choice‐style assessments, away from traditional fill‐in, for anatomy practical exams. Practical exams for medical students have long consisted of identifying anatomical structures and writing the answer on an answer sheet. This study reports several aspects of this change at two medical schools: New York Medical College and SUNY Downstate Health Sciences University. The report also documents the evolution of opinion of faculty, teaching assistants [TAs] and students with respect to the benefits of each testing modality. Student t‐tests were used to compare test score data between the two testing modalities employed at each institution. Results showed there were no statistically significant differences between fill‐in and MCQ mean scores for NY Med (5 years of fill‐in vs 5 years of MCQ) and Downstate (5 years of fill‐in vs one year of MCQ), 0.5 and 0.7 (p < 0.05), respectively. Resistance and support for the transition from faculty, TAs and students were common from both schools irrespective of constancy in student performance between the two testing formats. The quantitative results were surprising as an expected increase in the statistical mean in the MCQ format did not occur. Logistical and pedagogical merits of each exam format were assessed. Faculty and TAs resistant to MCQ exams predicted that offering choices would provide an advantage, as there would be a 25% chance of having a correct choice against the binary of a correct or incorrect written answer. TAs felt that a fill‐in format tapped into a higher cognitive level to reach the correct answer. Students with English as their second language, on the other hand, strongly supported the MCQ exam format. MCQ exams had the benefit of being corrected within 10 minutes of submission with Item Analysis accompanying the report allowing identification of poorly tagged questions and providing the opportunity to make corrections in real time for the entire class. Effectively assessing medical student competence must be addressed in contemporary curriculum design but is MCQ a reliable way to do so?
Hippo Signaling Pathway has a critical role in Zika Virus Replication and in the Pathogenesis of Neuroinflammation
Authors: Garcia Gustavo , Paul Sayan , Beshara Sara , V Krishnan Ramanujan , Ramaiah Arunachalam , Nielsen-Saines Karin , Li Melody M. H. , French Samuel W. , Morizono Kouki W. , Kumar Ashok , Arumugaswami Vaithilingaraja
Published: April 2020

Zika virus (ZIKV) is a reemerging human pathogen that causes congenital abnormalities including microcephaly and eye disease. The cell/molecular basis of ZIKV and host interactions inducing ocular and neuronal pathogenesis are unclear. Here we noted that the Hippo/SWH signaling pathway, which controls organ size through progenitor cell proliferation and differentiation, is dysregulated following ZIKV infection. In human fetal retinal pigment epithelial (RPE) cells, there is an early induction of transcriptional co‐activator, YAP, which is later degraded with a corresponding activation of the TBK1‐IRF3 Type I interferon pathway. YAP/TAZ silencing results in reduced ZIKV replication, indicating a direct role of Hippo pathway in regulating ZIKV infection. Using an in vivo Ifnr1−/− knockout mice model, ZIKV infection was found to reduce YAP/TAZ protein levels while increasing pYAP Ser127 in the retina and brain. Hippo pathway is activated in major components of the blood‐brain barrier (BBB), incuding endothelial cells and astrocytes. In addition, our data shows AMPK signaling pathway’s role in regulating YAP/TAZ in ZIKV‐infected cells. Our data demonstrates ZIKV infection initiates a crosstalk among AMPK‐Hippo‐TBK1 pathways, which regulate antiviral and energy stress responses during oculo‐neuronal inflammation.

Support or Funding Information

This work was funded by California Institute for Regenerative Medicine (CIRM) Quest – Discovery Stage Research Projects Grant (DISC2‐10188) to V.A. at the University of California, Los Angeles.
Electrochemical DNA Biosensors for Detecting Nontuberculous Mycobacteria
Authors: Poch Dylan , Sodia Tyler , Honda Jennifer R. , Bonham Andrew J.
Published: April 2020

Mycobacterial infection and mortality rates remain very high; it is estimated that 10 million cases of Mycobacterium Tuberculosis (M.Tb) emerge each year. However, a less‐studied class of mycobacteria is becoming a growing concern: Nontuberculous Mycobacteria (NTM). Over the last 30 years, NTM‐based lung disease has grown to outnumber M.Tb infections in many regions. The gold standard for positive identification of NTM in a patient sample involves a lengthy microbiological culture‐based method which frequently takes weeks to months in order to make a diagnosis. In this work, we propose a novel electrochemical biosensor that can be used to detect NTM using a DNA aptamer. Our biosensor changes conformational shape when binding to the molecular target mannosylated lipoarabinomannan (ManLAM). ManLAM is a complex lipoglycan abundantly present in the cell envelope of many NTM and M.Tb. This conformational change is measured by voltammetric analysis, and we observe a significant difference in electrochemical current upon binding of ManLAM. We are also expanding our NTM detection efforts through the design of a biosensor targeting glycopeptidolipids (GPL). This strategy will increase our specificity, as GPL is absent from M.Tb. The expected outcome of this project is to greatly improve patient outcomes for NTM infection by allowing rapid, point‐of‐care diagnostics that eliminate invasive sampling and lengthy culturing requirements. This would significantly reduce the time between correct diagnosis and subsequent treatment.
Optimization of Click Chemistry for Bioconjugation
Authors: Reilly Quinn , Mulllin Alissa C. , Oza Javin P.
Published: April 2020

Application of the expanded genetic code has created specialized proteins that enable new methods to investigate biological systems. Biomaterials and antibody‐drug conjugations and often employ “click chemistry” utilizing copper(I)‐catalyzed azide‐alkyne cycloaddition. We have functionalized green fluorescent protein (GFP) by incorporating a non‐standard amino acid (nsAA), p‐Azido‐phenylalanine. However, the literature on the rate and yield of this reaction remains incomplete. To enhance bioconjugation, we have explored and optimized reaction conditions and stoichiometry. We employed two corroborating assays to test single‐ and double‐click reaction events: size exclusion chromatography and a fluorescence polarization assay. We evaluated the efficiency of a single click reaction between a small molecule fluorophore and our functionalized GFP. Completed click reactions were run through a purification column and fluorescence readings of eluted samples were used to evaluate yield. Analysis of this single click event was also conducted through a fluorescence polarization assay. This allows the determination of the reaction rate and yield in real time. Additionally, yield of double click reactions between two functionalized proteins and a polyethylene glycol (PEG) molecule with two alkyne handles has been evaluated using SDS‐PAGE. Through these efforts, we report reaction yields that surpass previously reported values. These advances support the improved employment of click chemistry for broad applications in biofunctionalization such as the development of biomaterials and antibody‐drug conjugates

Support or Funding Information

Funding and support provided by the Cal Poly Chemistry and Biochemistry Department, the Center for Applications in Biotechnology, and the National Science Foundation.
Discovery of a Fibulotalocalcaneus (Peroneotalocalcaneus) Muscle – Rare Variant of Fibularis Quartus
Authors: Herrold Christina B. , Angilello Allison E. , Burkett Jesse T. , Zdilla Matthew J. , Lambert H. Wayne
Published: April 2020

The fibularis (peroneus) quartus muscle is a variant muscle located in the lateral leg compartment that, when present, resides posterior to the fibularis longus and brevis muscles. Only found in approximately 11.5% of legs, the fibularis quartus arises from the lower one‐third of the fibula and descends to wrap posteriorly around the lateral malleolus. Confusion in the nomenclature arises due to the variations seen in regards to the distal insertion point of this variant leg muscle. The fibularis quartus can insert into the foot at a variety of locations, leading to many variations in the naming of the subtypes of this muscle. If the fibularis quartus muscle inserts into the retrotrochlear eminence and fibular (peroneal) trochlea of the calcaneus, it is called the fibulocalcaneus externus, which are the most common insertion sites. More distally, the fibularis quartus can insert into the cuboid bone, which is called the fibulocuboideus, though this variant is seen less frequently. Even further into the foot, a variation of the fibularis quartus may insert into the fifth metatarsal of the foot, changing its name to the fibularis digiti minimi (quinti). In this study, we examined the lateral leg compartment to see if a fibularis quartus may be present. In three of 20 legs (15%), a fibularis quartus muscle was identified. The first two examples were the more common fibulocalcaneus externus variant, in that this muscle inserted into the calcaneus. The third example was unique in that its distal tendon split to insert onto the calcaneus as well as the talus bone. The rare example of a fibulotalocalcaneus (peroneotalocalcaneus) muscle has only been reported once previously. This rare variant of the fibularis quartus is only the second known example of this exceedingly rare fibularis quartus variant. This finding is important clinically due to the presence of a fibularis quartus muscle being related to chronic lateral ankle pain and ankle instability. It is also important for radiologists, surgeons, and clinicians to be aware of these lateral leg muscle variants when looking at radiological imaging or in advance of surgical procedures in the lateral leg or foot.

Support or Funding Information

T. Walley Williams Summer Anatomy Research Fellowship; WV Research Challenge Fund [HEPC.dsr.17.06]; WVU Research Apprenticeship Program (RAP)
SLC26A3 Deficiency Alters the Colonic Lamina Propria Lymphocytes
Authors: Jayawardena Dulari , Priyamvada Shubha , Kageyama Takahiro , Majumder Apurba , Sano Teuyuki , Dudeja Pradeep K.
Published: April 2020

SLC26A3 or Down Regulated in Adenoma (DRA) is the key anion transporter involved in electroneutral NaCl absorption in the mammalian intestine. DRA knockout (KO) mice exhibit diarrheal phenotype and greater susceptibility to experimental colitis. A recent genome wide association study (GWAS) identified SLC26A3 polymorphism leading to lower DRA expression as a risk factor for ulcerative colitis. Additionally, we have shown that genetic deficiency of DRA in mice results in compromised barrier integrity and increased gut permeability. These studies render DRA to have a more complex involvement in IBD, beyond its transporter function. Enhanced intestinal permeability is associated with activation of mucosal immune system and may contribute to alterations in lamina propria lymphocytes consisting of innate (innate lymphoid cells ILC1, ILC2, ILC3) and adaptive immune system (Th1, Th2, Tregs and Th17). Thus, we sought to determine the total levels of lamina propria lymphocytes both innate and adaptive, to decipher their involvement in intestinal pathology observed in DRA KO mice. Ileal and colonic lamina propria lymphocytes were isolated from Wild type (WT) and DRA KO mice (n=7). Fluorescence Assisted Cell Sorting (FACS) analysis was carried out in these mice to determine the various subtypes of CD127+ ILCs and TCRb+/CD4+ Th Cells. Isolated lymphocytes were also stimulated to secrete cytokines using phorbol 12‐myristate 13‐acetate/PMA (50 ng/mL) and ionomycin (500 ng/mL) and were further analyzed with FACS. Our results demonstrated that GATA3+ colonic Th2 cells were significantly increased along with the corresponding CD127+/GATA3+ ILC2 in DRAKO mice compared to WT mice. These cells partake in mediating intestinal fibrosis and collagen deposition which are highly upregulated in DRAKO mice colons. However, tuft cell hyperplasia which may contribute to increased ILC2 was not observed in DRA KO mice. Similarly, the anti‐inflammatory CD127+/RORgt+ ILC3, which are important in maintaining intestinal epithelial integrity and intestinal cell homeostasis were significantly down‐regulated showing lower secretion of the important cytokine IL‐22 in DRAKO mice colons. However, the FOXP3+ Treg levels increased in colon of KO mice. The differences in lymphoid cells were mainly detected in the colon (where DRA expression is higher) and were less evident in the ileum. Overall, our data indicate that loss of DRA transporter results in altered intestinal lamina propria lymphocytes in the colon which may in part contribute to the higher susceptibility of DRA KO mice to inflammatory and infectious insults.

Support or Funding Information

NIH/NIDDK, Department of Veterans Affairs
Isolation and Characterization of Antibiotic Resistant Bacteria from the Lane College Soil
Authors: McCullough Mariah , Baker Aliayah , Payne Quindarius , Van Stry Melanie
Published: April 2020

Since the late 1940’s antibiotics have been an asset to the treatment of bacterial diseases. Considering they are naturally occurring or synthetically made, they still slow down the growth of bacteria. β‐lactam antibiotics, like penicillin, kill sensitive bacteria by inhibiting the enzyme transpeptidase required for cell wall synthesis. The β‐lactamase producing bacteria are important contributors to resistant infections that have increasing concern in the battle against infectious diseases. Our goal for this project is to isolate and characterize antibiotic bacteria from the soil on the Lane College campus. In addition, we are characterizing beta‐lactamases present in these bacteria. We hypothesize that bacteria in the sample will be resistant to the beta‐lactam antibiotics. We collected a soil sample from Lane College campus and screened the presence of bacteria resistant to ampicillin. We selected three colonies for further analysis. We then used the β‐lactam antibiotics penicillin, amoxicillin, and ampicillin in the disk assay experiment to test for multidrug resistance. We also further characterized the bacteria with Gram staining. We concluded that our sample did contain multidrug antibiotic resistant bacteria. We will screen for the presence of the beta‐lactamases TEM‐1 and Bla‐1 with PCR followed by gel electrophoresis.

Support or Funding Information

This work is supported by NSF HRD 1623340.
Differential Expression of Murine Renal Genes in Olfactory Receptor 78 Null Mice
Authors: Sanchez Jason Castillo , Xu Biyang , Poll Brian , Gupta Kunal , Pluznick Jennifer
Published: April 2020

The microbiome has been implicated in blood pressure regulation and recent studies have found that gut‐derived microbial short chain fatty acids (SCFAs) modulate blood pressure. This is mediated, at least in part, by Olfactory receptor 78 (Olfr78), a G‐protein coupled receptor (GPCR) which responds to SCFAs to modulate renin release. Other renal GPCRs which respond to SCFAs include Olfr558, Gpr41, and Gpr43. Although we have previously studied the physiology of Olfr78 null mice, we do not know whether the expression of the other SCFA receptors, or other key renal genes, are altered in these animals. To address this, we extracted RNA from murine kidneys (8 Olfr78 WTs and 5 Olfr78 KOs) and performed reverse transcription and quantitative PCR using Taqman probe sets for 2 SCFA ORs (Olfr78, Olfr558), 2 SCFA GPCRs (Gpr41, Gpr43), as well as genes of interest (Renin1, Avpr2, Aqp2, ENaC, ROMK, BKA, and Wnt5a), and Gapdh as a control. From our screen, three genes were significantly decreased (p<0.05) in Olfr78 null mice when compared to WT: Olfr558 (mean fold change ± s.d., WT 1 ± 0.49, KO 0.34 ± 0.26), Aqp2 (WT 1 ± 0.51, KO 0.25 ± 0.11), and ROMK (WT 1 ± 0.36, KO 0.52 ± 0.20). The other genes assayed did not change between genotypes. Although we are not properly powered to examine sex, we did include both males and females and did not note any sex differences. Notably, novel changes in Aqp2 and ROMK expression imply potential changes in water or potassium handling in these animals. Future studies will include RNAScope and immunohistochemistry confirmation of these findings, as well as functional studies to determine the consequences of these changes.
PKA RIα Holoenzyme Structure Reveals ATP‐Dependent Dynamics and Allosteric Regulation in Isoform‐Specific Manner
Authors: Lu Tsan-Wen , Wu Jian , Aoto Phillip , Weng Jui-Hung , Ahuja Lalima G. , Sun Nicholas , Taylor Susan
Published: April 2020

Protein Kinase A (PKA) is the master switch for cAMP‐mediated signaling. PKA holoenzyme (R2C2) comprised a cAMP‐binding regulatory (R)‐subunit dimer and 2 catalytic (C)‐subunits, while cAMP binding to the R‐subunits unleashes the activity of the C‐subunit. Of the 4 functional non‐redundant R‐subunits (RIα, RIβ, RIIα, RIIβ), RIα and RIβ contain PKA pseudo‐substrate sites, in contrast to RIIα and RIIβ have substrate sites in their inhibitor sequences.

Understanding how protein kinases serve as molecular switches is one of the holy grails in kinase biology. We provide new molecular features of PKA RIα allostery by identifying two conformational states of the holoenzyme (R2C2), one with and the other without ATP.

RIα, in contrast to RIIβ, contains PKA pseudo‐substrate sequence, and our structures, with and without ATP, show how ATP functions as an allosteric inhibitor of RIα activation. The conformational changes of RIα holoenzyme are driven by ATP. ATP also facilitates RIα holoenzyme locks into inactive state and become more resistant to cAMP.

In contrast to the RIIβ holoenzyme, ATP has no effect on RIIβ holoenzyme conformations; however, it serves as a substrate and can facilitate the phosphorylation of the inhibitor sequence in RIIβ. When the RIIβ holoenzyme is phosphorylated, it is easier to be activated by cAMP and unleash kinase activity.

The isoform‐specific quaternary structures reveal a novel interface that helps to define domain crosstalk and a previously unappreciated allosteric pathway for cAMP activation. The results allow us to further appreciate the diversity of PKA isoforms and to understand the mechanism for allosteric activation.