bluuebunny/crossref_metadata_2025 huggingface dataset:
Concussive injury elicits JNK‐mediated human astrocyte retraction (651.10)
Authors: Augustine Claudia , Cepinskas Gediminas , Fraser Douglas
Published: June 2021

Background: The pathology of traumatic brain injury (TBI) is poorly understood, but likely involves blood‐brain barrier (BBB) dysfunction. The BBB is lined with microvascular endothelial cells, ensheathed by astrocyte endfeet. We investigated human astrocytes and endothelial cell responses to concussive injury in vitro.
Methods: Concussive injury in vitro was elicited by using a Cell Injury Controller II, a device which induces regulated and repeatable pulse of gas pressure to deform a flexible membrane. Human astrocytes (HA) or human immortalized cerebral microvascular endothelial cells (hCMEC/D3) grown on flexible culture membranes were subjected to a single (20, 35, or 55% deformation) or repeated (20% deformation; 6‐consecutive injuries spanned every 2 minutes) concussive injuries. Cells were assayed for morphological changes (retraction), detachment/cell death, and MAPK activation (phosphorylation of JNK1/2, ERK1/2, and p38).

Results: Significant HA retraction was elicited 30 min following either a single (55% deformation) or repeated concussive injury. HA retraction was not associated with cell death or detachment. HA recovered morphologically after 2 hrs post‐injury. Concussive injury increased phospho‐JNK1/2, but not phospho‐ERK1/2 or phospho‐p38. Pre‐treatment of HA with JNK inhibitors (SP600125 or SU3327) reduced injury‐induced HA retraction. In contrast, concussive injury failed to induce either morphological changes or MAPK activation in hCMEC/D3 Conclusion: Concussive injury induces JNK‐mediated astrocyte retraction in vitro, while sparing morphological changes in endothelial cells. In vivo, astrocyte retraction from endothelial cells may result in BBB dysfunction. JNK inhibition may be a potential therapeutic target for TBI.

Grant Funding Source: Supported by the Children's Health Foundation (London ON) and HSFO Grant NA6914
Characterization of electroporation for unbiased delivery of small molecules in high throughput screening applications (654.13)
Authors: Pihl Johan , Karlsson Anna , Karlsson Mattias , Lardell Susanne , Karila Paul
Published: June 2021

In cell‐based high throughput screening applications, the assumption is typically made that all molecules have equal and complete access to the target of interest. In the case of intracellular targets, this is most likely not the case, as the molecules in a diverse screening library will exhibit varying degrees of cell permeability, or even lack thereof. This is likely to result in in false negatives.

Electroporation is the use of brief electric pulses to overcome the barrier of the cell membrane. By the application of external electric fields, transient and reversible breakdown of the membrane can be induced. This transient state can be used to load cells with a variety of different molecules. Initially developed for gene transfer, electroporation can be used for delivery of a variety of other molecules ranging from ions to drugs, dyes, tracers and antibodies. Electroporation has proven useful both in vitro, in vivo and in patients, where drug delivery to tumors has been performed.

In this work, we have explored the use of a high throughput electroporation platform, Cellaxess Elektra, for unbiased delivery of small molecules into cells in a cell‐based assay. The molecules selected were fluorescent and were representative of those that can be found in a small molecule library. The cell permeability with and without electroporation was characterized by means of fluorescence microscopy, and in the selected collection of ~400 compounds, 20 exhibited low or no cell permeability which could be enhanced by means of electroporation. We believe that this could prove to be a valuable tool in identifying families of new leads previously undiscovered.
BEZ235, a selective PI3k/mTOR inhibitor, enhances the therapeutic efficacy of doxorubicin in pancreatic cancer (655.7)
Authors: Durrant David , Das Anindita , Kukreja Rakesh
Published: June 2021

Background: Pancreatic cancer is the fourth leading cause of cancer deaths in the U.S. and has the lowest relative survival rate (~6%) of all major cancers. Current treatment options have had limited success, therefore, patients are in desperate need for novel therapies which utilize both new drugs that target specific survival pathways as well as traditional therapeutics. Here we test whether BEZ235 (BEZ), a dual PI3k/mTOR inhibitor, in combination with doxorubicin (DOX) will enhance the killing of pancreatic cancer cells.

Methods and results: MiaPaca2 cells were treated with BEZ and DOX either alone or in combination for 24 to 48 hours. Cell viability, measured using the MTS assay, showed a significant reduction of cells in the combination group compared to either drug alone (Fig. A). Western blot analysis showed an increase in cleaved‐PARP, a marker for apoptosis, and p‐chk2 corresponding to decreased signaling through the PI3k/mTOR pathway (Fig. B). Interestingly, BEZ caused increased intracellular DOX accumulation which also paralleled the increase in cleaved‐PARP (Fig. C). A xenograph tumor model using Athymic nude mice injected with MiaPaca2 cells showed enhanced in vivo efficacy when combining BEZ and DOX (Fig. D).

Conclusion: Inhibition of survival signaling downstream of PI3k/mTOR with BEZ re‐sensitizes cells to the cytotoxic effects of DOX even in cancers not thought of to be dependent on this pathway for survival (i.e. KRAS mutants). That along with increased DOX accumulation results in enhanced cell killing. We propose that this unique combination could be a viable clinical option for patients with pancreatic cancer.

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Grant Funding Source: Supported by AHA 13PRE16910038, NIH HL051045
Role of efficacy at the mu opioid receptor on the interactions between mu opioid receptor agonists and cannabinoid receptor agonists: antinociception and drug discrimination (658.4)
Authors: Maguire David , France Charles
Published: June 2021

Cannabinoids such as Δ9‐tetrahydrocannabinol (THC) enhance some (analgesia) but not other (e.g., positive reinforcing) effects of mu opioid receptor agonists. Whether efficacy at the mu opioid receptor impacts those interactions is unknown. The effects of combining THC with agonists varying in efficacy at the mu opioid receptor were studied with antinociception and drug discrimination assays in rhesus monkeys. Etorphine, morphine, and nalbuphine increased tail withdrawal latency from warm water. THC increased the potency of high (etorphine) and moderate (morphine) efficacy agonists up to 16 fold but did not alter the potency of the low efficacy agonist nalbuphine. In monkeys discriminating fentanyl from saline, THC shifted the discrimination dose‐effect curves of etorphine, fentanyl, and nalbuphine rightward and downward. THC was 3‐fold more potent in attenuating the discriminative stimulus effects of the low efficacy agonist nalbuphine than those of the higher efficacy agonists etorphine and fentanyl. These data suggest the benefit of combining cannabinoids with opioids to treat pain is greatest with drugs having high efficacy at the mu opioid receptor and the subjective effects of high efficacy drugs are more difficult to disrupt than those of lower efficacy drugs. Given that other (abuse‐related) effects of opioids are not enhanced, these data support combining opioids with cannabinoids to treat pain.

Grant Funding Source: Supported by NIH grants R01DA05018, K05DA017918 (CPF), T32DA031115 (DRM), and F32DA035605 (DRM).
Effects of butter flavoring vapor exposure on cultured primary human tracheal/bronchial epithelial cell ion transport (660.11)
Authors: Zaccone Eric , Goldsmith Travis , Shimko Mike , Wells Ray , Thompson Janet , Fedan Jeffrey
Published: June 2021

In the microwave popcorn industry, inhalation of butter flavoring may result in “popcorn workers’ lung,” a disease resembling bronchiolitis obliterans. Rats exposed for 6 h to vapor from butter flavorings diacetyl and 2,3‐pentanedione demonstrate flavoring concentration‐dependent damage of the upper airway epithelium. Because epithelial ion transport is essential for maintenance of transcellular electric potential, fluid transport and cellular volume, we investigated the effects of flavoring vapor exposure on ion transport in HTBE cells. We hypothesized that epithelial ion transport may be among the earliest targets associated with the onset of butter flavoring toxicity. Using a novel exposure system designed for cultured cells, HTBE cells were exposed for 6 h to diacetyl or 2,3‐pentanedione vapors (25 ppm or >60 ppm). After exposure, cells were placed in an Ussing system to record short‐circuit current (Isc) at the 0 h or 18 h time‐point. Flavoring (25 ppm) exposure reduced amiloride (apical; 3.5 × 10‐5 M)‐sensitive Na+ transport as compared to controls. This reduction in Na+‐dependent Isc was not accompanied by changes in transepithelial resistance and recovered 18 h after exposure. Concentrations of diacetyl and 2,3‐pentanedione above 60 ppm resulted in cell death. Our results demonstrate that flavorings reduce apical Na+ conductance in airway epithelium.
Subcellular enrichment of calreticulin at myoendothelial junctions (664.2)
Authors: Biwer Lauren , Straub Adam , Isakson Brant
Published: June 2021

In the walls of resistance arteries, cellular projections from primarily endothelial cells (EC) make contact with smooth muscle cells (SMC), forming myoendothelial junctions (MEJ). The MEJ is a unique signaling microdomain responsible for multiple components of vasoreactivity. In MEJs, endoplasmic reticulum (ER) is often present; the significance of this has not been directly addressed. Using iTRAQ proteomic analysis, we discovered that calreticulin, a 50kDa calcium binding protein typically found in the ER, is highly enriched in the soluble fractions (i.e., ER) isolated from MEJs in a vascular cell co‐culture (VCCC). In the insoluble fraction (i.e., non‐ER, caveolin‐enriched lipid rafts), calreticulin was present in EC and SMC, but not MEJs. Using immuno‐TEM, mouse thoracodorsal arteries showed higher expression of calreticulin in EC (3.0 ± 0.76 beads/um2) and MEJ (2.3 ± 0.74 beads/um2) compared to SMC (0.6 ± 0.29 beads/um2). In the MEJ, beads could be identified within the ER. In order to determine a functional relevance of MEJ‐enriched calreticulin in soluble fractions, we successfully transfected EC in the VCCC with calreticulin siRNA. Next, SMC were stimulated with phenylephrine (PE) and cyclic guanosine monophosphate (cGMP) was measured. This method is known to produce a significant increase in SMC cGMP via inositol 1,4,5‐triphosphate‐based release of Ca2+ from ER at the MEJ, with subsequent activation of MEJ localized endothelial nitric oxide synthase (eNOS). The knockdown of calreticulin in EC/MEJ significantly decreased cGMP in SMC after PE stimulation. The mechanism behind this may be an interaction of calreticulin with SERCA that leads to less ER uptake of calcium, diminishing the localized calcium mobilization from the ER at the MEJ upon PE stimulation. Thus, we hypothesize that where the calreticulin is localized in cellular compartments, and not overall calreticulin expression, could be critical to MEJ function.

Grant Funding Source: Supported by NHLBI 2 T32 HL007284‐36
AKAP150‐dependent cooperative TRPV4 channel gating is central to endothelium‐dependent vasodilation and is disrupted in hypertension (678.10)
Authors: Sonkusare Swapnil , Dalsgaard Thomas , Bonev Adrian , Hill‐Eubanks David , Kotlikoff Michael , Santana Luis , Scott John , Nelson Mark
Published: June 2021

Endothelial cell (EC) dysfunction is a hallmark of hypertension. We recently discovered that local Ca2+ influx through clusters of functionally coupled TRPV4 channels (detected optically as “TRPV4 sparklets”) drives physiological vasodilation. Here, we show that stimulation of EC muscarinic receptors activated TRPV4 sparklets exclusively at discrete sites at myoendothelial projections (MEPs)—specialized regions of ECs that contact adjacent smooth muscle cells. This activation was dependent on protein kinase C (PKC) and was absent in mice lacking the PKC‐anchoring protein AKAP150 (A‐kinase anchoring protein 150), which was localized predominantly to MEPs. Cooperative gating of TRPV4 channels within a cluster amplified Ca2+ influx at MEPs by more than 2‐fold. This cooperativity was largely absent at non‐MEP sites and was virtually eliminated by chelation of intracellular Ca2+ or AKAP150 knockout, suggesting AKAP150‐dependent potentiation of TRPV4 activity by Ca2+ influx via adjacent channels. Notably, MEP‐localization of AKAP150 was disrupted in angiotensin II‐induced hypertension, leading to a complete loss of muscarinic activation of TRPV4 channels, much weaker coupling among TRPV4 channels at MEPs, and approximately an 80% reduction in carbachol‐induced vasodilation. Our results support the concept that endothelial‐dependent vasodilation of resistance arteries is enabled by MEP‐localized AKAP150, which ensures the proximity of PKC to TRPV4 channels and coupled channel gating necessary for efficient communication of the endothelium to the smooth muscle cells in arteries—a molecular configuration that is disrupted in hypertension.

Grant Funding Source: Supported by HL044455, 1P01HL095488, R37DK053832, R01HL098243 to MTN
Female mice are more sensitive to obesity‐induced vascular dysfunction than males (680.14)
Authors: Le Huy , Huby Anne‐Cecile , Anderson Ruchi , Belin de Chantemele Eric
Published: June 2021

Over the last two decades, a three fold increased in the prevalence of cardiovascular disease (CVD) has been reported in premenopausal women and attributed to the recent epidemic of obesity. While the mechanisms involved in obesity‐related CVD are beginning to be understood in males they remain unknown and unstudied in females. We tested the hypothesis that obesity negates the protection of women from CVD by submitting 6‐week old male and female Balb/C mice to a control (CD) or a high fat diet (HFD) for 12 weeks. On CD, females were smaller than males, but exhibited higher adiposity, fasted glycemia and leptin levels than males. On HFD, females had a higher food intake (HFDF: 3.1±0.5 vs. HFDM: 2.7±0.2g/day) but displayed lower increases in body weight (HFDF: +15% vs. HFDM: +35%), adiposity (HFDF: +12% vs. HFDM: +190%), glycemia and leptin levels than males, suggesting that female mice have a higher metabolic rate than males. Aortic ring reactivity was studied via wire myography at the end of the diet. Consistent with the human literature, at baseline, females displayed a better endothelial function than males (CDF: 82±3 vs. CDM: 49±3%, p<0.05). However 12 weeks of HFD impaired endothelial function (12% reduction in relaxation) and reduced the contractility of the aortic rings to phenylephrine (‐35%), serotonin (‐14%) and depolarization (‐13%), in female mice only, suggesting that females are more sensitive to obesity‐induced vascular dysfunction than males. While reduction in vascular contractility might initially protect females from hypertension, reduced endothelial function might predispose females to atherosclerosis and further cardiovascular events.

Grant Funding Source: 11SDG5060006
Blood pressure and heart rate measures: How well do pre‐clinical models translate? (681.7)
Authors: Steidl‐Nichols Jill , Bhatt Siddhartha , Hemkens Michelle , Heyen Jon , Marshall Catherine , Li Dingzhou , Flynn Declan , Wisialowski Todd , Northcott Carrie
Published: June 2021

Within research, animal models are commonly used to examine the physiology of disease, and evaluate the efficacy and safety of drugs. However, understanding of the translation of cardiovascular (CV) endpoints [specifically, heart rate (HR) and blood pressure (BP)] from pre‐clinical animal studies to the clinic is still limited. Internal databases containing BP and HR data from a broad range of compounds evaluated in pre‐clinical CV safety pharmacology animal models and Phase 1 human clinical trials were used to evaluate the translation across pre‐clinical findings from rodent (rat) to large animal (non human primate and dog), as well as the translation of findings from pre‐clinical animal models to humans. The database currently consists of >700 historical preclinical CV safety pharmacology studies and represents experiments across 5 species. The analysis of this information has indicated that many mechanisms modulate blood pressure and heart rate, further demonstrating the complexity of CV system. Moreover, the data from this wide range of compounds and mechanisms translates across species and also into the clinic, confirming the importance of pre‐clinical animal models in the examination of the efficacy and safety of drugs, and the usefulness of these models for selection of compounds for advancement to clinical testing and ultimately the treatment of disease.
Activation of P2X‐purinergic receptors in the lateral parabrachial nucleus increases renal sympathetic nerve discharge and mean arterial pressure in anesthetized rats (686.13)
Authors: Menezes Miguel , Favero Michele , Yamamoto Kenta , Menani Jose , de Paula Patricia , Mifflin Steve
Published: June 2021

The lateral parabrachial nucleus (LPBN) is an important area involved in cardiorespiratory control. ATP is an important neurotransmitter and purinergic receptors are present into LPBN. The involvement of purinergic mechanisms into LPBN in the control of mean arterial pressure (MAP) and renal sympathetic nerve discharge (RSND) is unknown. We investigated the effect of alpha, beta‐me ATP (P2X agonist) alone or following pyridoxalphosphate‐6‐azophenyl‐2',4'‐disulfonic acid (PPADS, P2 antagonist) injected into LPBN on MAP and RSND in rats. Anesthetized, vagotomized and mechanically ventilated male rats (n=3) were used. Unilateral injections of alpha, beta‐me ATP (2.0 nmol/50 nl) into LPBN increased MAP (Δ 12±2 mmHg, p<0.05) and RSND (Δ 32±8%), without changing heart rate (HR). Unilateral injection of PPADS (0.125 nmol/50 nl) into LPBN did not change MAP, HR and RSND, however, pre‐treatment with PPADS into LPBN (10 min before) abolished the pressor response (Δ 2±0.8 mmHg, p<0.05) and reduced the RSND increase (Δ 12±7%, p<0.05) produced by alpha, beta‐me ATP injected into LPBN. In 2 rats responses to alpha, beta‐me ATP recovered to control within 30 min while in the remaining rat the response had recovered 31% by 70 min. These results suggest that alpha, beta‐me ATP acting on P2X purinergic receptors into LPBN produces pressor and sympathoexcitatory responses in anesthetized rats.

Grant Funding Source: CAPES (18906‐12‐5); FAPESP; CNPq
Macula densa progenitor cells contribute to nephron formation during kidney development (692.6)
Authors: Burford James , Riquier‐Brison Anne , Villanueva Karie , Peti‐Peterdi Janos
Published: June 2021

Cells of the macula densa (MD) in the adult kidney are strategically localized in the distal tubule at the glomerular vascular entrance, which mirrors the position of nephron‐committed progenitor cells in the developing kidney, at the interface between the ureteric bud and the metanephric mesenchyme. Indirect evidence suggests that MD cells contribute to nephrogenesis. Here we aimed to assess the stem/progenitor identity of MD cells via in vivo lineage tracing. We developed MD‐Confetti mice with tamoxifen‐inducible expression of the confetti fluorescent reporter construct (CFP/GFP/YFP/RFP) specifically in MD cells by crossing nNOS‐CreERT2 and Confetti/fl mice. Confetti expression was induced by ip tamoxifen injections on postnatal days 2 and 3, and kidneys were harvested three weeks later. MD cells were identified by one of the four Confetti colors, expressing either membrane‐targeted CFP, nuclear‐GFP, cytosolic YFP or RFP. MD plaques usually contained individual MD cells of all four colors. However, in vivo multi‐color lineage tracing identified MD cells as a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb (TAL) of Henle’s loop. In addition to multicolor cell labeling in MD plaques, fluorescence image analysis revealed the presence of several, exclusively clonal (i.e. single‐color) multi‐cell tracing units in TALs. Our results highlight the new role and important contribution of individual MD stem/progenitor cells to nephron formation during kidney development.
Role of O‐GlcNAcylation in regulating mitophagy in cardiomyocytes (693.14)
Authors: Wright JaLessa , Chatham John
Published: June 2021

Acute increases in the post‐translational protein modification O‐linked‐N‐acetylglucosamine (O‐GlcNAc) have been shown to protect cardiomyocytes against oxidative stress and this has been associated with O‐GlcNAcylation of mitochondrial proteins. Mitophagy, a type of cargo‐mediated autophagy, protects against oxidative stress by removing damaged mitochondria. Therefore the goal of this study was to determine whether O‐GlcNAcylation plays a role in regulating mitophagy in AC16 immortalized human ventricular cardiomyocyte cells. Cells were treated with 2,3‐dimethoxy‐1,4‐napthoquinone (DMNQ, 20 µM), a redox cycling agent which increases mitochondrial reactive oxygen species (ROS) and FCCP (0.5 µg/mL), a mitochondrial uncoupler commonly used to induce mitophagy. Following 3 hour treatment with DMNQ or FCCP, O‐GlcNAc levels were decreased and this was associated with increased levels of total PINK1 and increased mitochondrial Parkin. Increasing basal O‐GlcNAc levels for 2 hours with thiamet‐G (3 µM) a highly selective inhibitor for O‐GlcNAcase, attenuated the loss of O‐GlcNAc induced by both DMNQ and FCCP treatment and appeared to increase basal PINK1 levels. These results support the concept that protein O‐GlcNAcylation plays a role in regulating mitophagy and that this could contribute to the protection against oxidative stress. Future studies will include quantifying the effects of DMNQ and thiamet‐G on mitochondrial function using XF24 Extracellular Flux Analyzer as well as determining whether PINK1 or Parkin are targets for O‐GlcNAcylation.

Grant Funding Source: Supported by NIH grants HL101192 and HL110366
T cell mitochondrial ROS in development of hypertension (693.15)
Authors: Nazarewicz Rafal , Dikalova Anna , Bikineyeva Alfiya , Egnatchik Rob , Young Jamey , Harrison David , Dikalov Sergey
Published: June 2021

T cell mitochondrial ROS in development of hypertension
Rafal R. Nazarewicz*, Anna Dikalova*, Alfiya Bikineyeva*, Rob Egnatchik#, Jamey Young#, David G. Harrison*, Sergey Dikalov* Division of Clinical Pharmacology*, Chemical and Biomolecular Engineering#, Vanderbilt University Medical Center, Nashville, TN Immunity plays a key role in the development of hypertension. Locally acting T cells secret pro‐inflammatory cytokines that have profound damaging effects on end‐organs and facilitate hypertension. Mitochondrial ROS may contribute to regulation of T cells activity and cytokine production by switching from aerobic mitochondria‐base metabolism to anaerobic glycolysis. The objective of this study was to establish the role of T cell mitochondrial ROS in development of hypertension. Increased mitochondrial superoxide and H2O2 in T cells from hypertensive mice was inhibited by overexpression of catalase in mitochondria or mitochondria targeted SOD mimetic mitoTEMPO. Angiotensin II‐induced hypertension led to impaired T cells respiration which was reversed by scavenging of mitochondrial H2O2. Despite impaired respiration, T cells from hypertensive mice synthesized more ATP compared to normotensive mice. Glycolysis was identified as the main source of ATP as indicated by increased lactate production. HPLC analysis showed higher glutamine uptake suggesting increased glutaminolysis. These metabolic changes facilitated T cell activity. Scavenging of mitochondrial superoxide prevented angiotensin II‐induced pro‐inflammatory cytokine production and ultimately hypertension in mice. Our data show that mitochondrial ROS‐dependent metabolic adaptation is necessary for T cell activation and their pro‐hypertensive responses. We suggest T cell mitochondrial ROS as a potential target for pharmacological intervention in hypertension.

Grant Funding Source: Supported by National Institutes of Health grant R01HL094469.
Regulation of reactive oxygen species in L6 myoblasts by glucose transporter 1 (693.5)
Authors: Andrisse Stanley , Koehler Rikki , Chen Joseph , Patel Gaytri , Vallurupalli Vivek , Ratliff Benjamin , Warren Daniel , Fisher Jonathan
Published: June 2021

Elevated reactive oxygen species (ROS) levels are associated with metabolic abnormalities such as insulin resistance. Glucose transporter 1 (GLUT1) transports glucose and dehydroascorbic acid, both of which play roles in antioxidant defense. We hypothesized that conditions that would increase GLUT1 activity would result in decreased ROS levels and conditions that would decrease GLUT1 activity would increase ROS levels. . L6 myoblasts loaded with H2‐DCFDA, a fluorescent ROS probe, were treated under various conditions to alter GLUT1 activity, and fluorescence readings were taken under basal conditions or after increases in ROS were induced by exposure to pyrogallol or antimycin A. GLUT1 inhibitors, fasentin and phloretin, increased induced ROS levels. Ataxia telangiectasia mutated inhibitors and activators, previously shown to inhibit and augment GLUT1 activity, increased and decreased basal and induced ROS levels, respectively. GLUT1‐S490A, previously shown to decrease GLUT1 activity, enhanced basal and induced ROS levels. GLUT1‐S490D, previously shown to increase GLUT1‐mediated transport, lowered basal ROS levels. Thus, the data suggest that GLUT1 plays a role in regulation of ROS levels.

Grant Funding Source: Supported by: William Townsend Porter Predoctoral Fellowship from the American Physiological Society
PTK6 mediates intestinal endothelial barrier dysfunction in response to TNFα (695.1)
Authors: Haines Ricci , Beard Richard , Yuan Sarah , Wu Mack
Published: June 2021

A key event in the progression of systemic inflammation resulting from severe trauma or shock involves hyperpermeability of the intestinal endothelium. The signaling events leading to increased endothelial permeability in these disease states are incompletely understood. Although the role of protein tyrosine kinase 6 (PTK6, also known as breast tumor kinase BRK) has been primarily studied in mechanisms underlying metastasis and cancer, it has also been shown that PTK6 participates in mediating colonic epithelial barrier dysfunction. In this study, we hypothesized that PTK6 is 1‐ expressed in intestinal endothelial cells, and 2‐ contributes to endothelial hyperpermeability in response to inflammation. To this end, we investigated the role of PTK6 in mediating the disruption of intestinal endothelial barrier function. Results showed that PTK6 was detected in intestinal endothelial cells at the level of protein and mRNA. In addition, PTK6 knockdown attenuated decrease in transendothelial electric resistance (TER) as measured by electric cell‐substrate impedance sensing (ECIS) of intestinal endothelial monolayers in response to inflammatory factors including TNFα. Furthermore, we showed that TNFα increased activity of PTK6 as evidenced by increased phosphorylation at Y342, supporting the hypothesis that this kinase plays a role in endothelial inflammation. We further studied the potential targets of PTK6 including junctional structure proteins to determine the molecular mechanism of its role in regulating endothelial barrier function. Our study explores a potential implication that targeting PTK6 may serve as a novel therapeutic strategy in treating diseases characterized by hyperpermeability of the intestinal endothelium.

Grant Funding Source: Supported by VA Merit Review 5101BX000799, the NIH grants HL96640, HL61507 and HL70752
Activation of transient receptor potential vanilloid 1 decreases endothelial nitric oxide synthase phosphorylation at Thr497 by protein phosphatase 2B‐dependent dephosphorylation of protein kinase C (696.1)
Authors: Su Kuo‐Hui , Lin Shing‐Jong , Shyue Song‐Kun , Lee Tzong‐Shyuan
Published: June 2021

We investigated the effects and underlying molecular mechanism of transient receptor potential vanilloid 1 (TRPV1), a calcium‐permeable non‐selective cation channel, on phosphorylation of endothelial nitric oxide synthase (eNOS) at threonine 497 (Thr497) in bovine aortic endothelial cells (BAECs) and in mice. In BAECs, treatment with the TRPV1 ligand evodiamine decreased the phosphorylation of eNOS at Thr497, protein kinase Cα (PKCα at Serine 657, and PKCβ2 at Serine 660. Evodiamine increased protein phosphatase 2B (PP2B) activity and promoted the formation of a PP2B‐PKC complex. Inhibition of TRPV1 activation, removal of extracellular calcium or pharmacological inhibition of PI3K/Akt/CaMKII/AMPK signaling pathway abolished the evodiamine‐induced alterations in phosphorylation of eNOS at Thr497, PKCα, PKCβ2 and PP2B activity, as well as the formation of a PP2B‐PKC complex. Inhibition of PP2B activation partially reduced the evodiamine‐induced NO bioavailability and tube formation in ECs and angiogenesis in mice. Moreover, evodiamine decreased the phosphorylation of eNOS at Thr497, PKCα, and PKCβ2 in apolipoprotein E (ApoE)‐deficient mouse aortas but not TRPV1‐deficient or ApoE/TRPV1 double‐knockout mice. TRPV1 activation in ECs may elicit a calcium‐dependent effect on PP2B‐PKC signaling, which leads to dephosphorylation of eNOS at Thr497 in ECs and in mice.

Grant Funding Source: National Science Council, Taiwan
Tracking and quantifying focal adhesion kinase and paxillin at lamellipodial protrusion in migrating endothelial cells (696.5)
Authors: Hu Ying‐Li , Lu Shaoying , Szeto Kai , Wang Yingxiao , Lasheras Juan , Chien Shu
Published: June 2021

Focal adhesion kinase (FAK) and paxillin are involved in focal adhesion (FA) disassembly and formation, which can regulate cell migration, intracellular signaling, and remodeling of actin filaments. We used time‐lapse double‐color imaging to monitor concurrently the spatial‐temporal dynamics of FAK and paxillin in live endothelial cells (ECs) co‐transfected with GFP‐FAK and DsRed‐paxillin, and the fluorescence intensities (FI) of paxillin and FAK at FAs were determined in cell front, center and rear. The mean FAK/Paxillin FI ratio is highest at cell front (mean±SEM: 4.73±0.48), near 1 at cell center (0.95±0.11), and lowest at cell rear (0.64±0.05) (P<0.01). Determination of the time difference between the assemblies of FAK and paxillin at FAs in lamellipodial protrusion (LP) showed that FAK assembly occurs ahead of that of paxillin at individual FAs. Computational analysis indicates that FAK reaches its maximum intensity earlier than paxillin by about 4 min. The assembly of FAK is at LP ahead of paxillin Indicates that, while both molecules play important roles in modulating FA dynamics during cell migration, FAK promotes FA formation ahead of paxillin at the cell leading edge. This work was supported by NHLBI Research Grants HL‐104402 and HL‐106579 (S.C.).
Focus on the demands of employers in the integrated management system of the medical university (719.18)
Authors: Raisova Karlygash , Zhuzhanov Ormanbek , DeLellis Nailya , Dalenov Erbolat
Published: June 2021

The purpose is to study and analyze the satisfaction, demands and interests of employers of graduates of Astana medical university in order to develop and revise its strategy on its basic and the selection of priority directions for improving the educational process at the university. The high level of the demand for graduates indicated 53,3% respondents, sufficient level of knowledge of graduates indicated 75% respondents, the level of practical skills indicated 70 % and 48,3% respondents indicated the conduction by graduates the scientific research at a satisfactory and high level, the compliance with a set of disciplines needs of the region indicated 65% employers in the university, 66.7% respondents said that the university knows and takes into account the requirements for specialists in their organizations. Identified the following employers’ requests:professional quality, which should have a specialist is a strong basic education(76.7%), knowledge of the latest technologies in health care (55%), ability to take initiative in work (55%); in employers’ opinion the most effective forms of knowledge and skills control is a clinical examination at the bedside of the patient (58.3%) and a clinical examination on phantoms and plaster casts in the conditions of educational and clinical center(51.7%).
Flipping the medical school classroom: student acceptance and student evaluation issues (719.4)
Authors: Osborne David , Janssen Herbert
Published: June 2021

Flipping the classroom has generated improvement on summative exam scores in our first‐year medical students; however, faculty members adopting this teaching methodology often receive lower satisfaction rating on student evaluations. In previous years, these same professors received outstanding evaluation ratings when the same topics were presented using standard didactic lectures. We feel that this decreased student satisfaction may be result of two distinct causes. First, students who have been accustomed to didactic lectures often come to class unprepared and therefore are incapable of the critical thinking and problem solving skills need in the flipped classroom. Second, the evaluation tool which was appropriate for didactic lectures is inappropriate for the analyzing the flipped classroom methodology. The student evaluations have improved in that last several years; however, the transition was not accomplished without difficulty. Anyone planning to pursue this teaching approach should be prepared to weather the storm of sub‐standard student evaluations and, if possible, prepare their administrators for this potential outcome. Our experience suggests that faculty persistence targeted at changing student culture and expectations can help in this process. Accurately determining student’s acceptance of this relative new teaching methodology is important. Improvements in the teaching methodology can be made only when the evaluation tool is valid. It is felt a new evaluation tool can be developed based on results obtained in student focus‐groups coupled with cognitive assessment outcomes.
Creation and implementation of a two year preclinical curriculum using multiple teaching approaches at a new osteopathic medical school (719.6)
Authors: Wright Bruce , Hernandez Mark
Published: June 2021

Curricular design and implementation for the pre‐clinical years of a new osteopathic medical school is being built on a number of curriculum innovations. Beginning with semester 1, a foundations of medicine course sets the stage for teaching of osteopathic principles and practices (OPP). Introductory clinical skills are taught using standardized patients, small groups, peer instruction and independent study modules. Semester 1 also includes anatomy and a molecular medicine course that includes biochemistry, genetics, immunology, and other introductory material that provides a biomedical sciences foundation. The other three semesters of years 1 and 2 are scheduled on a systems based curriculum with substantial emphasis on physiology, microbiology, and pharmacology taught in a clinical context, while clinical skills and OPP content is highly correlated with the content of the systems blocks. The curriculum includes both traditional lectures and flipped‐classroom formats combining one to three hours of Designated Student Assignments (DSA’s) with either a Basic Science Correlation (BSC) or a Clinical Integration Session (CIS). Through the 2nd to 4th semesters, systems course materials are linked to clinical applications courses. For example, in the 3rd semester cardiovascular systems course, physiological introduction to the EKG will be followed by applications on simulators in which students place leads and read strips. Finally, from day one, use of Surface Pro tablets by all students, electronic textbooks & electronic testing & attendance tracking has made the curriculum virtually paperless. Throughout years 1 and 2, all courses assure active engagement of students by using and audience response system (ARS) in lectures, BSCs and CISs.
Active learning: hands‐on activities add pizzazz to lecture‐based anatomy courses (721.10)
Authors: Bee Mary , Farrugia Jamie , Ottenbreit Mark
Published: June 2021

Educating students in the field of anatomy has been a challenge for decades. Bringing a “dead” science to “life” has been problematic for even the greatest of educators. We have examined several different group activities and approaches to teaching anatomy that incorporated a variety of teaching activities designed to keep the learning process engaging, stimulating, and self‐motivating. Developing novel approaches for the group activities in a lecture based class is essential for student engagement. Group activities that we have successfully implemented include the incorporation of clinically relevant video clips, which students assess and discuss as a group, construction of organs out of modeling clay and/or other craft supplies, using fabric muscles made of felt to attach to a skeleton which helped reinforce their understanding of muscle attachments, using yarn to depict the passageway of nerves in the brachial plexus and spinal cord tracts, and other approaches. The level of student satisfaction increased 23% and the majority of students felt that these activities enhanced their learning with an average of 4.74 out of 5 on the Likert scale. Moreover, grades increased 12% compared to another section of the same class where these techniques were not employed. Engaging teams of students is of great importance for understanding difficult concepts, retention of knowledge in anatomical sciences and student satisfaction.
A better anatomy experience through small team multimedia production and curation of anatomical specimens (721.18)
Authors: Fox Glenn , Alsup Kathleen
Published: June 2021

Plastinated anatomical specimens provide a useful learning experience for students. However, many learners find first encounters with a specimen intimidating and can be overwhelmed when dealing with this type of collection as an adjunct in learning anatomy. To address these issues, faculty introduced two initiatives: 1) the creation of a queryable database of anatomical specimens, and 2) the development and creation of multimedia associated with individual specimens (with an emphasis on plastinated specimens). Given the size and scope of the anatomical collections in the Division of Anatomical Sciences, University of Michigan Medical School, a queryable database is necessary for both proper curation of the specimen collections, as well as, general use and advanced self‐directed inquiry by students. We also evaluated how the development and creation of multimedia relating to specimens, as well as the use of the multimedia in the context of learning anatomy, affected student learning and students’ perceptions of anatomical learning. While these projects are in their early stages, we observe net beneficial effects on collection curation and student learning using mixed methods methodologies.
Design of a novel approach to teaching online neuroanatomical education (725.1)
Authors: Allen Lauren , de Ribaupierre Sandrine
Published: June 2021

E‐learning allows for the development of educational tools that present information in different modalities suited to each student's unique needs. However, the implementation of such tools is occurring in advance of adequate empirical research on how students utilize and learn from such instructional resources. Research suggests that the effectiveness of any instructional tool depends on how well its design reflects human cognitive architecture. We hypothesize the implementation of a neuroanatomy e‐learning curriculum that takes students’ different learning abilities and preferences into consideration by incorporating equivalent material presented in multiple modalities (2D, 3D, and text), and is congruent with the principles of Cognitive Load Theory, will be more effective, and will result in improved student learning. Learning outcomes will be assessed using a pre‐test/post‐test knowledge measure. The student’s visuo‐spatial abilities will also be measured, and will be compared with each student’s self‐directed “e‐learning path”. A qualitative measure will also be administered to assess student satisfaction and preference for different modalities. Results of this study may be used to help establish guiding principles that will facilitate the design and implementation of effective and efficient e‐learning curricula tools that complement students’ unique learning strengths and needs.
Evaluation of the scapula module from the interactive atlas of the human skeleton (725.10)
Authors: Casey Gregory , Cork R.
Published: June 2021

Last year we developed a three component module of the scapula which included a short instructional video, an interactive animation, and a self‐testing quiz. This semester undergraduate nursing students were provided with access to this module in Block 3 (upper extremity) of their anatomy course. In order to evaluate its effectiveness we compared the scores on this semester’s Block 3 exam with the scores last Fall when they did not have access to the scapula module. Both groups of students were asked the same 11 scapula‐related questions. These questions were divided between the practical exam (6) and the written exam (5) and ranged from identifying bony landmarks to knowing the innervation of muscles attached to the scapula.

In addition to analysis of their exam performance we also surveyed the students about their usage of the scapula module and their opinions of its usefulness. Overall, the scapula module received very positive reviews from the students with the interactive animations being the most popular feature. Most negative reviews related to technical issues arising from Browser differences and the inability of some features to work properly on tablets such as the iPad. Statistical analysis of the evaluation data is ongoing, but it does show a small but consistent improvement. The students who had access to the online scapula module did better on the scapula‐related questions than those who did not.
Three‐dimensional modeling of facial nerve VII (726.3)
Authors: Peterson Dani
Published: June 2021

Due to its long and complicated trajectory through the cranium, facial nerve VII (CN VII) can be damaged in surgeries, sometimes resulting in facial muscle paralysis. We created a three‐dimensional (3D) model based on data from dissection of the nerve in a human cadaver in order to give physicians a greater in vivo knowledge of the pathway of CN VII. We dissected the lateral side of the right half of the head to the level of the parotid gland, identified the parotid plexus of CN VII, and followed its five branches. In addition, we are currently following the nerve through the internal auditory meatus on its pathway through the temporal bone. We imaged the head using Magnetic Resonance Imaging (MRI). The resulting images served as the anatomical template for the reconstructed nerve model. After completing the dissection, we tracked the nerve trajectory using a MicroScribe 3D Digitizer in the context of other nearby structures. The resulting map will aid surgeons by increasing accuracy when performing removals of acoustic neuromas and parotid tumors and in repairing the temporomandibular joint.

Grant Funding Source: Supported by Mentoring Research Grant, College of Life Sciences, Brigham Young University
Comparative study of in vitro expanded somatic stem cells from different sources (732.3)
Authors: Danisovic Lubos , Bohac Martin , Novakova Zuzana , Varga Ivan , Bohmer Daniel , Polak Stefan
Published: June 2021

Mesenchymal stem cells (MSCs) represent a unique tool of regenerative medicine. They are undifferentiated cells with the ability of long‐term self‐renewing and plasticity. The main goal of this study was biological and morphological analysis of human MSCs derived from bone marrow, adipose tissue and dermis in respect to cartilage tissue engineering. MSCs were isolated and cultured in vitro using standard protocols. They were maintained in a‐MEM supplemented by 15% of fetal bovine serum and antibiotics. MSCs from all tissues were cultured up to the 5th passage. The kinetics of proliferation was analyzed by CEDEX XS and expression of selected markers was assessed by flow cytometry. They were morphologically analyzed by inverted microscope and TEM. Pellet cultures were used to induce chondrogenic differentiation and this event was proved by real‐time PCR. MSCs from all sources were attached on the Petri dishes after 24 h and started to proliferate in colonies. After 7‐10 days they reached 80% confluency and were passaged. In the next 5 passages they displayed fibroblastoid morphology. MSCs also showed similar kinetics of proliferation and shared the expression of CD29, CD44, CD73, CD90, CD105 and CD166 and were negative for CD14, CD34, CD45 and HLA‐DR. TEM showed similar morphology of all analyzed MSCs. We have demonstrated that MSCs from bone marrow and adipose had potential to undergo chondrogenic differentiation comparable with chondrocytes, but this potential was decreased in dermis‐derived MSCs. We can emphasize that MSCs isolated from bone marrow, adipose tissue and dermis possess similar biological and morphological properties, but differ in chondrogenic potential. Dermis‐derived MSCs have an inferior potential for chondrogenic differentiation. So this fact decreases their potential for cartilage tissue engineering. Hoverer, further studies focused on applying of appropriate exogenous factors must be carried out to clarify this issue.

Grant Funding Source: Supported by MZSR no. 2012/4‐UKBA‐4 and APVV no. 0434‐12.
Tumor suppressor properties and a role in maintaining genome integrity for the Meig1 gene (736.3)
Authors: Don Jeremy , Marciano‐Levi Iris
Published: June 2021

Meig1 is a highly conserved gene first identified in mouse pachytene spermatocytes undergoing meiotic recombination. In these cells the MEIG1 protein enters the nucleus and forms foci along the meiotic chromosomes, reminiscent of foci obtain by proteins known to be involved in the DNA damage response. Meig1 knockout (KO) male mice are infertile. These mice exhibit increased apoptosis of post recombination spermatogenic cells and significantly fragmented DNA in the few epididymal sperm cells that could be obtained. Given these findings the objectives of this study were to examine the hypothesis that MEIG1 plays a role in the system responsible for maintaining genome integrity. Immunocytochemistry analysis of γH2AX signaling in meiotic chromosomes of late pachytene spermatocytes showed that in KO mice they were highly “decorated” with γH2AX foci, compared to WT chromosomes. Moreover, analysis of post‐recombination meiotic chromosomes revealed the existence of numerous chromosomal fragments in KO meiotic preparations whereas no such fragments could be seen in preparations from WT spermatocytes. Regarding somatic cells, KO MEFs exposed to etoposide treatment exhibited significantly slower disappearance of the γH2AX foci compare to WT MEFs, suggesting delayed or aberrant repair. Interestingly, most Meig1 KO mice (more than 30 mice to date), and about one third of the Meig1 +/‐ heterozygotes developed huge and aggressive tumors between 6‐18 months of age, whereas none of the age‐matched wild‐type (WT) siblings developed tumors. These tumors were identified as being mainly malignant adeno‐carcinomas and lymphomas. These results imply that Meig1 has tumor suppressive properties and suggest that it is involved in maintaining chromatin integrity not only in spermatogenic cells but in somatic cells as well, rendering it as a new player in the general DNA damage response, especially the response to DSB.

This study was supported by grant No. 20072019B from the Israel Cancer Association.