bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of Learning Approaches Utilized by Osteopathic Medical Students
Authors: Eldeeb Khalil , Kuo Yen-Ping , Vaskalis Zachary T. , Dogbey Godwin Y.
Published: April 2020

Medical education curricula have been increasingly undergoing changes in recent years to respond to continuous transformations in the professional education landscape. Utilizing and promoting learning approaches that are most effective in helping learners navigate through these ongoing changes, while fostering optimal educational outcomes, are of particular importance. While there are various learning approaches, they can be categorized into three main types: Superficial, Deep, and Strategic. Although Strategic and Deep Learning approaches were often found in the literature to correlate with high performance among medical students, some researchers suggest that promoting the Strategic Learning Approach predominantly may be more effective. The Campbell University Jerry M. Wallace School of Osteopathic Medicine (CUSOM) opened its doors in 2013 with an innovative, hybrid curriculum, combining discipline‐based and organ system‐based courses, fully integrating biomedical and clinical skills‐based learning. With the ultimate goal in facilitating greater academic success among CUSOM students, as well as among all medical students, this Institutional Review Board (IRB)‐approved study aims to determine the predominant type of learning approaches utilized among medical students at CUSOM using a slightly‐modified Study Skills Inventory for Students (ASSIST) instrument. Descriptive statistics were used to describe participants’ demographic variables. Categorical variables were reported as frequencies or proportions/percentages. Univariate, multivariable, and multivariate statistical analyses were performed as relevant and necessary. Preliminary data were analyzed using the Statistical Package for Social Sciences (SPSS, version 24, Chicago, IL). All inferences will be considered statistically significant whenever P‐value ≤0.05 and will be reported with corresponding effect sizes as necessary. A total of 240 osteopathic medical students voluntarily participated in this study, which began at the start of this current academic year. The first‐year students comprised the largest subgroup of respondents, representing 65.8% of all completed instruments. Preliminary findings indicate that Strategic Learning was the principal approach utilized by participants, accounting for 61% among the fourth‐year, 58% among the first‐year, and 52% among the third‐year students. It is interesting to note that the current second‐year student participants utilize the Deep Learning approach predominantly. These preliminary results serve as an important basis for tailored study skills interventions that could facilitate students’ academic success. Further analysis will explore relationship between the predominant learning approaches and metrics of their academic performance.
Absorption, Distribution and Excretion of [14C] Zamicastat after Single Oral and Intravenous Administration in Rats and Dogs
Authors: Loureiro Ana I. , Araujo Francisca , Bonifacio Maria-Joao
Published: April 2020

Background

Zamicastat is a dopamine‐β‐hydroxylase inhibitor
(1), previously showed to increase the survival rate in the monocrotaline rat model of pulmonary arterial hypertension.

Objectives

The purpose of this study was to characterize the absorption, distribution and elimination of 14C‐zamicastat in rat and dog, after single oral (p.o.) or intravenous (i.v.) administration.

Methods

Wistar rats and Beagle dogs were administered with 14C‐zamicastat at 30 mg/kg orally or intravenously with 2.5 mg/kg (rat) and 0.1 mg/kg (dog). Plasma, urine and faeces samples were collected at different time‐points to measure the plasma exposure and the recovery of total radioactivity up to 168 h post‐dose. Whole body phosphor imaging (WBPI) was evaluated in rat after p.o. administration of 30 mg/kg of 14C‐zamicastat. The extent of plasma protein binding was determined by equilibrium dialysis and blood cell binding was determined, using centrifugation, on pooled male samples from each species.

Results

After single oral dosing of 14C‐zamicastat, more than 95% of total radioactivity was recovered at five and seven days from rats and dogs, respectively. The radioactivity peaked in plasma between 6 to 8 h and 2 to 4 h post‐dosing in rats and dogs, respectively, followed by a slow elimination over time. Zamicastat related radioactivity was mainly recovered in faeces with very low levels (less than 20% of total radioactivity) excreted in urine in both species. Following i.v. administration more than 80% of radioactivity was recovered in both species mainly via feaces excretion. The WBPI study indicates that radioactivity was distributed throughout the body with the majority of tissues achieving a tissue:blood ratio greater than 2. Elimination of radioactivity progressed over time with most tissues reaching the low limit of quantification at 168h. The highest tissue:blood ratios were noted in thyroid gland, lung and harderian gland at, respectively, 168h, 8h and 8h post‐dosing. Protein binding data indicates that zamicastat is preferentially bound to plasma proteins in a concentration independent manner, with 98% and 96% bound to rat and dog plasma proteins, respectively.

Conclusion

Zamicastat is a highly plasma protein bound compound after oral administration and it is extensively distributed to tissues. After oral administration to rats and dogs, radioactivity was almost completely recovered with most of zamicastat‐related radioactivity excreted through faeces.
Methamphetamine‐induced retinal hyperpermeability, hypoxia, and vascularization
Authors: Lee Minsup , Leskova Wendy , Pickett Haley , Eshaq Randa , Harris Norman
Published: April 2020

Purpose

Methamphetamine (METH), a highly addictive stimulant of neurotransmitters induces central retinal artery occlusion with retinal atrophy and neovascularization. We previously have identified METH‐induced loss of endothelial surface molecules including platelet and endothelial cell adhesion molecule‐1 (PECAM‐1) and glycocalyx in mice. However, the pathophysiologic pathway of METH‐induced retinal vascular dysfunction has been insufficiently investigated. The purpose of this study is to investigate the METH‐induced alteration of the retinal vascular circulation.

Materials and methods

C57BL/6J male and female mice were administrated progressively increasing doses of METH (0 to 6 mg/kg) by repetitive intraperitoneal injections (4 times per day) for 4 weeks. Norepinephrine levels in plasma were measured by ELISA. Cross‐sections of the retina were stained with hematoxylin and eosin. Levels of hypoxia in the retina were determined by immunostaining of pimonidazole adducts with Hypoxyprobe. Retinal blood vessels were visualized with fluorescent plasma tracers injected via femoral vein injection for live imaging, and immunostained with griffonia simplicifolia lectin 1 (GSL‐1) for retinal flatmounts. Expression of various proteins was determined by immunoblot.

Results

The level of pimonidazole adduct was increased in METH‐treated retinas, compared with saline‐treated retinas, indicating METH‐induced retinal hypoxia. Plasma norepinephrine levels, which can promote hypoxia‐inducible factor 1a (HIF‐1a) at the transcriptional level, were elevated by METH, compared with saline treatment at 1 week (2.2±0.9 vs 7.8±1.6 ng/mL, P<0.01), 2 weeks (0.8±0.3 vs 5.5±1.8 ng/mL, P<0.05), and 4 weeks (0.9±0.2 vs 2.0±0.4 ng/mL, P<0.05). Retinal protein expression levels of HIF‐1a and angiogenic proteins, such as vascular endothelial growth factor a (VEGFa), erythroblast transformation‐specific (ETS)‐related gene (ERG), and Protein C‐ets‐1 (ETS‐1) were increased in the METH‐treated retinas. Counts of retinal arterioles and capillaries were higher in METH‐treated mice than in saline‐treated mice. Moreover, protein concentration in the vitreous humor and immunoreactivity of immunoglobulin in retina, which are measures of retinal permeability, were increased by METH treatment.

Conclusion

Our present data suggest that METH is associated with retinal neovascularization and hyperpermeability with increases in hypoxia, plasma norepinephrine, and HIF‐1a activation.

Support or Funding Information

This work was supported by funding from the National Institute of Health (NIH) EY025632.
Ovarian Cancer Detection Using Targeted Plasma Metabolic Profiling
Authors: Turner Cassidy , Jasbi Paniz , Shi Xiaojian , Atlas Sarah , Wei Yiping , Gu Haiwei
Published: April 2020

Ovarian cancer (OC) is the second most common form of gynecological cancer and is the most fatal among all forms of gynecological malignancies. Despite the important role of metabolic processes in the molecular pathogenesis of OC, robust metabolic markers to enable effective screening, rapid diagnosis, accurate surveillance, and therapeutic monitoring of OC are still lacking. In this study, we present a targeted liquid chromatography‐tandem mass spectrometry (LC‐MS/MS)‐based metabolic profiling approach for the identification of metabolic biomarker candidates that could enable expedited, highly sensitive and specific OC detection. Using this targeted approach, 90 plasma metabolites from many metabolic pathways of potential biological significance were reliably detected and monitored in 218 samples taken from the three groups of subjects (78 OC patients, 50 benign samples, and 90 healthy controls). Multivariate significance testing and receiver operating characteristic (ROC) analysis revealed 8 significant metabolites (p<0.05) with high magnitudes of fold change (FC>2) and high predictive accuracy [area under the curve (AUC)>0.90] for distinguishing healthy controls from OC patients. For discrimination of OC patients from benign samples, the results of our model refinement pipeline informed the construction of a logistic regression classifier defined by a 5‐metbaolite panel of potential plasma biomarkers, exhibiting roughly 75% predictive accuracy using a 50% random‐split training and testing paradigm for external validation. Multivariate ROC analysis showed enhanced classification performance for identification of OC patients from benign samples, relative to individual metabolites. Pathway analysis revealed significant disturbances in glycine, serine, and threonine metabolism; glyoxylate and dioxylate metabolism; the pentose phosphate pathway; and histidine metabolism. The results expand basic knowledge of the metabolome related to OC pathogenesis relative to both healthy and benign samples, revealing potential pathways or markers that can be targeted therapeutically. This study also provides a promising basis for the development of larger multi‐site projects to validate our findings across population groups and further advances the development of improved clinical care for OC patients.
DNA‐Binding Properties of TBX5 Mutants Associated with Congenital Heart Defects
Authors: Cesar Brenda I. , Carrasquillo Emmanuel I. , Zazaboi Jasmin , Rodriguez Jose A.
Published: April 2020

Congenital Heart Diseases (CHD) affects over 150,000 persons every year. CHDs are characterized by abnormal structural heart formation during embryonic development. Previous studies have identified several TBX5 mutations in CHD patients. TBX5 is a transcription factor (TF) that has been identified as an important regulator during heart development. TFs are sequence‐specific DNA binding proteins that regulate gene expression and control cellular state. We selected 9 non‐synonymous TBX5 mutants discovered in CHD patients. The consequences on the interactions between these mutated cardiac TFs and DNA have not formerly been investigated. Evaluation of DNA‐recognition properties of TBX5 is fundamental for understanding its possible roles in controlling gene regulatory networks and could potentially help us know more about the role of TFs in the context of CHD. Our lab plans to determine the sequences our TFs prefer and how this specificity changes when it mutates. In order to test this, we have cloned TBX5 and its T‐Box domain that contain a His‐ and Strep‐Tag affinity tags that we will use for purifying after we overexpress it in competent E. coli. cells. We have also generated all 9 sequence‐verified TBX5 mutants using site‐directed mutagenesis. In site‐directed mutagenesis, mutations are introduced by the use of a PCR primer pair carrying the intended mismatch. We have also optimized experimental conditions for the overexpression and affinity‐purification of the TBX5 T‐Box domain. The DNA‐binding activity of TBX5 DNA binding domain T‐box has been confirmed through electrophoretic mobility shift assays (EMSA) using the TBX5 cognate site. This assay allows us to visualize DNA‐protein affinity through electrophoretic gel shifts. The mutation I54T shows more affinity towards the TBX5 cognate site and the mutation G80R shows no affinity towards it. Identifying the effects of non‐synonymous mutations on the DNA‐binding properties of TBX5 will contribute to our understanding of TBX5 role during heart development in congenital heart defects.

Support or Funding Information

This research was funded by SC1GM127231
A metformin‐methylglyoxal imidazolinone metabolite (IMZ) increases angiogenesis via HIF‐1 alpha activation in endothelial cells.
Authors: Nguyen Huong , Wang Jiemei , Monks Terrence
Published: April 2020

Deficient angiogenesis exacerbates the outcome of patients with peripheral arterial diseases, leading to critical limb ischemia, non‐healing ulceration or even amputation in lower extremities. These events progress more rapidly in patients with diabetes mellitus. Therapeutic strategies aiming at improving angiogenesis have been intensively studied in clinical trials, but with limited benefit. We previously discovered that metformin, the first‐line medicine for diabetes, scavenges a biologically reactive endogenous dicarbonyl, methylglyoxal, to form a novel imidazolinone metabolite (IMZ). However, the impact of IMZ on endothelial cell function is unknown. We hypothesize that IMZ increases endothelial cell‐mediated angiogenesis by activating hypoxia inducible factor (HIF)‐1 alpha, engaging key angiogenesis pathway(s). We examined the effects of IMZ on endothelial cell angiogenesis using functional assays (aortic sprouting assay, network formation, migration and proliferation assay) in vitro and characterized IMZ’s potential signaling pathways regulating HIF‐1 alpha, using western blot analysis.

Our findings revealed that 24‐hour treatment of IMZ at 1μM significantly increased endothelial sprouting, the formation of capillary‐like networks on matrix, and cellular migration in endothelial cells. These effects of IMZ occurred concomitantly with an upregulation of multiple pro‐angiogenic mediators, such as placenta and platelet‐derived growth factor, fibroblast growth factor 2 beta, vascular endothelial growth factor, angiopoietin 1, as revealed by protein arrays. Meanwhile, IMZ activated endothelial nitric oxide synthase, increased nitric oxide production, and engaged the PI3K/AKT and MAPK pathway in a concentration and time‐dependent manner. These pathways stabilize and upregulate HIF‐1 alpha expression in endothelial cells. Indeed, IMZ promoted HIF‐1 alpha expression as measured by western blot. More importantly, the effects of IMZ on angiogenesis were blunted by knockdown of HIF‐1 alpha with small interfering RNA. Collectively, our data uncovered, for the first time, the pro‐angiogenic effects of a metformin metabolite IMZ. Future studies will further characterize the therapeutic potential of IMZ in alleviating impaired angiogenesis under diabetic conditions.

Support or Funding Information

NIH/NIDDK R01 DK119222 (Wang & Monks) and NIH/NIDDK R01 DK109036 (Wang)
Nuclear Receptors CAR, ERα and RORα Communicate via Phosphorylation to Differentially Regulate the SULT1E1 Gene Expression in Diabetic and Nondiabetic Human Livers
Authors: Fashe Muluneh Markos , Negishi Masahiko
Published: April 2020

Studies showed that SULT1E1 is differentially expressed in nondiabetic and diabetic human as well as mouse livers. Here, we intended to investigate the molecular mechanism of the differential expression of SULT1E1 in diabetic and nondiabetic human livers. Utilizing chromatin immunoprecipitation (ChIP) in male human livers and coimmunoprecipitation (CoIP) assays, we report that nuclear receptors CAR, ERα and RORα communicated via phosphorylation of their respective DNA binding domains to differentially regulate the expression of SULT1E1 gene in nondiabetic and diabetic (type 2) male human livers. We studied the functional role of ERα and RORα with respect to SULT1E1 expression in human primary hepatocytes by treating the cells with siRNAs against ERα or RORα and SULT1E1 mRNAs were suppressed along with ERα or RORα mRNAs. Moreover, promoter analysis, electrophoretic mobility shift assays and cell‐based reporter assays in COS‐1 cells identified three DNA elements: ERα response element (h1E1‐ERE) and RORα response element (h1E1‐RORE) within the enhancer region and a TATA box within the proximal promoter that were critical for the regulation of SULT1E1 gene in human liver cells. ChIP assays using antibodies against ERα, p‐Ser212 ERα, RORα or p‐Ser100 RORα and primers targeting the proximal SULT1E1 promoter revealed that both receptors were phosphorylated and enriched on the proximal region of SULT1E1 gene promoter in nondiabetic but not in diabetic male human livers. Similar observations were also made in ChIP assays performed with antibody against CAR. Moreover, CoIP assay in HepG2 cells overexpressing ERα WT or its ERα S212D mutant and RORα WT, RORα S100A or RORα S100D displayed a stronger interaction between ERα S212D and RORα S100D and this interaction was further enhanced with CAR cotransfection. Altogether, phosphorylation driven interaction between nuclear receptors CAR, p‐Ser212 ERα and p‐Ser100 RORα was critical for a positive expression SULT1E1 gene in human livers and lack thereof might be responsible for the observed downregulation of SULT1E1 in diabetic livers.

Support or Funding Information

NIEHS ‐ NIH Intramural Research Program
5‐HT2 Receptor Activation Differentially Modulates Linker Histone Kinetics and Histone Deacetylase Activity
Authors: Flanagan Thomas Wayne , Billac Gerald Bernard , Nichols Charles David
Published: April 2020

Introduction

Alterations to chromatin dynamics and histone modification represent crucial processes metazoan cells employ to influence regulation and gene expression. As such, modification to the mobility of the linker histone, the chief component of chromatin compaction and higher‐order chromatin architecture, significantly impacts downstream transcriptional regulation and cellular processing. At present, the contributions of serotonergic signaling to not only heritable epigenetic modification but also higher‐order chromatin structure are a matter of intense research focus. Through both in vitro and in vivo assays, our lab has shown that serotonin 5‐HT2A receptor agonists (psychedelics) exhibit differential efficacy and potency in their ability to prevent inflammatory pathway activation. Modulation to both overall chromatin structure and epigenetic signaling may represent a potential mechanism that psychedelics employ to promote their long‐lasting, therapeutic effects.

Objectives

We sought to evaluate the impact of chromatin compaction and histone acetylation on 5‐HT2A functional selectivity in human embryonic kidney cells stably expressing the 5‐HT2A receptor.

Methods

We utilized fluorescence recovery after photobleaching (FRAP) to probe the kinetics of the linker histone H1c transiently transfected into the aforementioned HEKH2A cell line. We concurrently measured histone deacetylase (HDAC) activity following exposure to numerous 5‐HT2A agonists.

Results

5‐HT itself minimally impacts H1c mobility, whereas compounds such as psilocin and the highly selective 5‐HT2 agonist (R)‐2,5‐dimethoxy‐4‐iodoamphetamine [(R)‐DOI] significantly slow H1c kinetics. Interestingly, these changes to linker histone mobility do not correlate with putative epigenetic regulation, as 5‐HT2A compounds with anti‐inflammatory properties reduce histone deacetylase (HDAC) activity, whereas antagonism of the 5‐HT2A receptor induces HDAC activity.

Conclusion

Together, this data illustrates that agonism of 5‐HT2A receptors induce unique changes to chromatin architecture and HDAC activity, a conceivable mechanism psychedelics might employ to promote their potent therapeutic properties. Translationally, while HDAC inhibitors have emerged as attractive agents in cancer therapy and asthma treatment, their utility is limited by off‐target toxicity. 5‐HT2A receptor activation represents a potentially more promising therapeutic strategy to modulate histone acetylation patterns for therapeutic effects.

Support or Funding Information

Support from this work was provided in part by the Eleusis Benefit Corporation and the LSU Foundation.
Substrate 1,25(OH)2D3 and Adrenodoxin exert a coordinated change in CYP24A1 structure
Authors: KUMAR AMIT , Tu Chengjian , Fernando David Estrada
Published: April 2020

The mitochondrial cytochrome P450 24A1 (CYP24A1) is involved in deactivation of bioactive 1,25‐(OH)2‐vitD by side chain hydroxylation. Mutated CYP24A1 leads to idiopathic infantile hypercalcemia, characterized by elevated calcium, failure to thrive, vomiting, dehydration, and nephrocalcinosis. Deactivation relies on the protein‐protein interaction with the redox partner, Adrenodoxin (Adx), housing the iron‐sulfur cluster which provides two electrons for the hydroxylation step through reduction of heme iron in CYP24A1. We have previously used solution nuclear magnetic resonance (NMR) to demonstrate the long‐range allostery of CYP24A1 in response to redox partner recognition. Here we report a comprehensive multidisciplinary approach using chemical cross‐linking coupled to mass spectrometry, site‐directed mutagenesis, NMR, functional assays and molecular dynamics simulations. Chemical crosslinking using 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC) traps a 1:1 CYP24A1‐Adx complex by way of reaction with the carboxylate side chains of Adx and the primary amines of CYP24A1. Through chymotrypsin digest and mass spectrometry analysis of this product, we’ve identified a set of CYP24A1 peptides that are differentially modified, consistent with rearrangement of salt bridges in the presence or absence of substrate and Adrenodoxin. Affected sites include regions of the F‐G helices, the A‐helix, and the C/D elbow of the proximal surface. Interestingly, substrate‐induced differential cross‐linking at these sites only occurs when Adx is already present, suggesting a coordinated effect on CYP24A1 structure. This approach is combined with data from additional biophysical methods in order to inform a model of the functional 1:1 P450‐Adx complex that is required for metabolism of vitamin‐D.
Investigation of Ceramide involvement in Diesel Exhaust‐Induced Astrocyte Apoptosis
Authors: Vettrus Noah , Morgan Connor , Johansen-Sallee Anthony
Published: April 2020

Apoptosis is a complex process involving many different molecules that eventually leads to the programmed death of a cell. Ceramides are believed to function as a second messenger in this process; however, the full involvement of ceramides is under investigation. We are investigating a connection between airborne particulate matter and neurodegenerative processes, including cell death. The primary mission of our research is to better understand how ceramide levels change during apoptosis induced by exposure to diesel exhaust particulate matter (DEP). To track the changing levels of ceramides we performed total lipid extractions on our cell cultures at various timepoints after exposure to DEP. As we gain more understanding on this role of ceramides in apoptosis, we hope to make connections between Alzheimer’s disease and incorrectly functioning ceramide signaling networks. Our current results have indicated a change in ceramide levels during apoptosis at a long timepoint and we continue to work to understand how the ceramide signaling route works.

Support or Funding Information

Research reported in this publication was supported by grant number P20GM103443.
Does DUSP15 promote activation of ERK MAPK signaling after Hsp90 inhibition in the spinal cord to promote opioid anti‐nociception?
Authors: Gabriel Katherin , Duron David , Streicher John , Langlais Paul , Barker Nathalie
Published: April 2020

The misuse of opioids has become a prevalent problem for chronic pain relief, worsened by numerous side effects such as constipation, tolerance and addiction. Researchers have attempted to optimize opioids to decrease side effects and eliminate dependence by modulating the signal transduction cascades of the mu opioid receptor (MOR). Previous studies from our lab have demonstrated that the chaperone protein Heat shock protein 90 (Hsp90) is an important regulator of the ERK/MAPK pathway which is downstream of MOR. When an Hsp90 inhibitor is administered directly to the spinal cord, there is an increase in opioid antinociception in vivo, signifying that Hsp90 acts to repress the ERK/MAPK signaling pathway and reduce antinociception in the spinal cord. The exact molecular mechanism including the key players in activating opioid anti‐nociception in the spinal cord are still unknown and need to be further investigated. We thus performed quantitative proteomic analysis on the spinal cords of male and female CD‐1 mice treated with the Hsp90 inhibitor 17‐AAG. Network analysis of this data revealed that the phosphatase DUSP15 was upregulated; DUSP15 is known to be a key activator of ERK MAPK, suggesting this molecule could be the mechanism by which spinal Hsp90 inhibition activates ERK MAPK and thus opioid anti‐nociception. To test this hypothesis, we will perform CRISPR/Cas9 gene editing in the spinal cords of adult male and female CD‐1 mice to knock down DUSP15. We will then combine knockdown with Hsp90 inhibitors (17‐AAG, KU‐32) and opioids in the tail flick anti‐nociception assay, to reveal if DUSP15 knockdown will affect the enhanced anti‐nociception caused by spinal Hsp90 inhibition. These studies will thus reveal a key molecular mechanism by which Hsp90 regulates the spinal cord opioid system. Our other studies have shown that spinal Hsp90 inhibition causes enhanced anti‐nociception and decreased side effects like tolerance; these studies will also establish the basic science foundation for the use of Hsp90 inhibitors to improve opioid therapy during chronic pain.

Support or Funding Information

These studies were supported by an Arizona Biomedical Research Commission New Investigator Award (#ADHS18‐198875) and institutional funds from the University of Arizona to JMS. The authors have no other relevant conflicts of interest to declare.
Optimizing Ex Vivo Model of Traumatic Brain Injury to Test Biomolecules of Therapeutic Interest
Authors: Vig Parminder J.S. , Ryu Jung , Lee H. Joon , Warrington Junie P. , Janorkar Amol V. , Pal Pallabi , Lopez Mariper
Published: April 2020

Traumatic brain injury (TBI) is recognized as a major health concern due to its increasing occurrence, broad spectrum of symptoms and disabilities. TBI initiates a cascade of secondary pathophysiological events, such as excitotoxicity, neuroinflammation, and oxidative stress that worsen brain pathology from hours to days following the injury. Apoptotic and necrotic neurons have been observed in regions far from the lesion site in the days and weeks after TBI. To evaluate the complex secondary and severity‐specific injury response, investigators have relied on pre‐clinical rodent models. However, these in vivo TBI models have demonstrated intra‐ and inter‐laboratory variability of clinically relevant functional evaluations that were not consistently correlated with the severity of injury. Here, we describe a novel ex vivo model of traumatic cerebellar injury (TCI) using organotypic cerebellar slice cultures to study trauma‐induced injury, and also to test the effects of hydrogels and Elastin‐Like Polypeptide (ELP) based therapeutic peptides (TPs) on cerebellar injury. The cerebellar slice cultures were prepared from 9–11 day‐old GFP wildtype or parvalbumin‐BrainBow mouse pups. After 7 days of organotypic culture, TCI was induced at day 7 in vitro (7 DIV) using a weight drop device. Live imaging of slices showed a marked loss of GFP‐fluorescence at 24 h post‐injury. At the impact site, we observed a loss of Purkinje cells and interneurons. In some slices where we induced acute TCI, a small cavity‐like defect developed within 72 hr post injury. After 4 DIV post‐injury, the injured area was treated with V120‐collagen hydrogel and the medium replaced with experimental medium (with or without ELP‐TPs). Live slices were observed every 4 DIV. Some TPs were able to progressively reduce the diameter of injured area and the loss of GFP‐fluorescence, emphasizing that a combination of hydrogel and ELP‐TPs is protective. These data suggest that our ex vivo TCI model could be a useful tool to test different pharmacological compounds with potential therapeutic activity on TBI.
zDHHC9‐mediated palmitoylation promotes hypertrophic signaling in the heart
Authors: Essandoh Kobina , Subramani Arasakumar , Molkentin Jeffrey , Brody Matthew
Published: April 2020

Heart disease remains a leading cause of mortality in the United States, contributing to over 600,000 deaths per year in the United States. Notwithstanding, the molecular mechanisms underlying the pathophysiology of heart disease remain largely elusive. Receptors, ion channels, and signaling proteins that regulate cardiac contractility and physiology are modified by post‐translational modifications such as phosphorylation. Recent research into the roles of S‐acylation or palmitoylation, the reversible post‐translational modification of proteins with fatty acids, has demonstrated critical functions in the regulation of intracellular signaling in physiology and disease. Palmitoylation, which is catalyzed by the zinc finger Asp‐His‐His‐Cys (zDHHC) family of S‐acyl transferases, has been implicated in trafficking, stability, sorting and binding of proteins to cofactors. However, the roles of palmitoylation in cardiac signal transduction and cardiac pathogenesis are not well understood. In HEK‐293 cells, the Golgi‐localized acyl transferase, zDHHC9, was shown to palmitoylate and induce plasma membrane localization and activation of H‐Ras and N‐Ras, members of the Ras family of membrane‐localized GTPases. Activation of Ras enhances the activity of downstream pathways such as the ERK/MAP kinase pathway and c‐Jun N‐terminal kinases (JNK), which induce pathological cardiac remodeling in the heart. To investigate the potential roles of zDHHC9 in cardiac pathogenesis, we generated a transgenic mouse model with cardiomyocyte‐specific overexpression of zDHHC9 (Tg‐Zdhhc9). These mice exhibited cardiac hypertrophy with increased ventricular wall thickness at 6 months of age. At 8 months of age, Tg‐Zdhhc9 mice showed impaired cardiac contractile function and severe dilated cardiomyopathy. Interestingly, there were no significant differences in palmitoylation of H‐Ras in Tg‐Zdhhc9 hearts compared to control hearts, indicating that zDHHC9‐triggered cardiac hypertrophy may be due to palmitoylation of other substrates in cardiomyocytes. Ongoing work is utilizing unbiased quantitative palmitoyl proteomics to identify novel zDHHC9 substrates that mediate activation of hypertrophic signaling in the cardiomyocytes. Altogether, these data suggest zDHHC9‐mediated palmitoylation contributes to hypertrophic signaling and heart disease.

Support or Funding Information

This work was supported by National Heart, Lung and Blood Institute grant R00‐HL‐36695 (MJB).
The Gantzer Muscle: An Expanded Study of this Variant Forearm Muscle
Authors: Herrold Christina B. , Cook Ryan L. , Burkett Jesse T. , Hobeika Natalie A. , Zdilla Matthew J. , Lambert H. Wayne
Published: April 2020

Gantzer muscles are anatomical variations found within the flexor compartment of the forearm. The Gantzer muscle typically arises from the flexor digitorum superficialis (FDS) and inserts into the flexor pollicis longus (FPL) or flexor digitorum profundus (FDP). The presence of this muscle can cause various compressive injuries, including neuropathy of the median or anterior interosseous nerve and impingement of the ulnar, common, or anterior interosseous arteries. Despite its high prevalence, the Gantzer muscle is often excluded from the differential for acute compartment syndrome and should be further considered during treatment and surgical management. This study expanded upon a previous assessment of cadaveric specimen forearms to determine further the prevalence of origin, insertion, and innervation of Gantzer muscles, as well as possible compressions induced from impingement by the anomalous muscle. A total of 288 limbs were dissected in 144 cadavers. The Gantzer muscle was found in 54.6% (148:271) of limbs. Therefore, in general, the Gantzer muscle is more likely to be present than absent. This report also details common and uncommon origins and insertions of Gantzer variants.

Support or Funding Information

T. Walley Williams Summer Anatomy Research Fellowship; WV Research Challenge Fund [HEPC.dsr.17.06]
A new approach for analyzing pHi recoveries from NH4+‐induced acid loads, applied to rat hippocampal neurons and astrocytes
Authors: Ruffin Vernon , Boron Walter
Published: April 2020

Regulation of intracellular pH (pHi) in brain cells (neurons and neuroglia) is essential for optimal cell function and primarily maintained by acid/base transporters in the SLC9 and SLC4 families. Large deviations in pHi can lead to disrupted cellular function or even cell death, whereas small changes have been proposed as a signaling method due to the pH‐sensitive residues within protein structure. In the hippocampus (HC), tight regulation of pHi underlies the cascading reactions and impacts the structure of proteins necessary for learning and memory formation. The present work provides a detailed examination of the methods used in the analysis of steady‐state pHi and the recovery from NH4/NH3 induced acidosis by the SLC9s in the absence of CO2/HCO3−, when SLC4 (HCO3−‐sensitive) acid/base transporters are largely inactive. In the present study, we use the pH‐sensitive dye BCECF to examine HC neurons and astrocytes, co‐cultured from embryonic (E18–20) Sprague Dawley rats. In the protocol, two sequential NH4/NH3 pulses are delivered and the steady‐state pHi values is identified before (checkpoint C) and after (E for pulse 1 and F for pulse 2) the NH4/NH3 pulse. Typically, steady‐state pHi values at C were more alkaline than E and F, most likely due to the inactivity of HCO3−‐dependent acid loaders in the SLC4 family. The recovery from acidosis is fit with a double exponential (DExp) which we replot as dpHi/dt vs pHi. With this traditional approach, dpHi/dt, as it approaches the asymptotic pHi, becomes slightly non‐linear. To exploit the mainly linear portion of the dpHi/dt vs. pHi plot (from the DExp fit), we fit these dpHi/dt vs. pHi points with a single exponential (SExp) to produce a quasi–single‐exponential rate constant. This analysis—when transformed to the pHi vs. time domain—generally produces a very good fit to the original pHi vs. time data. We summarize the twin pHi recoveries from individual experiments in thumbnails in which we display the quasi–single‐exponential dpHi/dt line segments that represent the pHi recoveries from the first and second NH3/NH4+ pulses. For each line segment in the thumbnail, the slope represents the quasi–single‐exponential rate constant, and the projected pHi‐axis asymptote represents the predicted new steady‐state pHi. The thumbnails permit a quick assessment of the pHi recoveries from acidosis in many cells, including a sense of the variability between the first and second pulses, or the variability among cells. The advantage to this approach is that our method of analysis allows an entire population of cells to be examined individually but quickly.

Support or Funding Information

This work was supported by the NIH National Heart, Lung, and Blood Institute. Grant # R01 NS018400
Studies on the Interaction of Unfractionated Heparin and Sulodexide with Functional Antiheparin Platelet Factor 4 Antibodies as Studied in Platelet Aggregation Assays
Authors: Farooqui Ambar , Hoppensteadt Debra , Iqbal Omer , Bacher Cassandra , Lewis Mary , Walenga Jeanine , Kouta Ahmed , Jeske Walter , Fareed Jawed
Published: April 2020

Introduction

Sulodexide represents a glycosaminoglycan derived antithrombotic agent which is clinically used for the management of vascular disorders and is pharmacologically active after both parenteral and oral administration. The purpose of this study is to compare the relative effect of sulodexide in mediating heparin induced thrombocytopenia (HIT) antibodies induced aggregation of human platelets in a standardized test system which is developed to compare the effects of heparins and related drugs.

Materials and Methods

Active pharmaceutical ingredients (API versions of sulodexide), fast moving heparin and dermatan sulfate were obtained from Alfa Sigma (Bologna, Italy). API versions of UFH were obtained from Medefil, Inc (Glendale Heights, IL, USA). All agents were dissolved in saline at a stock concentration of 1 mg/ml. Working dilutions were prepared at 100, 10 and 1 mg/ml. The source of functional HIT antibody represented a frozen pool of aphresis fluid (AF) collected from five clinically confirmed heparin induced thrombocytopenia patients. The functionality of the HIT antibodies in this pool were confirmed in the platelet aggregation and 14C serotonin release assay. All results were compiled in terms of percent aggregation of platelets and represented as mean ± standard deviation.

Results

UFH produced a concentration dependent aggregation from 0 – 10 ug/ml reaching a peak aggregation of 60 ± 18.76%. Sulodexide produced a relatively weaker aggregatory response and produced mild aggregation in the same concentration range exhibiting comparable platelet aggregation at 10 ug/ml. The fast moving heparin component of sulodexide produced a concentration dependent response comparable to heparin reaching a maximum aggregation of 66 ± 30.67% at 10 ug/ml. The dermatan sulfate component did not produce any significant aggregation of platelets and no concentration dependence was observed (<10% aggregation). Wide intra variations were observed in the HIT antibody aggregation response with both the heparin and sulodexide but not with dermatan sulfate.

Discussion

These results clearly show that in comparison to heparin, sulodexide produces a relatively weaker response in the functional HIT antibody screening studies. However, the fast moving heparin component of sulodexide produces comparable responses to UFH whereas dermatan sulfate has much weaker aggregation response. The presence of dermatan sulfate may modulate the interaction between platelet factor 4 and sulfated polysaccharide change present in sulodexide preparations.
Influence of the eye on neurocranium development; Insights from Mexican cavefish (Astyanax mexicanus)
Authors: Gimhani Nuwanthika , Atukorallaya Devi
Published: April 2020

Astyanax mexicanus is being recognized as an intriguing teleost model in developmental biological studies. A. mexicanus exists as two morphs eyed, pigmented surface dwelling morph known as surface fish and an unpigmented blind cave dwelling cavefish. The midline bones of anterior neurocranium of A. mexicanus consists of parasphenoid, ethmoid and vomer bone. The detailed anatomical development of neuorocranial skeleton of this fish is unexplored thus far. The present comparative study is designed to understand the palatal bones, parasphenoid, ethmoid and vomer bone development among surface fish and in two population of cavefish Pachon and Tinaja. Cavefish and surface fish (Astyanax mexicanus) were included in to this study. Fish were fixed at different time point ranging from 10 dpf, 20 dpf, 35dpf, 60 dpf and 5 year old adults. Alizarin red bone stained were used to stain the skeleton. Dissected samples were examined under the stereo microscope for the ossification sequence of the palatal bones. The micro‐CT images of adult fish were taken using SkyScan micro‐CT imager (Bruker) and CT vox software (Bruker) was used for image analysis. First ossification center appears at the parasphenoid bone at 5 dpf. The anterior neurocranial bones ossified via endochondral ossification. Both cavefish and surface fish ossification starts from the ossification center locate in the center of the parasphenoid cartilaginous palate. Ossification progresses towards anterior direction and merge with the ossifying maxillary bone. The ossification extend up to the notochord in posterior direction. The Micro‐CT images showed that the morphology of parasphenoid bone is different in each eye morph. In surface fish parasphenoid bone curved and it has 20° angle of elevation towards the anterior direction. In contrast, parasphenoid bone found to be straight in Pachon and Tinaja cavefish. Absence of eye structures have an influence on determining the shape of parasphenoid bone.

Support or Funding Information

We would like to thank the Natural Sciences and Engineering Research Council of Canada (#2019‐05364) for funding this research. We acknowledge the support from University of Manitoba.
Interplay Between Whole Body Metabolism, Physical Activity, and Muscle Function Following Volumetric Muscle Loss Injury
Authors: Dalske Kyle A. , Basten Alec M. , Raymond-Pope Christiana J. , Call Jarrod A. , Greising Sarah M
Published: April 2020

Volumetric muscle loss (VML) injury occurs when a substantial volume of skeletal muscle is abruptly removed and results in significant long‐term functional limitations. Following VML injury, patients likely undergo periods of low or no mobility and are expected to have reduced physical activity. Immobility and lack of physical activity leads to impaired muscle function, and increased all‐cause mortality and risk for chronic disease. It is plausible that inactivity and immobility negatively affect outcomes following VML injury, exacerbating functional impairments. Our previous work supports an ~25% lower oxygen consumption rate in permeabilized muscle fibers of the muscle remaining after VML up to 4 months post‐injury, but how this may impact whole body metabolism is unclear. Therefore, the purpose of this study was to characterize whole body metabolic and physical activity following VML injury. We hypothesized that metabolism and physical activity would be impaired following VML. To elucidate the interplay between metabolism, activity, and muscle function following VML injury, adult male C57BL/6 mice (n=7) underwent a full‐thickness multi‐muscle VML injury (removal of ~22mg) to the gastrocnemius, soleus, and plantaris, or sham surgery (n=7). Twenty‐four hour whole body metabolic and physical activity was assessed pre‐, and 2 and 6 weeks post‐VML (or sham) injury. Contractile activity of the ankle plantarflexors was assessed terminally at 8 weeks post‐VML. Prior to randomization into experimental groups metabolic rate was ~18.5kcal/kg/hr and there was no difference in any metabolic or activity measure (p≥0.21), and both groups gained ~3g over the 8 week period. Whole body metabolism was evaluated for the 24‐hour period and isolated by the 12 hours of active and inactive time; respiratory exchange ratio (RER), an indirect measure of muscle oxidative capacity, was not different across experimental groups (p≥0.76) but was significantly decreased with time (main effect p≤0.01). Two weeks post‐VML mice tended to become hypermetabolic with a metabolic rate ~14% greater than sham. On average, mice ambulated ~1.2 km per 24‐hour period and contrary to the hypothesis, there was no difference in activity levels (p≥0.23) between sham and VML injured mice. Terminally the plantarflexor muscle group of the VML‐injured mice had ~1.4 fold greater maximal passive torque (indicator of muscle stiffness) and ~40% less maximal active isometric torque. These findings indicate limited changes whole body metabolic and physical activity levels in a model of VML injury, contrary to our hypothesis. Future studies will limit physical activity levels in this model to better recapitulate the low or no mobility expected following VML injury clinically.

Support or Funding Information

Supported by W81XWH‐18‐1‐0710 & T32AR050938
CD99L2 is a Major Regulator of Transendothelial Migration
Authors: Rutledge Nakisha S. , Sullivan David , Muller William
Published: April 2020

Transendothelial Migration (TEM), the process in which leukocytes leave the bloodstream to enter inflamed tissues, is a crucial step in the inflammatory response. Adhesion/signaling molecules such as PECAM and CD99 are important for the initiation and completion of TEM, respectively. We and others have shown that CD99‐like 2 (L2), a highly glycosylated 52 kD type‐1 membrane protein that is the only molecule in the genome related to CD99, is also important for TEM. However, L2 is different enough from CD99 that it is not clear whether it regulates TEM by the same mechanism as CD99. Using intravital microscopy, we show a critical role for L2 in recruitment of monocytes and neutrophils to sites of inflammation. An acute dermatitis model shows L2 specifically facilitates leukocyte extravasation across endothelial cell borders.

All of the published studies on L2 have been performed in mice. In order to study the mechanism used by L2 and the relevance to human inflammation, we studied the role of L2 on human cells in vitro. Our data show that similar to PECAM and CD99, human L2 is constitutively expressed at the borders of endothelial cells and on the surface of leukocytes. Blocking L2 using antibodies or knockdown of L2 significantly reduced transmigration of human neutrophils and monocytes across human endothelial cells. TEM is restored by re‐expression of L2. Blockade of either leukocytes or endothelial cells blocks equivalently to blocking both. TEM is significantly reduced in the presence of soluble L2‐Fc. These data are consistent with a homophilic interaction between leukocyte L2 and endothelial cell L2 being essential for TEM. Our data also show that L2 regulates a step in TEM after PECAM and before CD99. Consistent with this, preliminary data suggest that L2 can promote TEM by engaging either the signaling pathway used by PECAM or the one used by CD99. Similar to PECAM and CD99, L2 promotes transmigration by recruiting the lateral border recycling compartment to the site of TEM, downstream of PECAM‐mediated initiation of TEM. Ongoing studies are focused on identifying signaling pathways used by L2 to promote TEM and validating them in vivo using intravital confocal microscopy to study TEM in real time.

Support or Funding Information

T32‐5T32AI007476‐19,R01‐HL064774, and F31‐HL131355‐01