bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurocognition of Teaching and Learning Clinical Reasoning in Veterinary Pathology Using Eye‐tracking and Electroencephalography
Authors: Anderson Sarah J. , Abdullayeva Nia , Hecker Kent , Warren Amy
Published: April 2020

Introduction

Examining clinical reasoning skill acquisition in health professions education is key to optimize the teaching of this skill. Insights from neural (electroencephalography) and biometric (eye‐tracking) data could provide novel proximal measures that generate quantitative empirical evidence representing a learner’s (and expert’s) cognitive processing. Visual diagnostic reasoning in particular relies on data acquired from visual images to recognize anatomic pathologies. Eye‐tracking research has shown distinct differences in expert versus novice eye tracking patterns in visual diagnostic reasoning, where novices are expected to have more disorganized eye‐movement patterns resulting in longer times to reach diagnosis and less time is spent focusing on important areas of interest. Employing eye‐tracking in conjunction with neural and behavioural measures could yield more complete evidence for analysis to understand nuances in progression of learning.

Aim

The aim of this work is to determine whether learners acquire the same neural signatures as expert pathologists and whether learners are attending to the clinically significant features of an image.

Methods

Novice learners (n = 27) were taught to accurately visually diagnose twelve distinct bovine liver pathologies through a trial and error process where feedback was provided using a computerized module. This learning module consisted of 288 fast paced (5 seconds each), self‐advancing, interactive questions. Expert veterinary anatomic pathologists (n = 8) also completed this module. Behavioural performance (accuracy, response time), EEG, and eye‐tracking data were collected for each participant as they progressed through the module. This study has been approved by the University of Calgary Conjoint Faculties Research Ethics Board (REB16‐0925).

Results

All data has been collected and is presently under analysis.

Discussion and Conclusion

Based on the results of this work we can determine whether training module is appropriately stimulating learner attention to clinically appropriate visual cues and whether similar neural signatures in competent learners (as defined by behavioural accuracy) compared to experts are observed. The findings of this work will also contribute to generating a neurobiometric profile of expertise and expertise development. By defining a profile, we can better assess how teaching strategies support or hinder the transition of learners along the novice to expert continuum. While advances in the development of tools to measure neurophysiological data continue to make neuroeducational research more accessible and practical in an educational setting, the challenge will be to meaningfully design and communicate research in a way that is applicable to educationalists.

Support or Funding Information

University of Calgary Veterinary Education Research Fund
Finite element analysis and β‐catenin Signaling at the Alveolar Bone after Tooth Extraction – study in rats
Authors: Rossi Ana Cláudia , Ferreira Beatriz Carmona , Freire Alexandre Rodrigues , Araujo Rafael , Okamoto Roberta , Prado Felippe Bevilacqua
Published: April 2020

β‐catenin signaling is critical for remodeling bone. Finite element analysis studies showed that there is an alveolar bone adaptation associated with chewing. The aim of this study was to evaluate the expression levels of β‐catenin and the alveolar bone deformation by finite element analysis in rats with tooth extraction. Twenty‐four male rats (Wistar linage), 2 months old, were used. The tooth (right upper incisor) extraction procedure was performed. In the control group (n = 6) the normal dentition was maintained. The euthanasia occurred at 5 (n = 6), 7 (n = 6) and 14 (n = 6) days after day 0 of extraction. The three‐dimensional model of the rat skull was constructed in each group, and the computational simulation by finite element analysis was performed to simulate the bite in the incisors. The left maxilla of all rats was removed and submitted to histological processing. The immunohistochemistry analysis was performed for β‐catenin labeling. The finite element analysis of the experimental group showed a smaller amount of deformations relative to the control group and a distribution pattern uniform. In osteocyte count, positive β‐catenin markings concentrated on the lingual alveolar bone and the ANOVA test showed that there were differences 14 days compared to the other groups (p = 0.00303). The findings of this study showed that the condition of tooth extraction can cause β‐catenin expression and altered regimens of alveolar bone deformation.

Support or Funding Information

The São Paulo Research Foundation – FAPESP (grant: 4674‐1/2017) and The Institutional Internationalization Program (PrInt) of the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES).
Tryptophan Depletion through a Low Protein Diet Alters Renal Structure and Function in Young Male Mice
Authors: Irsik Debra L. , Xu Jianrui , Kang Baolin , Zhong Qing , Ding Kehong , McGee-Lawrence Meghan , Bollag Wendy B. , Isales Carlos M.
Published: April 2020

Low protein diets have been used in patients with chronic kidney disease to decrease uremic burden, but its use is controversial due to concerns about malnutrition. Long term use of a low‐protein diet may also impact bone health through reduction of the essential amino acid, tryptophan. We have previously shown that feeding a low‐protein diet with increased levels of its metabolite, kynurenine (a change that also occurs in aging) to adult mice leads to bone loss. The kidney regulates many important factors for bone health such as Vitamin D, and in conjunction with parathyroid hormone, phosphate and calcium. Since aging is known to also impact the kidney, we hypothesized that alterations in the normal ratio of tryptophan to kynurenine would impact kidney structure and function. We fed 3‐mo‐old male mice one of four diets [normal 18% protein (NP) or low 8% protein (LP) with or without kynurenine added] for 8 weeks. Mice were housed in metabolic cages for baseline measurements and again at the end of the feeding for 24‐hr urine collection. At sacrifice, kidneys were weighed to detect changes in renal mass, frozen for Western blotting or formalin‐fixed for histological analysis. As expected, we found decreased albuminuria with LP; however, there was a further reduction with KYN treatment in LP mice. There was significant diuresis (2.2±0.31 vs. 0.8±0.1 ml/day) in LP compared to NP mice that was attenuated in KYN mice (1.0±0.2ml/day); this effect may be due in part to a reduction in AQP2 levels. Natriuresis followed a similar trend but did not reach significance (0.12±0.01 vs. 0.08±0.01 mmol/day). Renal mass alone (0.17±0.00 vs. 0.20±0.01 g) and kidney weight/body weight (0.0055±0.0001 vs. 0.0059±0.0001) was decreased in LP mice with no further impact of KYN. Thus, while changes in the tryptophan to kynurenine ratio seem to impact the kidney, the changes vary with the renal parameter investigated.

Support or Funding Information

AG036675
A Phosphorylation Dependent Regulatory Switch at the Gα12 N‐terminus Modulates Cell Growth Signaling
Authors: Nance Samantha R. , Meigs Thomas E.
Published: April 2020

The G12/13 subfamily of heterotrimeric guanine nucleotide binding proteins (G proteins) is composed of Gα12 and Gα13, which play important intracellular signaling roles including activation of serum response factor (SRF). SRF activates transcription of serum response element (SRE) regulated genes involved in cell growth and tumorigenic events. Although crystal structures for Gα12 and Gα13 have been reported, very little is understood about the structure and function of these proteins at the N‐terminus. Previous reports have suggested serine phosphorylation occurs near the N‐terminus of Gα12. To identify the target serine in Gα12 and its possible role in SRF signaling, a series of point mutations were engineered to either abolish or mimic serine phosphorylation. Replacing a Gα12‐specific N‐terminal serine with the phosphomimic aspartic acid disrupted Gα12 signaling to SRF, as measured by firefly luciferase assays. Signaling by Gα13 was unaffected by phosphomimics of potentially homologous serines, suggesting a Gα12‐specific mechanism. Insertion of the Gα12 N‐terminus harboring the phosphomimic into Gα13 bestowed negative regulation of SRF signaling upon this chimeric protein. These results point to a kinase‐dependent switching mechanism at the N‐terminus of Gα12 that governs its growth signaling. Currently, we are investigating protein interactions affected by the phosphomimic and screening potential kinases for phosphorylation of Gα12.

Support or Funding Information

NC GlaxoSmithKline Foundation; UNC Lineberger Comprehensive Cancer Center; NC Biotechnology Center
The Leadership Institute for Faculty Development at the Oakland University William Beaumont School of Medicine
Authors: Gould Douglas J. , Hammond Michelle
Published: April 2020

The purpose of the present project is to describe a cohort‐based Department‐based leadership program at the Oakland University William Beaumont School of Medicine. Leadership development programs are commonplace in the corporate world and becoming more popular in the academic sphere as institutions of higher learning continue to seek increasingly business‐like solutions to problems such as skyrocketing student debt, decreasing state support and competition for top students and faculty. Adapting the plentiful corporate leadership training models to academia is not always straightforward and even less so in the complex milieu of a medical school, which tend to have an even more bottom up structure than the main campuses with which they are affiliated. A review of the literature reveals that leadership training increases leadership behaviors and increases job performance. The various members of medical school departments have many lessons to apply from leadership training and development that can help with issues such as developing a vision, leading teams, negotiation, budgeting and difficult conversations. Our cohort‐based program was developed loosely on Wenger’s theory of communities of practice, which has the potential to increase dialogue, enhance performance and improve peer and team relationships. We developed and implemented our small, cohort‐based leadership program within the Department of Foundational Medical Studies at the Oakland University William Beaumont School of Medicine in order to not only develop our next cadre of leaders, but also to spread and disseminate the principles of leadership and enhance the recognition by all Department members of the various leadership roles each holds.
TNFα Increases Mitochondrial Biogenesis in Motor Neurons
Authors: Brown Alyssa Denise , Rana Sabhya Denise , Fogarty Matthew J. , Mantilla Carlos B. , Sieck Gary C.
Published: April 2020

Pro‐inflammatory cytokines, like TNFα, mediate a cellular stress response to inflammation that may target mitochondria. In the present study, we hypothesize that in motor neuron‐like NSC‐34 cells, TNFα induces mitochondrial fragmentation and biogenesis in order to maintain overall O2 consumption of these cells. NSC‐34 cells were differentiated by serum depletion (~24 h) until neurite structures (>50 μm) were observed. Cells were then exposed to TNFα (20 ng/ml) for 24 h. Mitochondria were labeled using MitoTracker Green and imaged using confocal microscopy. Mitochondrial morphometry was assessed by measuring aspect ratio (ratio of long to short axis) and form factor (perimeter2/4p·Area). Expression of mitofusin 2 (Mfn2 – fusion protein) and dynamin related protein 1 (Drp1 – fission) was determined by Western blot. From 3D reconstruction of optical slices of NSC‐34 cells, cell and mitochondrial volumes were determined and mitochondrial volume density calculated. Mitochondrial biogenesis was assessed by measuring mitochondrial DNA, and expression of PGC1α was measured. An Agilent Seahorse XF Analyzer was used to measure cellular O2 consumption. A quantitative histochemical method was used to measure the maximum velocity of the succinate dehydrogenase (SDHmax) reaction within NSC‐34 cells to determine the functionality of the tri‐carboxylic acid (TCA) cycle and the electron transport chain (ETC). Finally, reactive oxygen species (ROS) formation was measured using MitoSox fluorescence. After exposure to TNFα, aspect ratio and form factor of mitochondria decreased by ~15% compared to untreated controls with consistent changes in Mfn2 and Drp1 expression. After TNFα exposure, mitochondrial DNA expression increased as did mitochondrial volume density, together indicating increased mitochondrial biogenesis. After TNFα exposure, O2 consumption per mitochondrion (normalized for mitochondrial volume density) decreased. This was consistent with a TNFα‐induced decrease in SDHmax. Despite the decrease in O2 consumption and SDHmax per mitochondrion after TNFα exposure, ROS formation increased reflecting mitochondrial dysfunction. We conclude that mitochondrial fragmentation, biogenesis and increased mitochondrial volume density are a homeostatic mechanism to decrease O2 consumption per mitochondrion thereby damping ROS formation, while maintaining overall O2 consumption to meet ATP demand.

Support or Funding Information

Supported by NIH grants AG44615 and HL105355.
Effects of Dihydroxylated Fatty Acid Mediators on Ethanol‐Induced Liver Injury in a Mouse Model of Alcoholic Liver Disease
Authors: Warner Jeffrey Barr , Hardesty Josiah , Warner Dennis , Song Ying , McClain Craig , Kirpich Irina
Published: April 2020

Background

Alcoholic liver disease (ALD) is the most common chronic liver disease and a major contributor to liver disease burden worldwide, and a disease for which there is no FDA‐approved treatment. The mechanisms of ALD pathogenesis are not completely understood. Data from our group indicate that dihydroxylated fatty acids (dihydroxy‐FAs) are increased in the livers of human alcoholic hepatitis patients. Dihydroxy‐FA lipid mediators are produced via cytochrome P450/epoxide hydrolase‐mediated metabolism of polyunsaturated fatty acids and are generally pro‐inflammatory. Despite their increase in human ALD, the role of dihydroxy‐FAs in ALD has not been explored.

Hypothesis

Exogenous administration of dihydroxy‐FAs will exacerbate liver injury in a mouse model of ALD.

Methods

Male C57BL/6 mice were provided ethanol (EtOH) using a chronic‐binge Lieber DeCarli ad libitum EtOH feeding model. Mice were administered either 8,9‐DiHETE (arachidonic acid metabolite), a combination of 9,10‐DiHOME and 12,13‐DiHOME (linoleic acid metabolites), or a vehicle control by i.p. injection once daily for two days prior to sacrifice. Liver injury, inflammation, and steatosis were measured. To further elucidate the effects of dihydroxy‐FAs on liver cells, primary mouse hepatocytes or HepG2 cells were treated with 8,9‐DiHETE, 9,10‐DiHOME, 12,13‐DiHOME, or a vehicle control. Effects on baseline or palmitic acid (PA)‐induced cell death were measured by MTT assay and apoptotic cell death was identified by Annexin‐V staining (cellomics).

Results

In vivo, 8,9‐DiHETE treatment modestly while 9,10/12,13‐DiHOME significantly increased liver injury as determined by elevated plasma ALT levels (1.3‐fold and 2.2‐fold p=0.04, respectively). While 8,9‐DiHETE had no effect on steatosis, 9,10/12,13‐DiHOME decreased liver triglyceride/free fatty acid levels compared to vehicle control. Dihydroxy‐FA treatment decreased the hepatic expression of several pro‐inflammatory chemokines at the mRNA level, including Mip2a and Mcp1. In vitro, 8,9‐DiHETE treatment of HepG2 cells or primary mouse hepatocytes significantly exacerbated PA‐induced cell death as determined by MTT assay, whereas 9,10‐DiHOME, and 12,13‐DiHOME had no effect on PA‐induced or baseline cell death or apoptosis.

Conclusions

Different dihydroxy‐FAs have diverse effects in vivo, in a mouse model of ALD, and in vitro. In vivo, dihydroxy‐FAs exacerbated liver injury and was associated with down‐regulated several pro‐inflammatory cytokines. In vitro, 8,9‐DiHETE increased hepatocyte cell death, while 9,10/12,13‐DiHOME showed no effects. Future studies are warranted to dissect the effects of individual dihydroxy‐FAs in ALD.

Support or Funding Information

This work was funded by grants from the National Institutes of Health and the Department of Veterans Affairs.
PaeR, a Multiple Antibiotic Resistance Regulator Homolog from Clostridium difficile is a Sequence‐Specific DNA Binding Protein that Responds to Small Molecule Effectors
Authors: Thompson Kara , Garcia-Ortiz Ruben , Wilkinson Steven
Published: April 2020

Proteins belonging to the multiple antibiotic resistance regulator (MarR) family are ubiquitous amongst species in the bacterial and archaeal domains. These proteins function as transcriptional regulators of a wide variety of cellular processes including catabolic pathways, oxidative stress response mechanisms, efflux or degradation of toxins, and virulence factor expression. DNA binding by MarR proteins is mediated by a characteristic winged helix‐turn‐helix (wHTH) motif which makes sequence‐specific contacts with both the DNA major and minor grooves. For a number of MarR homologs, DNA binding is abrogated by ligand binding. The natural ligands of MarR proteins are structurally diverse but generally fall into three main categories: small aromatic compounds, metals and reactive oxygen species. In this study, we report the first biochemical investigation of a MarR homolog from the pathogenic bacterium Clostridium difficile which we have named PaeR (putative antibiotic efflux regulator). Biophysical investigations of the structural properties of PaeR indicate that this protein exists predominantly as a homodimer in solution and unfolds in a manner consistent with a simple, two‐state transition. PaeR binds at two sites in its own promoter/operator region, suggesting autoregulation. Analysis of binding at a single site indicates a high‐affinity interaction with a microscopic dissociation constant of 3.3 ±0.4 nM. Competitive electrophoretic mobility shift assays (EMASs) indicate that PaeR interacts with DNA in a highly sequence‐specific manner. Ligand binding studies indicate that PaeR binding to its cognate DNA binding sequence is attenuated by small aromatic compounds. These findings, in conjunction with the genetic context of paeR in the C. difficile genome, suggests a possible role of this MarR homolog in regulating antibiotic resistance in this pathogen.

Support or Funding Information

Bill and Linda Frost Fund
Characterization of the Dual Routes for Bacillus subtilis Aspagainyl‐tRNA Synthesis
Authors: Cassello Natalie , Sheppard Kelly
Published: April 2020

Attaching asparagine to its cognate transfer RNA (tRNAAsn) is an essential step in protein synthesis. The bacterium Bacillus subtilis encodes two distinct routes for the formation of asparagingyl‐tRNAAsn. In the first pathway, asparaginyl‐tRNA synthetase catalyzes the direct esterification of asparagine tRNAAsn. In the second pathway, a non‐discriminating aspartyl‐tRNA synthetase misaminoacylates tRNAAsnwith aspartate. The aspartyl‐tRNAAsnformed is then transamidated by the amidotransferase GatCAB to form asparaginyl‐tRNAAsn. The non‐discriminating aspartyl‐tRNA synthetase, GatCAB, and tRNAAsnform a complex known as the transamidosome to synthesize Asn on tRNAAsn. Intriguingly, Bacillussubtilis also codes for two distinct tRNAAsnisoacceptors in its genome. Why Bacillus subtilis acquired an asparaginyl‐tRNA synthetase is not clear. We hypothesize that it enables the organism to adapt to different environmental conditions. To test this hypothesis, we have purified the Bacillus subtilis asparaginyl‐tRNA synthetase and transamidosome. We will report on characterizing in vitro the two pathways for asparaginyl‐tRNA synthesis under different physiologically relevant conditions as well as knocking out asnS in the Bacillus subtilis genome that encodes asparaginyl‐tRNA synthetase. The work will provide insight into how bacteria have adapted amino acid metabolism and use in protein synthesis for different environmental niches.

Support or Funding Information

National Science Foundation (MCB‐1615770)
Investigating the Activity of Novel Sigma‐2 Receptor Analogs in Pancreatic Ductal Adenocarcinoma Cell Lines
Authors: Inbody Laura R. , Hollenbacher Courtney A. , Hickey Mikaela C. , Kinder David H. , Schneider Ryan A.
Published: April 2020

Pancreatic cancer is an aggressive cancer that is highly resistant to most chemotherapy agents and has an estimated 5‐year survival rate of less than 10%. Sigma‐2 receptors (S2Rs) are highly upregulated in many cancers, including pancreatic. Specifically, S2Rs are overexpressed in pancreatic cancer cells, while minimally expressed in normal pancreatic cells, which makes them a potentially selective drug target. Additionally, pancreatic ductal adenocarcinoma cell (PDAC) lines have been found to be sensitive to SR2‐ligand binding, which initiates an apoptotic response. The selective estrogen receptor modifier, tamoxifen, has been shown to have affinity for S2Rs. We developed a series of analogs based on tamoxifen’s scaffold, with the intent to increase sigma‐2 receptor activity while decreasing estrogen receptor affinity and activity.

Previously, we have shown the analogs in our sigma receptor library presented here have low micromolar IC50 values in glioblastoma, melanoma, breast, and colorectal cancer cell lines. These analogs do not display selectivity between cancerous and noncancerous cell lines of normal human astrocytes and mammary epithelium.

Here we present additional screening data of SP‐1, SP‐5, SP‐6, and SP‐9 in three PDAC lines, ASPC‐1, BXPC‐3, and Su86.86. SP‐1, SP‐5, and SP‐9 all demonstrate similar efficacy with IC50 values ranging from 30 to 50 μM in each of the three PDAC lines. These findings are in line with previous studies with this library in the other cell lines listed above. Interestingly, the SP‐6 analog was much less active with IC50 values of 100 μM or greater in all three PDAC lines.

In summary, we found that our sigma‐2 analogs have low micromolar IC50 values in multiple PDAC lines. In the future, we would like to assess selectivity against non‐cancerous pancreatic cells and measure binding affinity of these analogs for S2R and estrogen receptors. Lastly, we would like to explore the mechanisms by which these analogs induce cell death in pancreatic cells.
The Role of Autophagy in Hydrogen Peroxide‐ or Methylglyoxal‐induced Cellular Damage in H9c2 Cells
Authors: Sehgal Amogh , Onal Halil , Martorana Rose , Wastella Catherine , Pjetergjoka Adona , Barsotti Robert , Young Lindon , Chen Qian
Published: April 2020

Oxidative stress (e.g., increased hydrogen peroxide [H2O2] levels) and dicarbonyl stress (e.g. increased methylglyoxal [MG]) serves as an initiator for the pathogenesis of cardiovascular diseases and diabetic complications. Increased H2O2‐derived free radicals and MG can be highly reactive and attack cellular components resulting in cell damage and even cell death. Normally, endogenous antioxidant mechanisms and glyoxalase degrade H2O2 and MG, respectively. Additionally, autophagy facilitates the break‐down of damaged cellular components allowing the components to be recycled by the cells. Researchers are still unclear about whether autophagy exacerbates cell damage or protects cells under higher doses of H2O2 and MG. This study investigated cell damage and autophagy changes when rat cardiac H9c2 myoblasts were treated with H2O2 or MG. Moreover, autophagy enhancing or inhibiting drugs were tested to evaluate their effects on H2O2‐ or MG‐induced cell damage. We found that H2O2 (300–900 mM, n=4) dose‐dependently decreased cell viability via measuring live cell dehydrogenase activity. H2O2 (600 mM, n=4) decreased cell viability to 16 ± 1% of the control. Similarly, MG (400–1400 mM, n=5) decreased cell viability in a dose‐dependent manner. MG (1200 mM, n=5) decreased cell viability to 29 ± 6% of the control. These results were further confirmed with the use of double staining, which contained calcein‐AM for labeling live cells and propodium iodide for labeling dead cells. By using Cyto ID autophagy detection dye, we found that H9c2 cells exhibited higher autophagy fluorescence after incubation of H2O2 (300–600 mM, n=2) or MG (400–1200 mM, n=2). Furthermore, trehalose (0.1–100 mM, n=3), an autophagy enhancer, reduced MG (1200 mM) induced cell damage. Trehalose (100 mM, n=3) increased cell viability by 69 ± 27% when compared to MG (1200 mM). Our preliminary results suggest that higher doses of H2O2 and MG can cause cell damage accompanied with increased autophagy. The autophagy enhancer trehalose provided cell protection against MG. Further studies are needed to confirm the effects of autophagy modulation on H2O2 and MG‐induced cell damage by using different autophagy modulators.

Support or Funding Information

This research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine.
Role of focal adhesion kinase in burn induced gut epithelial barrier dysfunction
Authors: Wang Fang , Eitnier Rebecca A. , Aponte Alexandra M. , Ewing Thomas J. , Wu Mack H.
Published: April 2020

Mucosal barrier failure is a critical pathological step in the development of multiple organ failure during severe burn injury and trauma. The opening of the epithelial barrier leads to increased leukocyte infiltration into the lamina propria and microbial translocation from the intestinal lumen. These events augment intestinal inflammation and contribute to the severity of trauma‐associated complications. The aim of the study was to characterize the role of focal adhesion kinase (FAK) in intestinal barrier dysfunction following thermal injury. Our data demonstrated that severe thermal injury significantly increased the gut lumen‐to‐blood transport of FITC‐dextran in mice. The effect was significantly attenuated by the inhibition of FAK with PF573228. Stimulation of mouse intestinal epithelial cells with TNFα/IFNγ caused a decrease in transcellular electric resistance coupled with downregulation of junctional proteins. The responses were prevented by the inhibition of either PTK6 or FAK. Western blot analysis showed that knocking down PTK6 in cells reduced TNFα/IFNγ mediated FAK activation via diminished detection of FAK phosphorylation at Y397. Reintroduction of active PTK6 to PTK6 deficient cells rescued FAK phosphorylation in response to TNFα/IFNγ. The results suggest that PTK6 dependent FAK phosphorylation plays an important role in gut barrier dysfunction during inflammatory injury.

Support or Funding Information

VA BX000799, NIH HL120954
Genetic Background influences Endothelial Function along the Mouse Vascular Tree
Authors: Holly Dylan Stone , Massett Michael , Gaytan Samantha , Kim Hyoseon , Shin Songyi , Woodman Christopher
Published: April 2020

The endothelium is crucial for its role in maintaining vascular integrity and protecting against cardiovascular disease. Endothelial dysfunction is associated with a host of disease states including cardiovascular disease, diabetes, chronic kidney disease, and atherosclerosis. However, it is unclear whether endothelial function or dysfunction varies throughout the vasculature. In addition, accumulating evidence suggests that endothelial function, and more generally vasomotor function, is genetically regulated. The purpose of this study was to determine the influence of genetic background on vasomotor function in blood vessels of differing size. Vasomotor performance was measured in aorta (thoracic (TA), and abdominal (AA)) and conduit arteries (carotid artery (CA) and femoral artery (FA)) from four inbred strains (SJL/J (SJL), DBA/2J (DBA), NZW/LacJ (NZW) and C57BL/6J (B6)) of mice. Arteries were dissected, cut into 2 mm segments, and mounted in a wire myograph system. Increasing concentrations of phenylephrine (PE, 1×10−9 – 1×10−5M) were used to measure contractile responses, while increasing concentrations of the endothelium‐dependent vasodilator acetylcholine (ACh, 1×10−9 – 1×10−5M) and endothelium‐independent vasodilator sodium nitroprusside (SNP, 1×10−9 – 1×10−5M) were used to assess relaxation responses. Two way ANOVA revealed a significant main effect of strain in response to PE for AA (P = .0032) and CA (P = .0098). Post hoc analysis for max contractile response showed impaired vasoconstriction in NZW AA compared to B6 (P = .0004). In response to ACh, a significant main effect of strain was found for all vessels (TA: P = <.0001, AA: P = .0156, CA: P = .0069, FA: P = <.0001). Post hoc analysis showed that TA max vasorelaxation was impaired for all strains (NZW: P = < .0001, DBA: P = .0140, SJL: P = < .0001) and that NZW FA were impaired when compared to B6 ( P = .0015). A significant main effect of strain in response to SNP was found in TA (P = .0011), AA (P = .0086), and CA (P = < .0001). Relaxation responses to SNP were not significantly different between strains. These findings indicate a strong influence of genetic background on endothelial function and could be important in understanding the implications that genetics have on vascular response to vasoactive agents.

Support or Funding Information

This work was supported by a Texas A&M Triads for Transformation grant (MPM), a Texas A&M Merit Fellowship (DH), a Texas A&M College of Education Strategic Research Award (SYS), and J.L. Huffines Institute of Sports Medicine and Human Performance (SYS).
SPARC: Development of Pigs with mCherry‐CRE Recombinase Fusion Proteins in Cholinergic Neurons
Authors: Sponchiado Mariana , Liao Yan-Shin , Collins Emily , Whitworth Kristin M. , Wells Kevin G. , Prather Randall S. , Reznikov Leah R.
Published: April 2020

Large animals are increasingly utilized in biomedical research and offer an advantage in that their anatomy and physiology closely parallel humans. Yet despite these advantages, rodent models dominate many areas of research, including neuroscience. This might be due in part to the greater number of technologies available to rodent researchers. Here we describe the development of pigs with mCherry and CRE‐recombinase proteins expressed in cholinergic neurons. We first cloned a 5,972 bp DNA fragment containing the predicted choline acetyltransferase (ChAT) promoter from porcine genomic DNA and placed into a pGlow TOPO™ TA expression system. We then tested for promoter activity by transfecting the expression system into porcine hippocampal slices and assessed for green fluorescent protein (GFP). We observed GFP at 60–72 hours post transfection. Confirmation of specificity was achieved by using antibody detection of ChAT. To optimize the expression system, we relied upon proven strategies used in rodents, and added an additional 535 bp of predicted promoter to the 5’ end. We then used this sequence to drive production of a mCherry‐P2A‐CRE recombinase fusion protein (ChAT‐mCherry‐P2A‐CRE). We transfected brain slice cultures from the pig hippocampus, striatum and brainstem with ChAT‐mCherry‐P2A‐CRE. Approximately 60 hours later, we isolated RNA and froze brain slices from transfected and non‐transfected samples. qRT‐PCR revealed mCherry and CRE recombinase mRNA in all transfected tissues. Expression of mCherry protein was also visible in discreet subsets of neurons in transfected samples. Studies are ongoing to confirm mCherry expression in cholinergic neurons. Key next steps include engineering our ChAT‐mCherry‐P2A‐CRE to contain GGTA1 homology arms for placement into the GGTA1 safe harbor in the pig genome, providing in vivo validation of ChAT‐mCherry‐P2A‐CRE using lentivirus pseudotyped with rabies glycoprotein, and targeting porcine fetal fibroblasts for somatic cell nuclear transfer. If successful, this model will allow for simultaneous visualization of neurons (and their innervation), and the ability to control or monitor neural activity through CRE‐mediated recombination.

Support or Funding Information

HL119560, 1OT2OD026582
Intraspecific and intrafamilial variation in primate forearm muscle architecture
Authors: Hartstone-Rose Adam , Boettcher Marissa L. , Seelye Jason , Worden Nikole , Dickinson Edwin
Published: April 2020

Recent studies have examined locomotion and postural correlates of primate forearm muscle architecture. However, these have done so using relatively small sample sizes – usually one or two specimens per species, and relatively few species per genus and family. Thus, little is known about intraspecific variation in forearm muscle fiber architecture in primates, and whether there are notable differences in these variables between closely related taxa. The current study addresses these deficits by assessing both qualitative (presence/absence of specific muscles) and quantitative (physiological cross‐sectional area [PCSA], fascicle length [FL] and muscle mass [MM]) differences in forearm muscle architecture of larger intraspecific and samples with greater coverage of specific primate families than had been included in previous work. Namely, we studied 30 callitrichid specimens from 11 species, 15 lemurid specimens from 7 species and 9 cebids from two species ‐ a total sample size of 54 specimens from 20 species. Within species, FL varied the greatest among architectural variables and MM varied the least. We also found that there is great variation in both the extent of separation and fusion of flexor digitorum superficialis and profundus, and in the extent of accessory extensors in most species of callitrichids and Saimiri, but no variation in the gross forearm muscle architecture within lemurids. More than a quarter of the callitrichid specimens had forearm muscle configurations that deviate from their standard published description, suggesting that blanket characterizations of anatomical configurations at the genus or family level may obscure potentially important intra‐ and inter‐specific trends.

Support or Funding Information

National Science Foundation: IOS‐15‐57125, BCS‐14‐40599
Autophosphorylation of the CK1 kinase domain regulates enzyme activity and function
Authors: Cullati Sierra , Chen Jun-Song , Gould Kathy
Published: April 2020

CK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; misregulation of CK1 contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Despite this, little is known about how CK1 activity is controlled.

CK1 kinases have highly similar catalytic domains, plus a conserved extension to that kinase domain that is important for enzyme stability and activity. In contrast to the catalytic domains, the C‐terminal tails of CK1 family members diverge in sequence and length; however, they all appear to serve as substrates of autophosphorylation. The autophosphorylated tails are proposed to inhibit kinase activity by acting as pseudosubstrates. In addition to C‐terminal autophosphorylation, autophosphorylation of the CK1□ kinase domain has been detected, but its effect on CK1 activity and cellular function has never been explored.

Here, we addressed the role of kinase domain autophosphorylation in human CK1□ and CK1□, as well as Hhp1 and Hhp2, their homologues in Schizosaccharomyces pombe. In each case, we found that autophosphorylation of a conserved threonine residue in the kinase domain inhibited enzyme activity. This site resides in the mobile L‐EF loop proximal to the active site, distinct from the well‐characterized T‐loop autophosphorylation that occurs in other kinase families. We found that yeast and human enzymes with phosphoablating mutations at this site are hyperactive in vitro. In vivo, mutation of this site protects yeast cells from heat shock, indicating a change in substrate profile, a prediction we are currently testing using quantitative phosphoproteomics. We have also found that autophosphorylation of the kinase domain may affect autophosphorylation of the C‐terminus, so there is likely interplay between the two different modalities of CK1 autoregulation that ultimately determines the extent to which substrates are phosphorylated.

We propose that phosphorylation on the L‐EF loop prevents substrate docking with the kinase domain by shielding the positively charged substrate binding pocket and/or sterically hindering the active site. Due to the strong sequence conservation of this autophosphorylation site and the functional importance of the L‐EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo.

Support or Funding Information

This work was supported by R35‐GM131799 (to KLG) and T32‐CA119925 (to SNC).
Investigating CRISPR Spacer Sequences of Escherichia coli in NJ/NY Metropolitan Area
Authors: Shah Rameen , Hassan Sherouk , Wei Yufeng
Published: April 2020

Clustered regularly interspaced short palindromic repeats (CRISPR) is a DNA sequence, found in bacteria, that functions as protective system against bacteriophages. CRISPR DNA sequence is characterized by the presence of short, identical nucleotide repeats that are interspaced by spacers. The spacer sequences are unique in that they are records of viral infections experienced by the bacterium and act as an “immunization card”. If the same virus attempts to invade again, the spacer sequence recognizes it via the help of CRISPR‐RNA (crRNA) and CRISPR‐associated (Cas) proteins. Subsequently, Cas proteins cut the viral DNA to eliminate the threat. Thus, CRISPR/Cas system provides an effective mechanism for bacteria, such as Escherichia coli (E. coli), to resist infections from bacteriophage, but it could also pose challenges to phage therapy, which utilizes bacteriophages to kill bacteria. The focus of this research is to examine spacer sequences from soil samples collected around NY/NJ metropolitan area at various timepoints, locations, and weather conditions. To accomplish this goal, the genomic DNA was extracted from E. coli bacteria from the soil and purified. The CRISPR locus was amplified and purified using polymerase chain reaction (PCR) and gel electrophoresis, respectively. The sequences were run through the CRISPR Finder program to identify spacer sequences, and NCBI’s Blast alignment was carried out to determine the specific E. coli strain associated with the sequence. We have identified several new spacer sequences, which could putatively link the history of bacteriophage infections of the E. coli bacteria to the unique environments the bacteria are located.

Support or Funding Information

We would like to thank the Chemistry Department at NJCU. This research was funded by National Institutes of Health (NIH), grant number R21DA046223.
Membrane Substrate Transporters as Target to Re‐balance Cardiac Energy Metabolism to Mend the Failing Heart
Authors: Glatz Jan F.C. , Luiken Joost J.F.P. , Nabben Miranda
Published: April 2020

Background

There is growing recognition of the importance of energy metabolism in the cardiovascular system, and that disturbances therein are at the basis of causing cardiac disease. Specifically, chronic fuel shifts towards a single type of substrate are known to induce cardiac dysfunction. For instance, lipid overload due to Western life style or obesity eventually elicits diabetic cardiomyopathy, while excess glucose utilization is associated with hypertrophic heart failure (and vice versa). Therefore, interventions aimed at re‐balancing a tilted substrate preference toward an appropriate mix of substrates (in particular fatty acids and glucose) may result in restoration of cardiac contractile performance. Because the key‐governing kinetic step of substrate utilization is their sarcolemmal transport, we hypothesize that manipulating their recruitment to the sarcolemma is an effective approach to mend the failing heart.

Results

Inhibition of the vesicular translocation of the fatty acid transporter CD36 to the sarcolemma by targeting endosomal H+‐ATPase in rat cardiomyocytes subjected to lipid overload completely restored both the fatty acid–glucose fuel balance and contractile function. Likewise, decreasing GLUT4‐mediated glucose uptake in experimental cardiac hypertrophy restored both the fuel balance and contractile function.

Conclusion

Re‐balancing cellular fuel supply, in particular with respect to fatty acids and glucose, appears an effective strategy to treat the failing heart. For such metabolic modulation therapy membrane substrate transporters, especially their subcellular recycling, are a promising target. Because vesicular trafficking involves >50 proteins and is highly (tissue‐)specific, manipulating transporter recycling would allow a heart‐specific approach.

Support or Funding Information

MN is supported by the Dutch Heart Foundation, grant 2019T041.
Role of AQP1 and RhAG proteins in the deoxygenation of mouse red blood cells during aging
Authors: Zhao Pan , Taki Sara , Popple Jacob , Radford Thomas S. , Meyerson Howard J. , Occhipinti Rossana , Moss Fraser J. , Boron Walter F.
Published: April 2020

The traditional view had been that all O2 moves through red blood cell (RBC) membranes by dissolving in and diffusing through the membrane lipid. In a previous abstract, we reported data obtained using stopped‐flow analysis of hemoglobin (Hb) absorbance spectra on RBCs from wild‐type (WT) vs. double‐knockout (dKO) mice genetically deficient in both aquaporin‐1 (AQP1) and the rhesus (Rh) protein RhAG. We found that the rate constant of Hb deoxygenation (kHbO2)—which includes intra/extracellular diffusion and membrane permeability—was ~31% lower for RBCs from dKO than for WT mice. From these data, we computed that, together, AQP1 and the Rh complex account for ~60% of membrane O2 permeability. We now examine the effect of aging on kHbO2 of RBCs from WT vs. dKO mice, studying mice at six ages (2–3 months, 6 months, 12 months, 18 months, 24 months, and 29 months). The kHbO2 of RBCs from WT mice are indistinguishable from age 2–3 months through 12 months. However, compared to 2–3 months, kHbO2 is 9% lower at 18 months (p<0.05, N = 6), 12% lower at 24 months (p<0.05, N = 5), and 13% lower at 29 months (p<0.05, N = 3). For RBCs from dKO mice, kHbO2 starts off ~30% lower than that of WT mice at 2–3 months (see above), and remains at this same depressed level through 29 months (N = 5 to 6 at 6–24 months, 4 at 29 months). In the case of WT mice at 24 months, our hematology data—which show decreases for both mean corpuscular volume and mean corpuscular hemoglobin—cannot explain the observed decreases in kHbO2. DIC microscopy of living RBCs and microscopic observation of stained blood smears with RBCs from 3 months and 29 months with both WT and dKO groups confirm that RBCs are normal biconcave disks. Preliminary data from mass spectroscopy of RBC ghosts from WT mice (N = 1 each for 2–3 months and 29 months) suggests that, compared to 2–3 months, membrane protein levels at 29 months are lower by 11% for AQP1, 19% for RhAG, 27% for mRh, and 22% for AE1. We conclude from our dKO data that the non‐AQP1/RhAG component of RBC membrane O2 permeability is independent of aging. We hypothesize that the 12–13% decrease of kHbO2 at 24–29 months for WT mice is due in part to decreased expression of AQP1 and the Rh complex (i.e., the AQP1 and RhAG components of O2 membrane permeability decline with aging).

Support or Funding Information

N00014‐15‐1‐2060 Gas Channels, ONRand N00014‐16‐1‐2535 ONR‐MURI
Why Does Nkx6.1 Fail to Increase Functional Beta‐cell Mass in Aged Islets?
Authors: Crabtree Jacqueline , Littlefield Connor , Tessem Jeffery Sivert
Published: April 2020

Diabetes Mellitus is a major health problem across the world. The number of people diagnosed is predicted to reach 642 million by 2040. The major forms of diabetes mellitus, type 1 and type 2, have the shared characteristics of hyperglycemia, impaired insulin production, and decreased beta‐cell mass. A possible cure for diabetes mellitus is transplantation of the beta‐cell containing islets of Langerhans. A major constraint to greater use of islet transplantation is the relative scarcity of transplant ready islets. Understanding that molecular pathways that result in increased beta‐cell proliferation could be used to increasing the number of transplant ready islets. We have shown that the beta‐cell transcription factor Nkx6.1 is sufficient to enhance glucose stimulated insulin secretion and induce beta‐cell proliferation in primary rat islets. However, Nkx6.1 overexpression in aged rat islets is unable to drive expression of key target genes that result in increased glucose stimulated insulin secretion and proliferation. This dichotomy is extremely important, given that most islet donors are beyond adolescence. Therefore, understanding why aged beta‐cells are refractory to these Nkx6.1 mediated signals is essential. We hypothesize that Nkx6.1 is unable to drive expression of genes necessary for beta‐cell proliferation and insulin secretion in aged islets due to lack of accessibility to chromatin due to changes in Nkx6.1 binding partner expression. Here we present data demonstrating the difference in Nkx6.1 promoter localization and binding partners as a process of aging. These data begin to describe age dependent changes that leave the aged beta‐cell refractory to proliferative signals.
Carotid Artery Stiffness and Elasticity in Angiotensin II Treated Mice
Authors: Pardo Paulina V. , Chambers Laura C. , Yen Ting-Chieh , Jackson William F. , Dorrance Anne M.
Published: April 2020

Hypertension, a risk factor for dementia, can impair the structure and function of cerebral arteries. Carotid arteries supply approximately 80% of the of blood required by the brain; increased stiffness and reduced distensibility in these arteries contribute to cerebrovascular disease. Angiotensin II (AngII)‐induced hypertension negatively affects the cerebral vasculature; it is possible that this is in part a response to increased carotid artery stiffness because stiffer arteries allow more of pulse pressure to be transmitted to the downstream vasculature. We hypothesized that AngII treatment in mice would lead to an increase in carotid artery wall stiffness, a loss of distensibility and impaired cognitive function. Sixteen‐week‐old C57BL/6 female mice were treated with AngII (1200 ng/kg/min via osmotic mini pump) for four weeks. Shams served as controls. Blood pressure was measured using tail‐cuff plethysmography. Barnes Maze was used to assess spatial memory and a nesting test was used to assess executive function. At euthanasia, arteries were isolated and mounted in a pressure myograph. To assess passive structure carotid arteries were incubated in Ca2+ free physiological salt solution (PSS) containing EGTA (2μM) and sodium nitroprusside (100μM). A pressure‐response curve was constructed; the intraluminal pressure was increased from 3 to 140 mmHg in 20‐mmHg increments and lumen diameter (μm), outer diameter (μm), and wall thickness (μm) were measured at each pressure. These data were used to calculate wall‐to‐lumen ratio, distensibility, circumferential wall stress (dynes/cm2), and wall strain. b‐coefficient, a measure of artery stiffness was calculated from stress/strain curves. Data are presented as means ± SEM (n=5–12). AngII infusion resulted in elevated systolic blood pressure (Sham: 142 ± 7; AngII 179 ± 7 mmHg; p<0.05). Barnes Maze tests showed that spatial memory was not impaired by AngII treatment (Sham: 25.2 ± 3.2; AngII 30.1 ± 3.4 seconds spent in target quadrant; p>0.05). Nesting test showed that executive function was impaired by Ang II treatment (Sham: 41.6 ± 7.7; AngII 10.5 ± 1.3 % nestlet shredded; p<0.05). There was no change in b‐coefficient in the AngII treated group (Sham: 4.91± 0.84; AngII 5.73 ± 1.20 p>0.05). Distensibility was also unaffected by Ang II treatment (Sham: 0.39 ± 0.01; AngII 0.39 ± 0.01; p>0.05). The change in executive function does not appear to be related to changes in carotid artery stiffness. Future studies will assess cerebral artery function. These data support the hypothesis that young female hypertensive mice are protected from changes in spatial memory, however, the impaired executive function is suggestive of initial negative changes in cognition.

Support or Funding Information

RO1‐HL‐137694‐01, AM Dorrance and WFJackson