bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating Cellular Interactions Between the Rho GTPase TCL/RhoJ and Coronin Proteins
Authors: McRoss Jasmine M. , Grigoryants Angelina J. , Hamann Michael J.
Published: April 2020

The Rho‐family GTPase TCL/RhoJ enhances pro‐angiogenic and pro‐metastatic cellular processes; however, contributing protein interactions are not well characterized. TCL is part of the Cdc42 subfamily of Rho GTPases but has a short, unique N‐terminal amino acid sequence that is important for GTP‐loading and plasma membrane localization. Additionally, we have evidence that TCL binds vesicular membranes through a protein/protein interaction when it is GDP‐loaded. We are evaluating whether Coronin proteins selectively interact with GDP‐loaded TCL, as Coronin proteins contain CRIB motifs that bind other Rho family GTPases when they are GDP‐loaded. To determine if the Coronins co‐localize with TCL at vesicular membranes, mCherry‐fusions of the seven Homo sapiens Coronin proteins will be transfected into HeLa cells along with YFP‐tagged TCL and mutants bearing constitutively active (Q79L) and dominant negative (T35N) mutations. These TCL mutations will mimic GTP/GDP bound forms of TCL and help identify nucleotide‐dependent interactions with Coronin proteins. Coimmunoprecipitation assays will also be performed to detect interactions between Flag tagged‐TCL and myc tagged‐Coronin proteins. These results will provide additional insights into binding partners that influence the role of TCL in pro‐angiogenic/metastatic processes.

Support or Funding Information

This work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).
Methodology for Efficient and Biosecure Genetic Engineering of Biomedical Swine Models
Authors: Meudt Jennifer J. , Reichert Jamie L. , Lopez Ana Cecilia Escobar , Krentz Kathleen J. , Rubinstein Clifford Dustin , Lehman Brent P. , Schomberg Dominic T. , Monson Ricky L. , Willenburg Kilby , Frank Jennifer M. , Graff Keri D. , Shanmuganayagam Dhanansayan
Published: April 2020

The genetic proximity of swine to human, the overwhelming anatomical, physiological and pathophysiological similarities, and the ability to mimic external factors in a practical manner make swine the ideal preclinical model for many human diseases. While the value of swine in biomedical research is well recognized, their use has been limited by the lack of tools to easily edit the swine genome to model human disease, and the lack of facilities, resources and expertise at most institutions for supporting large‐scale swine research. The recent implementation of new genetic editing tools such as the CRISPR/Cas9 platform make the creation of novel genetic swine models possible and practical. However, conventional approaches to genetic engineering of swine rely on ova obtained from commercial abattoirs, significantly compromising biosecurity, and often offering little control over the background genetic of the swine breeds upon which the genomic editing is performed. Here, we describe methods and workflow that we have developed that allow for efficient genetic engineering of swine, while maintaining control over background genetics and minimizing the biosecurity risk to the animals and the facility. The process centrally relies on the refinement of reproductive cycle synchronization and superovulation of swine and time‐precise coordination of the entire genetic engineering workflow.

Support or Funding Information

The research was supported by the Biomedical & Genomic Research Group Discretionary Fund (University of Wisconsin‐Madison), NF North Central, NF Network, and NF Team Foundation
Household Air Pollution Indicators and Epigenetic Changes in the Renin Angiotensin System Genes ACE and AGTR1
Authors: Swann Sellers , Beach Kayman , Ehmke Sarah , Rice Nancy A.
Published: April 2020

Cardiovascular diseases (CVD) are the world’s leading cause of mortality and disproportionately affect individuals in low and middle income countries (LMICs). Hypertension (HTN) is a multifactorial disease that is the current leading cause of CVD. In this study, we investigated the epigenetic regulation of two key genes involved in regulation of blood pressure, angiotensin‐converting enzyme (ACE) and angiotensin II receptor type 1 (AGTR1), from a cohort of Kenyans known to have both a high prevalence of HTN and exposure to household air pollution (HAP). Genomic DNA, isolated from saliva samples, was used to quantify methylation of cytosine‐phosphate guanine (CpG) islands in the promoter regions of ACE or AGTR1 by pyrosequencing following bisulfite conversion. In preliminary samples (n=29) analyses indicate that when stratified by blood pressure, both ACE and AGTR1 are hypomethylated in hypertensive versus normotensive individuals (1.43 ± 0.316 vs. 1.524 ± 0.35 and 2.841 ± 1.35 vs. 2.104 ± 0.591, respectively). However, specific CpG3 dinucleotide in the AGTR1 promoter showed a significant 3% increase of methylation (5.652 ± 3.069 vs 2.638 ± 0.734; p=0.028) in hypertensive versus normotensive individuals. In this preliminary work, no significant associations were found between methylation and indicators of HAP, although a strong positive correlation exists between CpG2 and CpG4 methylation in AGTR1 (R=0.990; p<0.001). Our findings indicate epigenetic regulation of AGTR1 may have a role in modulating high blood pressure incidence in rural LMICs where HAP is epidemic.

Support or Funding Information

We acknowledge funding form the University of South Alabama to N.A.R.
Peer Assisted Learning Experience as an Extracurricular Activity during a Gross Anatomy course.
Authors: Pabon Daniela , Muñoz Nicolas Stozitzky , Rueda-Esteban Roberto Javier
Published: April 2020

Multiple medical schools have modified classical teaching methods introducing new tools to improve learning processes
1. Amongst these, extra academic activities defined as out of the curriculum, of voluntary participation
2, and without grading, have been described as activities requiring auto discipline, teamwork, commitment and knowledge
3 as these are mostly carried out by an academic peer. These and other group activities in and out of campus should not be overlooked, as they also influence student performance in medical school. Additionally, peer assisted learning has many benefits, which include preparing students as future educators, improving their communication skills and reducing staff workload.

The aim of this study is to describe the effect of a peer assisted learning based extracurricular activity on student performance during a Gross Anatomy course in an integrated curriculum medical school and the students’ perception on their own learning process.

Quantitative and qualitative data acquisition tools were used to determine the existence or not of an association between the activity and students summative performance. Grades from two groups without the activities n=162 and two with these n=127 were considered with a total of 22 interventions. Additionally, quantitative data was triangulated with qualitative data obtained from a focal group with the student’s perception on their own learning process.

No positive association (p > 0.05) between the grades of each group were found. However, as each exam is composed by a theoretical and a practical component, a decrease in the difference between theoretical and practical grades when peer assisted extracurricular activities were applied was evident. When qualitative data was analyzed, there was a general perception by students that they had less stress levels during the practical component after the activity.

Peer‐assisted extracurricular activities show to be beneficial for both: those who attend and peers. Students consider this activity as helpful in reducing other factors such as stress and anxiety that may also be influencing their performance on the exam. However, not finding an association may occur because the students who are more academically able tend to seek out teaching and/or research opportunities and participated at these activities
2. This and other factors need to be addressed and evaluated in order to restructure this tool, so it can be improved in a way it can be implemented and fully analyzed in the future.

Support or Funding Information

Anatomy Laboratory, Universidad de los Andes School of Medicine
Mitochondrial Fragmentation in Skeletal Muscle Derived Cells from an Old Male Donor May Relate to Decreased Oxygen Consumption Rates
Authors: Arevalo José Adan , Miller Marvin L. , Vazquez-Medina José Pablo , Brooks George A.
Published: April 2020

With age, skeletal muscle mitochondria lose their ability to fuse and regenerate following stress and exercise leading to fragmentation. These phenomena can lead to reduced oxygen, muscle atrophy, reduced maximal O2 consumption, loss of metabolic flexibility and risk of developing age‐related diseases such as sarcopenia. Skeletal muscle cells derived from human donors can be used to investigate these physiological capacities in primary culture. Our objective is to investigate mitochondrial morphology and cellular maximal oxygen consumption rates (OCR) of skeletal muscle cells derived from healthy young and old male donors. Primary skeletal muscle cells derived from the Rectus abdominis muscle of healthy active eighteen and sixty‐nine year old men (SKM18M and SKM69M, respectively) were obtained from Cook MyoSite Inc. (Pittsburgh, PA). Cells were stained with MitoTracker Red (Cell Signaling; Danvers, MA) and mitochondrial morphology was observed using a Zeiss LSM 710 AxioObserver confocal inverted scanning microscope (Carl Zeiss; White Plains, NY). The mitochondrial network was analyzed using the Mitochondrial Network Analysis tool in ImageJ (MiNA, FIJI) to estimate mitochondrial footprint from a binarized image. Oxygen consumption rates were measured in intact cells using Seahorse Cell Mito Stress Tests on a XFp extracellular flux analyzer (Agilent Technologies; Santa Clara, CA). Primary cells derived from the young donor (SKM18M) had a larger mitochondrial footprint, longer branch length, and a greater number of network branches compared to the old donor (SKM69M) (Footprint: 34.65 ± 25.30 vs. 11.64 ± 9.53 μm2; Branch Length: 20.59 ± 7.23 vs. 12.10 ± 6.84 μm; Network: 17.25 ± 0.16 vs. 7.67 ± 4.97 counts). SKM18M also showed higher Basal and Maximal OCR compared to SKM69M (Basal: 38.78 ± 8.34 vs. 12.82 ± 2.07; Maximal: 60.09 ±10.84 vs. 20.52 ± 2.36 pmol/min/protein). We observed differences morphologically and metabolically between the primary skeletal muscle cells derived from young and old donors. These preliminary results give us an insight into human skeletal muscle‐derived cellular physiological capacity. Technology to observe human muscle mitochondrial fragmentation in vitro will help us elucidate the effects of aging, illness and injury on skeletal muscle mitochondrial fragmentation, dysfunction, and loss of metabolic flexibility.

Support or Funding Information

Funded by the National Institute of Aging of the National Institute of Health; Grant Number R01‐AG059715.
Evolutionary Variation and Biomechanics of the Avian Furcula
Authors: Sullivan Samuel Patrick , Middleton Kevin M. , Holliday Casey M.
Published: April 2020

The avian wishbone, or furcula, serves as the cranial‐most attachment of the m. pectoralis in most birds, variably connects to the coracoid and sternum, and is morphologically diverse across the clade. Furcula shape and microstructure covary with flight style, suggesting its functional importance to flight. Several overlapping hypotheses have been proposed for furcular morphology, such as aiding wing protraction or increasing ventilation efficiency, but the bone remains an enigmatic part of the avian pectoral girdle. In particular, we have little understanding of how furcula morphology and material properties relates to other pectoral structures, including flight muscles such as m. sternocoracoideus. Despite the hypothesized significance of the furcula in the origin of flight and diversification of flight styles, further work needs to be done to understand its functional properties among extant taxa.

In order to explore variation in the furcula and pectoral structures relative to flight style, we studied members of 12 bird families using a three‐pronged approach. Specifically, we examined 1) the shape of the furcula in relation to its in situ orientation using non‐contrast computed tomography (CT), 2) the relative size of m. pectoralis and its subdivisions using contrast‐enhanced CT (DiceCT), and 3) the functional potential of the m. sternocoracoideus to effect or respond to furcula deformation using muscle lever analysis, material properties testing, and muscle spindle histology.

We found substantial differences in furcular orientation, muscular proportions, and furcula material properties across the sample. Whereas earlier studies found a relatively weak correlation between flight style and furcula shape, we found that accounting for in situ furcula orientation improves the correlation between shape and flight style. Birds with more anteriorly‐curved furculae also show a relatively larger cranial division of m. pectoralis. These results support the hypothesis that furcula curvature mediates the protractive capability of m. pectoralis during downstroke. Notably, muscular differences between furculate and afurculate species of parrots (Psittaciformes) were insignificant despite similar flight styles, suggesting that other musculoskeletal structures may take on the functional role of the furcula in certain clades. We also found little variation in m. sternocoracoideus regarding spindle density and muscle moments about the coracosternal joint. The small size and suboptimal location of m. sternocoracoideus suggests that it serves a proprioceptive role, perhaps necessary to assess interglenoid distance during flight, rather than to laterally spread the furcula via the coracoids. Nevertheless, its morphological disparity across our sample remains unexplained.

Support or Funding Information

University of Missouri Life Sciences Fellowship
The ErbB3 receptor tyrosine kinase regulates expression of Notch target genes and intestinal stem cell markers.
Authors: Hsieh Jonathan J. , Almohazey Dana , Frey Mark R.
Published: April 2020

Background

Stem cell‐driven regeneration of the intestinal epithelium is important for recovery of damage from insults including inflammation, chemotherapy, and radiation. This process is controlled through tight regulation of intestinal stem cells (ISCs). Our lab has previously reported that intestine‐specific knockout of the receptor tyrosine kinase ErbB3 leads to precocious development of Paneth cells and increased Paneth cell numbers in mice the mouse intestinal epithelium. Since Paneth cells can play a supportive role for ISCs, we tested the hypothesis that altered ErbB3 signaling results in changes in the ISC compartment.

Methods

We crossed ErbB3flox/flox mice with mice expressing villin promoter‐driven Cre to generate an intestinal epithelial‐specific ErbB3 knockout mouse model (ErbB3‐IEKO). Ileal samples were collected from these mice, fixed, and embedded in paraffin for immunostaining. Tissue samples from the ileum were also processed for RNA and protein for qPCR and immunoblots. Samples were probed for Notch target genes and Notch ligands due to the importance of Notch signaling in maintaining the intestinal stem cell compartment. Enteroids were generated from ileum of C57Bl/6 mice and treated with the ErbB3 ligand Nrg‐1β. To activate and inactivate Notch experimentally, enteroids and HT29 cells were treated with valproic acid (VPA) and the γ‐secretase inhibitor DAPT respectively.

Results

qPCR of ErbB3‐IEKO ileum showed increased expression of Lgr5 (rapid‐cycling ISCs) and Bmi1 (facultative ISCs or secretory progenitors capable of regenerating ISCs). Ileal sections also showed increased numbers of Lgr5+ cells by in situ hybridization, and western blots showed increased protein levels of Bmi1 in ErbB3‐IEKO ileum. qPCR of ErbB3‐IEKO ileum showed increased expression of Notch target genes Hey1, Hey2, and Heyl, as well as expression of Notch ligands Dll1, Jag1, and Jag2. Enteroids treated with Nrg‐1β had decreased expression of Hey1, Lgr5, and Bmi1. Enteroids and HT29 cells treated with VPA to activate Notch had decreased expression of ErbB3 RNA; this was abrogated by DAPT treatment.

Conclusion

ErbB3 negatively regulates expression of Lgr5 and Bmi1, markers for fast‐cycling and quiescent ISCs, respectively, indicating a possible role for ErbB3 in the ISC niche. Furthermore, ErbB3 and Notch appear to negatively regulate one another, suggesting a possible mechanism for ErbB3’s effects on ISCs and intestinal cell differentiation.

Support or Funding Information

This work was supported by NIH award R01DK095004.
Cellular Fractionation Identifies the N‐ and C‐ Termini Contributions Toward Membrane Association of the Rho GTPase TCL
Authors: Erickson Jeffery A. , Esoe Dave-Preston I. , Tader Brooke R. , Hamann Michael J.
Published: April 2020

TCL is a poorly characterized Rho‐family GTPase; however, our lab has performed detailed structure/function analysis of TCL to gain insight into its cellular biochemistry. We have found that its unique N‐terminal extension promotes GTP‐loading of TCL and plasma membrane localization. Deletion of this TCL‐specific sequence promotes localization of TCL to intracellular vesicles. To further explore the role of the N‐terminal and C‐terminal sequences on TCL localization, plasmids encoding Venus were manipulated to include amino acids 1–24 of TCL at the N‐terminus of Venus and amino acids 198–214 at the C‐terminus of Venus. Additional N‐ and C‐terminus deletion mutants of YFP‐tagged TCL were also produced. These plasmids have been transfected into HeLa cells, and subcellular localization of the expressed proteins assessed by fluorescence microscopy and a fractionation procedure. Our results show a clear role for the C‐ terminus of TCL in facilitating membrane localization, but no significant localization directly related to the N‐terminus. Defining how TCL localizes to different cellular membrane environments will help us to understand more clearly its role focal adhesion dynamics, as well as how these dynamics may relate to melanoma development and tumor‐associated angiogenesis.

Support or Funding Information

This work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).
The ciliary localized GPCR, MCHR1, modulates odor responses in the olfactory bulb
Authors: McIntyre Jeremy C. , Jasson Kalene , Ross Jordan , Fletcher Max , Reed Randy
Published: April 2020

The sense of smell is essential for maintaining full human health and quality of life. It plays an important role in the detection of environmental dangers as well guiding decisions such as what foods to eat. However, olfactory processing is influenced by the physiological state of an organism. Physiological changes such can be integrated in the hypothalamus, through populations of neurons expressing various neuropeptides. One of these peptides is melanin‐concentrating hormone (MCH) which functions in both promoting feeding and sleep. Neurons expressing MCH project to several areas of the brain including the olfactory bulb (OB), where the MCH receptor, MCHR1, a g‐protein coupled receptor, is expressed. MCH signaling has been implicated in olfactory driven behaviors, however the mechanism by which it does so is unknown. In rodents, there is a single MCH receptor, MCHR1, which is enriched in neuronal primary cilia. To better understand the mechanism by which MCHR1 signaling affects olfactory behaviors we have first analyzed its distribution in the OB. Using antibody labeling, we find that MCHR1 localizes to cilia of interneurons in the granule cell and glomerular layers. To further confirm this, and generate a potential tool for future studies, we used CRISPR/CAS9 to fuse mCherry to the n‐terminus of MCHR1 (mCherry:MCHR1). Expression of mCherry:MCHR1 matches that of endogenous MCHR1, localizing to the primary cilium of granule cells and a subset of periglomerular cells. MCHR1, and mCherry:MCHR1, is not detected in the primary cilia of mitral cells, the primary output neurons of the olfactory bulb. To test the neuromodulatory role of MCHR1 in olfactory signaling, we have performed functional imaging experiments of the OB in response to odorant stimulation. Using epifluorescence calcium imaging, we find that glomerular responses of mitral/tufted cells are enhanced following administration of an MCHR1 antagonist, SNAP‐94847, (t(157) = 5.425, p <0.0001) while application of MCH decreases glomerular responses (t(99) = 10.98, p <0.0001). Together these data suggest that the ciliary GPCR, MCHR1 contributes to modulation of olfactory function. Furthermore, our newly generated mouse line will aid in studying the role of MCHR1 in other brain regions and will aid in determining the importance of ciliary localization of specific GPCRs in modulating neuronal function.

Support or Funding Information

Support was provided by R00DC013555 and pilot grans from the University of Florida Center for Smell and Taste, and a UF MBI pilot project (JCM), T32DC015994 (KRJ) F31DC 016485 (JMR) and R01DC004553 (RRR)
Mathematical Modeling of Lymph Flow in a network of Lymphatic Vessels
Authors: Dabiri Yaghoub , Lu Xiao , Guo Xiaomei , Patel Bhavesh , Kassab Ghassan
Published: April 2020

Introduction

Lymphatic system is an important part of the circulation system. Assessment of alterations in lymphatic flow in diseased conditions could lead to better treatment strategies. In vivo determination of the flow and pressure in the lymphatic vessels can be challenging. Computational modeling can be used to simulate the lymph flow in silico. The goal of this study was to develop a platform whereby the lymphatic flow in a network of vessels can be simulated. The mathematical model was based on animal experiments.

Methods

We conducted swine experiments whereby the connections between lymphatic vessels in the hind limb were visualized. The length and dimeter of the vessels were also determined from animal experiments. Finite element (FE) method was used to discretize one dimensional lymph flow equations. The lymph vessels were considered as extensible cylinders where the relation between pressure drop and flow was based on experimental data. Also, for valves, the relation between pressure drop and flow was based on experimental data. At junctions we assumed pressure drop is negligible and the flow in the proximal vessel is equal to the sum of flow in distal vessels. The inflow velocities at the distal vessels and the pressure at the proximal vessel were used as boundary conditions. We conducted a time‐dependent analysis where the initial conditions were obtained from a steady state solution.

Results and Discussion

When number of the elements and time steps were increased the solution converged. For a steady‐state rigid vessel, the results were in agreement with the Poiseuille's law. Also, for a branch with rigid vessels the results were in agreement with analytical solutions. For models with extensible vessels, the computed pressure and velocities corresponded to the experimental data. The platform was applicable to junctions with different number of vessels. Because the dimension of lymphatic vessels is noticeably small, measurement of diameters, flow and pressure is challenging, which limits the data that can be fed to the numerical model. As a future direction, this platform could be used to predict flow characteristics in different disease conditions, and possibility to perform more efficient amputations.

Conclusion

Computational modeling was utilized to simulate flow and pressure in lymphatic vessels. There are limitations in measurements of flow characteristics in the lymphatic vessels. Our computational framework could be used to better understanding of lymphatic system functionality in health and disease, and to simulate outcomes which are difficult to determine experimentally.

Support or Funding Information

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs, through the Defense Medical Research and Development Program under Award No. W81XWH‐19‐1‐0087. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense
Metabolic Alterations in Cardiomyocytes are Associated with Methamphetamine‐Induced Cardiomyopathy
Authors: Von Lotten Mallory , Abdullah Chowdhury S. , Aishwarya Richa , Alam Shafiul , Morshed Mahboob , Remex Naznin Sultana , Orr A. Wayne , Goeders Nicholas E. , Kevil Christopher G. , Hartman Brandon , King Judy A. , Traylor James , Bhuiyan Md. Shenuarin
Published: April 2020

Introduction

Illicit use of Methamphetamine (METH) in humans is associated with cardiac dysfunction known as METH‐induced cardiomyopathy. Specifically, the stimulant inhibits mitochondrial bioenergetics, resulting in energy deprivation, which may ultimately lead to the activation of the adaptive autophagy process.

Objectives

We aim to determine the METH‐induced metabolic alterations in cardiomyocytes leading to the development of the METH‐associated cardiomyopathy.

Methods and Results

Autopsy heart samples from humans with a positive toxicology for METH use showed substantial fibrosis in the heart. ‘Binge’ METH‐administration in mice for four weeks developed cardiac hypertrophy and fibrosis in the heart. Mitochondria isolated from the METH‐treated heart showed defects in mitochondrial respiration. Western blot analysis of autophagy regulatory protein expression ‘Binge’ METH‐treated mice showed increased expression of LC3B‐II and p62 proteins. To determine the direct non‐catecholamine dependent effect of METH on cardiomyocytes, we treated primary cardiomyocytes with different doses of METH (100 and 250 μM). Western Blot analysis showed a dose and time‐dependent increase in the expression of LC3B‐II and autophagy‐related protein 7 (Atg7). We also observed increased phosphorylation of cellular metabolism‐related proteins, i.e., phosphorylated FOXO1 (at Ser 256) and FOXO3a (at Ser 253). We conducted autophagy flux assay to confirm the METH‐induced activation of autophagy exposing cardiomyocytes with METH (250 μM for 24 hours) followed by treatment with lysosomal inhibitor BafilomycinA1 (50nM for 3 hours). Western Blot analysis showed increased expression of LC3B‐II in BafA1 treated cardiomyocytes suggesting activation of autophagy following METH‐exposure.

Conclusions

We report that METH use causes adverse fibrotic remodeling in hearts from both human and pre‐clinical mouse model. We found that METH suppresses mitochondrial respiration in cardiomyocytes that elicit adaptive activation of autophagy and autophagy regulatory proteins as a response to METH‐induced energy deprivation.

Support or Funding Information

This project was performed as part of the CURIOUS program supported by grant R25HL147665 from the NIH/NHLBI. This work was further supported by NIH/NHLBI grants R00 HL122354, R01HL145753, R01HL145753‐01S1 and PGM121307A to Dr. Bhuiyan. American Heart Association postdoctoral fellowship to Dr. Alam; Malcolm Fiest post‐ and pre‐doctoral fellowships to Dr. Abdullah and Aishwarya by CCDS, LSUHSC‐S.
Dexamethasone and Phosphoinositide 3‐Kinase Inhibitor Attenuate PUMA‐mediated Apoptosis in Lovastatin‐treated Glioma Cells
Authors: Choi Jae Won , Shin Hyoshik
Published: April 2020

Lovastatin is an antihyperlipidemic drug, which inhibits 3‐hydroxy‐3‐methyl glutaryl‐coenzyme A reductase, the rate‐limiting enzyme of cholesterol biosynthesis, to block mevalonate biosynthesis. Mevalonate is a precursor of various metabolic intermediates, which are responsible for maintaining normal cellular functions. In our previous study, we have reported that lovastatin induces apoptosis in C6 glioma cells. In this study, we examined the effects of glucocorticoid and/or phosphoinositide (PI)‐3‐kinase inhibitors in lovastatin‐induced apoptosis in C6 and U251 glioma cell lines. When C6 cells were cultured in the medium containing lovastatin at a concentration of 25 μM, trypan blue exclusion assays showed that approximately 40% and 70% of cells were dead at 36 and 48 hours after treatment, respectively. Western blot assays and reverse transcription quantitative polymerase chain reaction (RT‐qPCR) data showed that lovastatin treatment led to increased p53 upregulated modulator of apoptosis (PUMA) expression. Lactate dehydrogenase (LDH) assays showed that dexamethasone, wortmannin, or LY294002 significantly attenuated lovastatin‐induced apoptosis in a dose‐dependent manner until 48 hours after lovastatin treatment in C6 cells. In fact, lovastatin‐induced apoptosis was almost completely suppressed in the presence of dexamethasone (100 nM) or wortmannin (30 μM). Serum deprivation has also been reported to induce PUMA‐mediated apoptosis in C6 cells. In our study, we found that serum deprivation‐induced cell death was attenuated by dexamethasone, but not by wortmannin. Interestingly, RT‐qPCR data showed that the lovastatin‐induced increase of PUMA mRNA levels was further enhanced by either wortmannin or LY294002 treatment; however, it was not affected by dexamethasone treatment. In L6 and AGS cell lines, LDH assays showed that dexamethasone (100 nM) treatment could attenuate lovastatin‐induced apoptosis at 48 hours after lovastatin treatment, while PI‐3‐kinase inhibitors could not. In U251 glioma cells, lovastatin‐induced cell death was attenuated by dexamethasone or PI‐3 kinase inhibitors. In all cell lines examined in this study, the preventive effects of dexamethasone on lovastatin‐induced apoptosis were blocked by RU486. Our data suggest that dexamethasone and PI‐3 kinase inhibitors could prevent PUMA‐mediated apoptosis in lovastatin‐treated glioma cells not by suppressing the increase of PUMA expression, but by activating unique anti‐apoptotic pathways.
Understanding fundamental aspects of respiratory control in dystrophin‐deficient mice
Authors: Burns David P.
Published: April 2020

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease associated with respiratory‐related morbidity and mortality. DMD is characterised by severe skeletal muscle weakness, which extends to the striated muscles of breathing. Our recent research has explored deficits and compensation in the respiratory control network governing respiratory homeostasis in a pre‐clinical model of DMD, the mdx mouse. Deficits at multiple sites of the network provide considerable challenges to respiratory control, including carotid body hypo‐activity (sensory deficit) and diaphragm dysfunction (
Burns et al., 2017). Despite profound diaphragm weakness and lower muscle activation during maximum non‐ventilatory efforts, inspiratory pressure‐generating capacity is preserved in young adult mdx mice, revealing compensation in support of respiratory system performance that is adequate, at least early in dystrophic disease (
Burns et al., 2019). Our findings point to a considerable reserve in the capacity of the respiratory neuromuscular system to perform a broad range of function, resulting from the recruitment of auxiliary muscles in support of peak respiratory‐related performance. We propose that a progressive loss of compensation during advancing disease, combined with diaphragm dysfunction, underpins the development of respiratory morbidity in dystrophic diseases. Studies utilising anti‐inflammatory, anti‐stress and anti‐oxidant pharmacotherapy strategies indicate that interventions aimed at preserving muscle fibre complement and promoting muscle fibre quality in mdx respiratory muscles can alleviate breathing and muscle functional deficits (
Burns et al. 2018). Dr. Burns will discuss current knowledge gaps and the need to better appreciate fundamental aspects of respiratory control in pre‐clinical models so as to better inform intervention strategies in human DMD.

Support or Funding Information

Department of Physiology, University College Cork and The Physiological Society.
Effect of Cerebral Blood Flow on Cognition across Healthy Adulthood
Authors: Shoemaker Leena N. , Wilson Luke C. , Lucas Samuel J.E. , Machado Liana , Walker Robert J. , Cotter James D.
Published: April 2020

Both cognitive function and cerebral blood flow (CBF) decline across the lifespan. However, the relation between them remains unclear, particularly in the context of aging. Furthermore, fitness improves both cognition and CBF, and may be associated with preserved cognitive and cerebrovascular function in an older population.

Purpose

The aims of this study were to 1) clarify the effect of CBF on cognition across healthy adulthood, and the extent to which age‐related reductions are modulated by the usually‐observed impairment in cerebrovascular function, and 2) examine the association of aerobic fitness on baseline CBF and cognitive performance in healthy young and older adults.

Method

Using a placebo‐controlled, single‐blinded, randomized cross‐over design, we tested the hypothesis that an acute reduction in CBF (using indomethacin [1.2 mg/kg oral dose]) would impair cognition in both young (n = 13; 25 ± 4y) and older (n = 12; 58 ± 6y) adults. Cerebrovascular function was assessed using blood velocity in the middle cerebral artery (MCAv; Transcranial Doppler Ultrasound). Cerebrovascular CO2 reactivity (CVRCO2) was calculated as the change in MCAv for a given change in end‐tidal CO2 (mm Hg). Cognitive function was measured using visuomotor, inhibitory, and mental switching response time tasks. Aerobic fitness was predicted using a submaximal exercise test (i.e., V̇O2peak). Linear and rank‐based mixed models were used to assess the individual and interactive effects of cerebrovascular function and cognition, while accounting for age, sex, and aerobic fitness.

Results

Older adults had 11% lower MCAv and 15% worse response time overall (all p ≤ 0.049), but not reliably lower CVRCO2 (p ≥ 0.16). Indomethacin decreased MCAv by 26 ± 11% and CVRCO2 by 69 ± 25% (all p < 0.01), regardless of age. The degree of indomethacin‐induced reductions in CVRCO2 was strongly associated with baseline CVRCO2 (r = 0.87, p < 0.01). Visuomotor response time was 6 ± 13% slower after indomethacin (p = 0.04), but this impairment was not linearly associated with decreases of MCAv (r = −0.26). Finally, aerobic fitness was not associated with baseline CBF in either group (r ≤ 0.14), and was only weakly associated with response time performance in young (r ≤ −0.34), but not older (r ≤ 0.21) adults.

Conclusion

The impairments in cognitive function from indomethacin were not evidently associated with the 26% reduction in cerebral perfusion, even in older adults. Compared to young, older adults had worse cognitive performance and decreased CBF but did not have impaired cerebrovascular function. Aerobic fitness was only weakly associated with resting CBF and cognition in both young and older adults. Therefore, cognitive performance may not be influenced by a ≤26% reduction in CBF per se in healthy young and older adults.

Support or Funding Information

Funding support from the School of Physical Education, Sport and Exercise Sciences, and Department of Medicine.
Actigraphic Measures of Sleep and White Matter Volume in a Bi‐Racial Cohort
Authors: Hossain Sharmin , Gamaldo Alyssa , Shiroma Eric J. , Katzel Leslie I. , Davatzikos Christos , Erus Guray , Gullapalli Rao P. , Seliger Stephen L. , Evans Michele K. , Zonderman Alan B. , Waldstein Shari R.
Published: April 2020

Objectives

About 50% of adults report some form of sleep disturbance with deleterious impact on cerebral white matter (WM). Here, we investigated interactive relations of sleep duration or quality and race on cerebral WM volumes in a largely middle‐aged, bi‐racial cohort.

Methods

The cross‐sectional Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) Scan study yielded 119 stroke and dementia‐free participants (mean age ~51y; 37% African American; ~48% women) with complete actigraphy, magnetic resonance imaging (MRI), and covariate data. A triaxial accelerometer (ActiGraph GT3X+, ActiGraph Corp) worn on the wrists for up to seven consecutive days, provided measures of total sleep time (TST), sleep efficiency (SE), and wake after sleep onset (WASO). Self‐rated sleep quality was assessed via the Pittsburgh Sleep Quality Index (PSQI). Brain MRI, acquired by 3.0 Tesla scanner, yielded quantitative measures of average WM volumes for total brain, total lesion, frontal, occipital, temporal, and parietal lobes. Multivariable linear regression analyzed interactive relations of each sleep variable and race to WM outcomes: model 1‐adjusted for age, sex, and race; model 2‐model 1+ body mass index, smoking, alcohol use, and depressive symptoms. Results are expressed as regression coefficients (β), p‐value.

Results

Following adjustments, TST*race interactions were noted for total (17.15, <0.05), frontal (13.17, <0.05), and parietal (7.77, <0.05) WM volumes. Shorter sleep duration was related to lower volumes in African Americans but greater volumes in Whites. PSQI*race interactions for total (59.62, <0.01), frontal (40.07, <0.01) and parietal (17.84, <0.05) WM showed relations of poorer sleep quality to lesser WM volumes in African Americans only.

Conclusion

Shorter sleep duration and lesser self‐reported sleep quality were related to lower total and regional WM volumes among African Americans. The mechanisms underlying these racial differences warrant further investigation and may help explain neuropsychological health disparities.

Support or Funding Information

Supported by National Institutes of Health grants ZIA‐AG000195 (MKE), R01‐AG034161 (SRW), and the University of Maryland Claude D. Pepper Older Americans Independence Center (P30 AG028747).
Progressive Collecting Liver Lymphatic Vessels Dysfunction over the Course of Extrahepatic Cholestasis
Authors: Gasheva Olga Yu. , O’Brien April , White Tori K. , Alpini Gianfranco D. , Zawieja David C. , Glaser Shannon , Gashev Anatoliy A.
Published: April 2020

Liver produces a large fraction of total lymph flow (25–50%). Transport function of lymphatics is altered during inflammation, which impacts the pathogenesis of numerous diseases. Despite their importance in liver function, the functional characteristics of liver lymphatic vessels (LLVs) have been never evaluated in health and disease. We evaluated liver lymphatic vessel (LLV) function in normal and cholestatic rats following extrahepatic bile duct ligation (BDL). LLV contractility/pumping characteristics were determined in LLVs isolated from Sprague‐Dawley rats in control, 1, 3 and 7 days after BDL (BDL1, BDL3, and BDL7) and sham groups. LLV pumping/contractility was analyzed in all groups. Control LLVs had outer diameters of ~500 um, typical lymphatic tone (~7%), moderate contraction amplitude (15–35%), high contraction frequency (12–21 contractions/min) and strong active pumping (5–7.5 vol/min). Over the course of its duration, the BDL significantly altered LLV contractile/pump function, inducing an enlargement of LLVs; their decreased tone; lowering of phasic contractions frequency responses to increases in stretch. Cumulatively, BDL induced progressive decline in minute pumping of LLVs with complete cessation of phasic lymphatic contractions after seven days of BDL. In addition, BDL even after one day completely ablated the light imposed flow‐dependent relaxatory/inhibitory reactions of LLVs observed in control. Progressive dysfunction of LLVs correlates with cholestasis‐induced alterations in liver and bile ducts. We believe that observed BDL‐induced changes in LLVs are likely the result of the liver inflammation and will further promote the inflammatory edema known to occur in the liver during BDL. Thus, LLV dysfunction may play important roles in the ensuing pathogenesis of cholestasis and merit further study.

Support or Funding Information

NIH/NIDDK R01 DK110035; NIH/NHLBI 1U01HL123420; funds from TAMU HSC COM and Dept. of Medical Physiology.
Adiponectin rescues the newborn rat lung from lipopolysaccharide (LPS)‐induced inflammation and injury
Authors: Despotovic Nikola , Ivanovski Nikola , Ivanovska Julijana , Belik Jaques , Gauda Estelle B.
Published: April 2020

Introduction

Preterm infants born during the canalicular and saccular stages of lung development are at high risk for lung injury from oxidative stress and inflammation. Low adipose tissue mass is associated with low serum levels of protective anti‐inflammatory and anti‐oxidative adipokines, such as adiponectin (APN). The modulatory role of APN on inflammatory cytokines and tissue remodeling has unveiled APN as a novel therapeutic target for developmental lung pathologies. We have previously shown that prophylactic administration of exogenous APN protects the newborn rat from LPS‐induced lung injury. In this study, we investigate the ability for systemic APN given 20 hours after LPS aspiration to reverse lung injury in newborn rats.

Methods

Sprague‐Dawley rats at postnatal day (PND) 4 were treated with 5 mg/kg intrapharyngeal (IPh) LPS or saline; 20 hours later, 2 mg/kg recombinant APN (rAPN) or equal volume saline was administered intraperitoneally (IP). The three treatment groups included: rats exposed to LPS (IPh)/rAPN (IP), LPS (IPh)/saline (IP) and saline (IPh)/saline (IP). Lungs were harvested 6 hours after administration of rAPN and processed for gene (qPCR) and protein (SDS‐PAGE Western blot) expression. In another cohort of PND4 rats, rAPN was administered at 20 and 44 hours, with lungs removed and assayed for histopathology with haemotoxylin and eosin staining 72 hours after LPS. n=4 in each treatment group for gene/protein expression and histopathology.

Results

Differences were observed in gene and protein expression between treatment groups (ANOVA, p<0.05). In animals treated with IP rAPN 20 hours after IPh LPS, mRNA expression of macrophage inflammatory protein‐1α (MIP‐1α), monocyte chemoattractant protein‐1 (MCP‐1), IL‐1β, IL‐6, and IL‐10 was reduced 7‐, 8‐, 3.6‐, 1.6‐ and 13‐fold, respectively, relative to saline treatment. Protein expression of tumor necrosis factor α (TNF‐α), IL‐1β and IL‐10 was also relatively reduced in rAPN treated rats. No change was detected in the mRNA expression of TNF‐α, or in the protein expression of IL‐6. Lungs treated with rAPN had a marked reduction in histopathological changes relative to saline‐treated controls.

Conclusions

Our novel in vivo study demonstrates that beyond the preventative role of systemic APN in neonatal lung injury, APN can also attenuate previously induced acute, inflammatory lung injury. We speculate that increasing serum APN levels in preterm infants might protect from acute and chronic inflammatory lung injury.

Support or Funding Information

Women’s Auxiliary Board at SickKids
Essential Anatomy for Radiology in the Undergraduate Medical Curriculum
Authors: Keim Sarah , Lopez Lisa , McNary Thomas , Zumwalt Ann , Harrell Kelly , Davies David , Cotter Meghan , Martindale Jim , Harmon Derek , Hankin Mark
Published: April 2020

Introduction

To prepare medical students for clinical training and practice, it is critical to understand the anatomical knowledge considered most important for different clinical specialties.

Aim

To address this issue, a consortium of anatomists in the US and Canada is collecting data from clinical educators in Radiology clerkships and electives to identify the anatomy they consider essential.

Methods

An IRB‐approved, online survey (Qualtrics, Seattle, WA) was used to assess the importance of 98 anatomical topics in seven body regions. The study first examined the percentage of Radiology clinical educators (clerkship/elective directors and attending physicians) that considered each anatomical region important to their specialty. Second, the study examined the rank assigned to each anatomical topic using an ordinal scale from 1 (not important) to 4 (essential).

Results

At the time of abstract submission, data had been collected from 15 Radiology clinical educators at 9 medical schools. The percentage of Radiology clinical educators that considered each anatomical region important to their specialty were (highest‐to‐lowest): Back (100%), Thorax (100%), Abdomen (100%), Pelvis & Perineum (100%), Head & Neck (100%), Upper Limb (93%), and Lower Limb (93%). Further data analysis has identified the highest ranked anatomical topics in each body region for the Radiology clerkship/elective.

Discussion and Conclusion

This database provides detailed information regarding the most clinically relevant anatomical topics as identified by Radiology clinical educators. This information can aid in focusing preclinical learning to best prepare medical students for success in their undergraduate and graduate clinical experiences.
Identifying the epigenetic drivers of an accelerated aging process
Authors: Xu Yi , Liu Zhenfeng , Ollar Joe , Billings Emma , Booher Keith , Chew Yap Ching , Yang Xiaojing , Jia Xi Yu
Published: April 2020

Aging impacts many important attributes of human beings such as the onset of diseases and the length of lifespans. Previous research demonstrated that DNA methylation based epigenetic clocks can be used to estimate biological age in both humans and mice. Interestingly, an accelerated aging process was observed in a short‐lived laboratory mouse strain (AKR/J, median lifespan = 288 days). By comparison, the more common laboratory mouse strain C57BL/6J lives substantially longer (median lifespan = 901 days). To elucidate the underlying mechanisms linking the accelerated aging process to a shortened lifespan, we used reduced representation bisulfite sequencing (RRBS) to profile DNA methylation in the cortex and the hippocampus extracted from these two strains, respectively (4 biological replicates per brain region for the C57BL/6J strain, 6 biological replicates per brain region for the AKR/J strain). At a 5X read depth cutoff, we detected 2,376,777 ± 195,194 unique CpG sites in the C57BL/6J strain samples and 2,288,300 ± 137,243 unique CpG sites in the AKR/J strain samples. Samples from the same brain region of the same strain clustered tightly based on their distinctive DNA methylation patterns. Using a publication‐supported R package, “DSS”, to compare between the two strains, we identified 14,625 differentially methylated cytosines (DMCs, Benjamini‐Hochberg FDR <0.05; methylation difference ≥0.15) and 257 differentially methylation regions (DMRs, p<0.05; methylation difference ≥0.15) from the cortex samples. Similarly, we found 14,215 DMCs (Benjamini‐Hochberg FDR <0.05; methylation difference ≥0.15) and 304 DMRs (p<0.05; methylation difference ≥0.15) from the hippocampus samples. Subsequent annotation of the DMRs from both sample groups revealed that 106 genes had significantly different methylation levels in their promoter regions. Pathway analysis demonstrated differential promoter methylation of genes (for example, Psat1, Smarca5, Rnf152 and Hspg2) enriched in the regulation of chromatin remodeling, cellular development, and metabolism. Importantly, Rnf152 and Hspg2 are associated with mouse aging. In summary, our study discovered potential epigenetic features involved in the accelerated aging process of a short‐lived mouse model. Future work should focus on the detailed functions of the identified genes and further understand their link to altered mice lifespans, eventually leading to strategies for improved healthcare in human lives.