bluuebunny/crossref_metadata_2025 huggingface dataset:
Lactate and Short‐Chain Fatty Acids on Ca2+ Oscillations in Rat Carotid Body Type 1 Cells
Authors: Hogan James O. , Kang Dawon , White Carl , Kim Donghee
Published: April 2020

Carotid body (CB) Type 1 (glomus) cells express Olfr78, a Gs‐coupled receptor that binds lactate and short chain fatty acids. Lactate is produced in all cells under anaerobic conditions, and short chain fatty acids are produced by bacterial fermentation in the colon. Lactate‐Olfr78 signaling has been proposed to be involved in hypoxia‐induced excitation of CB, but this idea is currently controversial. Ca oscillations are low‐level Ca signals in glomus cells. Here we compared the effects of lactate/short chain fatty acids and hypoxia on Ca oscillations in isolated glomus cell. Lactate ≈ acetate > propionate (1–20 mM) produced a concentration‐dependent increase in the frequency of Ca oscillations with no overall effects on the background TASK K current and resting cell membrane potential. Verapamil abolished Ca oscillations with or without lactate. Forskolin (2–10 μM) produced no significant effect on Ca oscillations, suggesting that adenylyl cyclase/cAMP may not be involved in lactate‐induced changes in Ca signaling. H‐89 (1 μM) did not block lactate‐induced increase in Ca oscillations, questioning the role of PKA. Comparison of lactate and hypoxia effects on Ca oscillations indicated that 5–10 mM lactate and 2–5%O2 (mild‐moderate) hypoxia produce similar effects on Ca oscillations. Thus, an increase of blood lactate to 5–10 mM may be necessary to mimic the effect of mild‐moderate hypoxia on Ca signaling in CB glomus cells.
The Role of AMPK in Neuromuscular Junction Biology
Authors: Ng Sean Y. , Desjardins Eric M. , Steinberg Gregory R. , Ljubicic Vladimir
Published: April 2020

The neuromuscular junction (NMJ) is the signaling apparatus between a motoneuron and the corresponding muscle fibers that it innervates. While its stability is vital for the neuromuscular system, the NMJ is susceptible to remodeling across the lifespan. Adenosine monophosphate‐activated protein kinase (AMPK) is a primary modulator of skeletal muscle phenotype, however the precise role of AMPK at the NMJ has yet to be investigated. Thus, the purpose of this study was to examine the role of skeletal muscle AMPK on NMJ biology across the lifespan. We employed 3‐ and 12‐month‐old muscle specific AMPKβ1β2 knock out mice (mKO, n = 3), age‐matched wild‐type (WT, n = 3) mice, as well as 22‐month‐old WT animals. Extensor digitorum longus (EDL) and soleus (SOL) muscles were dissected to represent fast glycolytic and slow oxidative tissues, respectively. These muscles were then whole‐mounted and immunolabelled for presynaptic neurofilament and synaptic vesicle 2A proteins, as well as postsynaptic acetylcholine receptors (AChR) revealed by □‐bungarotoxin. Approximately 25 NMJ images per muscle were acquired via confocal microscopy. In total, we conducted 3D analysis on over 400 NMJs to determine metrics of NMJ morphology. Postsynaptic analyses revealed an effect of age (p < 0.05) on the total amount of fragmented and ectopic NMJs in the SOL and EDL muscles of WT animals. Interestingly, mKO animals exhibited a significantly greater count of fragmented and ectopic NMJs in the SOL and EDL muscles, compared to their WT littermates. In fact, NMJs positive for postsynaptic abnormalities in EDL muscles of 12‐month‐old mKO animals were significantly greater than 22‐month‐old WT animals (p < 0.05), while SOL muscles were comparable. An effect of age (p < 0.05) on axonal blebbing and sprouting was observed in the EDL and SOL muscles, respectively. Additionally, NMJs with axonal blebbing were observed more frequently (p < 0.05) in the mKO animals compared to their age‐matched WT littermates. Quantitative real‐time PCR was also utilized to examine genes important for NMJ development and maintenance in the tibialis anterior muscles of 3‐month‐old WT and mKO animals (n = 10). Specifically, AChR□/ɣ, low‐density lipoprotein receptor‐related protein 4, rapsyn, and muscle‐specific kinase mRNA expression was significantly higher in the mKO animals relative to their WT counterparts. Our results suggest that AMPK is critical to the determination and maintenance of a healthy pre‐ and post‐synaptic apparatus in motoneurons and muscles of disparate phenotypes. Additionally, the loss of muscle‐specific AMPK exacerbates age‐induced postsynaptic alterations in faster, and more glycolytic muscle fibers.

Support or Funding Information

Canadian Institutes of Health Research, Canada Research Chairs, and Ontario Graduate Scholarship.
Sfrp4 Expression in Thyroxine Treated Calvarial Cells: A Novel Target
Authors: Cray James , Durham Emily , Grey Zachary , Black Laurel , Howie R. Nicole , Barth Jeremy L. , Lee Beth S.
Published: April 2020

Evidence suggests alterations of thyroid hormone levels can disrupt normal craniofacial development. Most data suggest the major targets of thyroid hormones to be the Htra1/Igf1 pathway. Recent discovery by our group suggests involvement of targets of the WNT pathway, specifically overexpression of antagonist Sfrp4 in the presence of excess thyroid hormone. Here we aimed to model these interactions in vitro to determine if thyroid hormone directly drives Sfrp4 expression in cells relevant to craniofacial development. Affymetrix transcriptional profiling found Sfrp4 to be overexpressed in cranial suture derived cells stimulated with thyroxine in vitro. Bioinformatics interrogation of the SFRP4 promoter identified multiple putative binding sites for thyroid hormone receptors. Experimentation with several cell lines demonstrated that thyroxine treatment induced Sfrp4 expression. However, protein levels did not correlate with induction of gene expression, demonstrating that Sfrp4 mRNA and protein levels are not tightly coupled. Transcriptional and protein analyses demonstrate thyroid hormone receptor binding to the proximal promoter of the target gene Sfrp4. Interestingly functional analysis of stem cells, fibroblasts, or primary culture cells after thyroxine stimulation for alkaline phosphatase activity shows only pre‐osteoblasts are pushed to become positive after thyroid hormone stimulation, suggesting cell type susceptibility. Finally, we added recombinant SFRP4 to pre‐osteoblasts in combination with thyroxine treatment and observed a significant decrease in alkaline phosphatase positivity, suggesting SFRP4 may be a key regulatory molecule that prevents thyroxine driven osteogenesis. The implications for this relationship indicate SFRP4 as a potential diagnostic or therapeutic target for hyperostotic craniofacial disorders.

Support or Funding Information

The authors would like to thank Xing Ming Shi for isolation of the bone marrow mesenchymal cells. The following funding sources were used for this study a grant from the Cleft Palate Foundation, NIH/NIDCR R03DE023350A, 5T32DE017551, and F31DE026684, NIH/NCATS UL1 TR000062, NIH/NIGM P30GM103331, GM103342 and GM103499, and MUSC’s Office of the Vice President for Research.
Cancer mutations inhibit the coactivator activity of p300 core
Authors: Hall Meghan , Shanle Erin K.
Published: April 2020

p300 is an important transcription coactivator that enhances gene expression by acetylating histones through its catalytic core domain. p300 is mutated in some cancers, and cancer mutations in the histone acetyltransferase (HAT) domain can inactivate the coactivator function of the protein. However, many mutations in the p300 catalytic core that have been observed in human tumors have not been characterized for functional effects on p300 coactivator activity. The goal of this study was to screen cancer mutations using a previously published system in which the p300 catalytic core is fused to deactivated Cas9 (p300core‐dCas9). Cancer mutations in the bromodomain, PHD/RING domain, and histone acetyltransferase (HAT) domain were created in the p300core‐dCas9 protein. After the addition of guide RNA targeting p300core‐dCas9 to the promoter of a given target gene, quantitative PCR was performed to measure the coactivator function of the wild‐type and mutant proteins. Mutations in the bromodomain showed variable effects on gene expression activation, while mutations in the PHD/RING domain and HAT domain were inhibitory. These results demonstrate that cancer mutations in the p300 core can alter p300 function and provide insight into the effects of cancer mutations that occur outside of the HAT domain.

Support or Funding Information

Longwood University Faculty Research and Development Grant
DUAL ACTING COX‐2 AND SOLUBLE EPOXIDE HYDROLASE INHIBITOR ATTENUATES GLOMERULAR INJURY IN FOCAL SEGMENTAL GLOMERULAR SCLEROSIS
Authors: Stavniichuk Anna , Khan Md Abdul H. , Jankiewicz Wojciech K. , El-Meanawy Ashraf , Yeboah Michael M. , Savchuk Olexiy , Hwang Sung H. , Hammock Bruce D. , Imig John D.
Published: April 2020

Proteinuric glomerular diseases like focal and segmental glomerulosclerosis (FSGS) are the leading cause of chronic and end‐stage kidney diseases. FSGS is characterized by glomerular injury, and, at the clinical level, a classic hallmark of FSGS is proteinuria. Although FSGS is associated with high morbidity and mortality, treatments are limited that emphasize the need for novel therapies. We developed a novel dual modulator, PTUPB that concurrently acts as a soluble epoxide hydrolase (sEH) inhibitor as well as a cyclooxygenase‐2 inhibitor (sEHi/COXi). PTUPB has previously demonstrated robust kidney actions including reduction of glomerular injury in diabetic nephropathy. In the current study, we investigated renal actions of PTUPB in a genetic FSGS model, ROP Os/+ mice. ROP Os/+ mice developed proteinuria and glomerular injury between 16–20 weeks of age (8‐fold higher albuminuria vs. WT). We treated 20 weeks old male wild type (WT) and 20 weeks old ROP Os/+ mice with vehicle or PTUPB (10 mg/kg/d, i.p.; n=4–5/group)for 4 weeks. At the end of the 4 week treatment period urine, blood, and kidney tissue were collected for biochemical and histological analysis. The glomerular injury was assessed from albuminuria, histopathology, mRNA expression of proteins critical for glomerular structural integrity and function. Vehicle treated ROP Os/+ had 10‐fold higher albuminuria compared to WT and PTUPB treatment markedly attenuated albuminuria in ROP Os/+ mice by 70%. The ROP Os/+ mice developed glomerular injury with a 3‐fold higher glomerular injury score compared to WT and PTUPB treatment decreased glomerular injury by 28%. We further demonstrated that there was 20–30% lower renal mRNA expression of nephrin, podocin, and synaptopodin in vehicle‐treated ROP Os/+ mice, and PTUPB treatment improved the increased expression for these glomerular proteins in ROP Os/+ mice. These findings demonstrate that the dual modulator PTUPB that concurrently inhibits sEH and COX‐2 has exciting potential to treat FSGS and related glomerular diseases.

Support or Funding Information

DK103616 and Dr. Ralph and Marian Falk Medical Research Trust Bank of America, N.A. to John D. Imig
The Effects of Ezh2 Mutation on Kras‐Driven Adenocarcinoma
Authors: Mandias Hannah Caroline Rattu , Mao Suifang , Xue Bin , Fuziwara Cesar Seigi , He Lin
Published: April 2020

Lung cancer is the deadliest form of cancer worldwide. Non‐small cell lung cancer (NSCLC) is the most common type of lung cancer, constituting an estimated 84% of diagnoses. Conducting research regarding the metastasis of lung adenocarcinomas is especially pertinent, as the 5‐year survival rate for localized NSCLC is an estimated 50% versus 6% in metastatic lung cancer. Here we investigate the activation effects of Polycomb Repressive Complex 2 (PRC2), an epigenetic regulator commonly found to be dysregulated in human lung cancer, as a driver of metastasis. We employed genetically engineered mouse models harboring activating point mutations in Kras and inactivating mutations in the p53 pathway (KP model) which develop lung adenocarcinomas but exhibit low penetrance and long latency for metastasis. As a resolution, our lab has generated a KPE mouse model which carries a mutation that induces Ezh2 activation, a core component of PRC2. We hypothesize that KPE is an effective and robust metastatic model which displays a shorter latency period than previously established lung adenocarcinoma models. In this project we used a histological approach to characterize morphological properties of primary tumors as well as distinct lymph node metastases in KPE mouse models. By visualizing cellular phenotypes through Hematoxylin & Eosin staining, we intended to quantify tumor area and composition of lymphatic micrometastases. The results illustrated that mice with the Ezh2 mutation in addition to the Kras and p53 mutations experienced a shorter latency period as well as a higher occurrence of lymph node metastases as opposed to the KP model.

Support or Funding Information

This project is supported in part by National Science Foundation Grant 1758007.
Regulation of DNA methyltransferase 3a Expression by Prostaglandin E2 in Human Fibroblasts
Authors: Seutter Sara , Mercando Jason , Sen Neil , Penev Boris , Auker Bennet , Shamseddin Seyed M. , Green Kevan , Borghaei Ruth
Published: April 2020

Introduction

Periodontitis is common chronic inflammatory condition that not only results in loss of teeth, but contributes to a variety of systemic conditions, including cancer. Previously we showed that exposure of human gingival fibroblasts (HGF) derived from patients with periodontitis to PGE2 decreases expression of DNMT1, DNMT3a and TET1, but increases global levels of methylated and hydroxymethylated DNA, acting predominantly through the EP4 receptor. The current study compares the effects of PGE2 on expression of these genes in non‐inflamed fibroblasts and aims to further elucidate the signaling pathways involved.

Methods

Real time PCR and ELISA are used to measure expression of DNMT1, DNMT3a, TET1 and EP receptors 1–4 in untreated HGF and IMR90 lung fibroblasts and those treated with PGE2. The effects of actinomycin D, as well as EP receptor agonists and antagonists and signaling pathway activators and inhibitors on mRNA expression of these genes will also be assessed by qPCR.

Results

Results so far show that PGE2 decreases expression of DNMT1 in IMR90 cells to an extent similar to that observed in HGF, but levels of DNMT3a are increased rather than decreased, and levels of TET1 are unaffected in IMR90 cells. Although all four EP receptors are expressed in both HGF and IMR90, the relative levels of EP4 expression are lower.

Conclusion

The ability of this inflammatory mediator to influence DNA methylation is consistent with the association of chronic inflammation with increased risk of cancer. However, the fact that the effects are cell and context dependent indicate that more information about the specific signaling pathways involved in regulating expression of these important enzymes is needed. Further studies will be aimed at elucidating the roles of the PGE2 receptors and their signaling pathways in mediating the cell‐specific effects.
Essential Amino Acid Supplement lowers Intrahepatic Lipid in Individuals with Alcohol Use Disorder
Authors: Coker Melynda S. , Ladd Kaylee , Kim Josh , Murphy Carl J. , DeCort Ryan , Newcomer Bradley R. , Wolfe Robert R. , Coker Robert H.
Published: April 2020

Excess alcohol consumption has been ranked as a top risk factor for death and disability. Supervised treatment for alcohol use disorder (AUD) is unlikely for most individuals; fatty liver will likely develop and the risk of liver disease increases. Essential amino acid supplementation (EAAS) has been demonstrated to reduce intrahepatic lipid (IHL) in pre‐clinical and clinical trials for nonalcoholic fatty liver disease. Therefore, the purpose of our study was to evaluate the influence of EAAS on IHL, body composition, and blood lipids in individuals with mild to moderate AUD. Following consent, determination of eligibility and medical screening, 18 young to middle‐aged participants were randomly assigned to a low dose (LD: 8 grams of EAAS BID) or high dose (HD: 13 grams of EAAS BID) group and consumed the supplement for 4 weeks. Pre‐ and post‐EAAS administration, IHL was determined using magnetic resonance imaging/spectroscopy, body composition was analyzed using dual energy x‐ray absorptiometry, and blood parameters were measured by LabCorp. T‐tests were used for statistical analysis and considered significant at P<0.05. While there was no change in IHL (Δ−0.1±0.0 %) in the LD group, there was a reduction in IHL (Δ−0.2±0.0 % water peak) in the HD group (p=0.02). Fat mass, lean tissue mass, bone mineral content, and blood lipids were not altered. Post‐EAAS phosphatidylethanol was unchanged and remained elevated in LD @ 422±224 ng/ml and HD @ 429±196 ng/ml, indicating chronic and excessive alcohol consumption. Based on these results, we suggest that 13 grams of EAAS may lower IHL in individuals with mild to moderate AUD and potentially provide additional opportunities for effective long‐term treatment.

Support or Funding Information

Research reported in this publication was supported by Essential Blends, LLC and by an Institutional Development Award (IDeA) (P20GM103395) and the Biomedical Learning and Student Training Program (UL1GM118991, TL4GM118992, or RL5GM118990) from the National Institute of General Medical Sciences under the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Elucidation of Terpenoid Biosynthesis in Sclerotinia
Authors: Lemire Colin Andrew , Mafu Sibongile
Published: April 2020

Terpenes are a large class of specialized metabolites found in plant and fungus species that have been widely studied for their medicinal and cytotoxic properties. The plant pathogenic fungus Sclerotinia homoeocarpa was previously shown to produce eleven nortetralabdane compounds that exhibit strong antiplasmodial and growth inhibitive properties. Our objective is to elucidate the biosynthesis of these unique terpene compounds in S. homoeocarpa. Genome mining identified two terpene cyclases as well as multiple clustered cytochromes P450 (CYPs). Combinatorial expression in Escherichia coli and GC‐MS analysis of extracts revealed that one cyclase, ShTPS1, is a bifunctional diterpene synthase that produces the 20‐carbon compound pimara‐8(14),15‐diene. This diterpene likely acts as a scaffold to be modified by downstream CYPs into nortetralabdanes. The second cyclase, ShTPS2, produces several 25‐carbon sesterterpenes, one of the least well‐characterized subsets of terpenes and a frontier for pharmaceutical research. The discovery of ShTPS2 is uniquely interesting due to its promiscuity and the rarity of characterized bifunctional sesterterpene synthases ‐ terpene synthases with a chimeric prenyltransferase domain. Homology modeling of ShTPS2 identified targets for site‐directed mutagenesis in the prenyltransferase and terpene cyclase domains. Mutations in these areas reveal both the functional plasticity of the enzyme and that the promiscuity may result from cationic intermediates of a single terpene cyclase pathway. This research contributes to understanding the diversity of terpene biosynthesis in fungi and allows for the biosynthesis of compounds of interest that are naturally produced at low levels, enabling future work to investigate their function.

Support or Funding Information

This work was supported by the Massachusetts Agricultural Experiment Station as well as the University of Massachusetts Amherst Commonwealth Honors College and Department of Biochemistry and Molecular Biology.
Transfer RNA Abundance Alters Modification Levels in Response to Peroxide Stress in the Radiation Resistant Fungus Cryptococcus neoformans
Authors: Kelley Melissa S. , Yoshida George M. , Myers Ryan A. , Jora Manasses , Limbach Patrick A.
Published: April 2020

Cryptococcus neoformans is a radiation resistant fungus that has been found growing at the site of the nuclear reactor meltdown in Chernobyl, Ukraine. The Defense Threat Reduction Agency (DTRA) funds research on this organism to gain fundamental knowledge on radiation survival. Many DTRA projects cause soldiers to be exposed to radiation, therefore, gaining this information may have future therapeutic implications. Radiation causes cellular damage through the radiolysis of water into reactive oxygen species (ROS) – such as hydrogen peroxide. Cells have mechanisms such as antioxidants to regulate ROS levels. When ROS is too high, nucleic acids may be damaged. Transfer RNA (tRNA) – the molecule that carries the amino acid to the translational machinery – directly affect protein production. Hence, the integrity of tRNA is critical to antioxidant defenses and general protein synthesis. Chemical modifications to tRNA are abundant and crucial in all domains of life. Peroxide is known to cause changes in tRNA modification 5‐methylcytidine (m5C) levels in the model fungus S. cerevisiae. This work sets out to evaluate the effects of peroxide exposure on tRNA modification and tRNA transcript abundance in C. neoformans. Using liquid chromatography coupled with mass spectrometry (LC‐MS), modifications were relatively quantified and found to respond differently than in S. cerevisiae. Moreover, RNA sequencing information indicated that there are changes in tRNA abundance in response to peroxide. Since the modifying enzyme transcripts were not altered, the tRNA transcript abundance was reflected in the detected modification levels. Therefore, C. neoformans exhibits an increase in certain tRNA transcripts which then causes an increase in those tRNAs’ modifications. In conclusion, C. neoformans responds to peroxide stress via regulation of the tRNA pool – consequently altering the modifications within it.

Support or Funding Information

This work was funded by the Defense Threat Reduction Agency (DTRA) ‐ a branch of the US Department of Defense (DoD). Further support has been provided by the University of Cincinnati.
A Murine Model of the Exercise Pressor Reflex
Authors: Li Qinglu , Garry Mary
Published: April 2020

The exercise pressor reflex (EPR) is an involuntary response to exercise. It is defined by a rise in mean arterial pressure (MAP) and heart rate (HR) in response to muscle contraction and is necessary for providing adequate blood flow to the exercising muscle to match metabolic demand and prevent premature fatigue. Proper function of this reflexive response is essential to healthy cardiovascular responses to exercise. Animal models available to test the EPR such as cat, dog, and rabbit have been utilized extensively to gain physiological information across species but generating disease or translating observations to humans has been challenging in these models. While this reflex can be tested rapidly and non‐invasively in humans, an elucidation of the mechanisms that regulate this reflex at the cellular and molecular level limit the utility of such studies. To test the EPR in the setting of heart failure, we previously developed a reliable and reproducible decerebrate rat model that allowed us to study the EPR in cardiomyopathic animals. We observed that this model yielded similar responses to exercise to those observed in humans. Here, we have developed a novel murine model of the EPR to allow for unlimited exploration of physiological and pathological cellular and molecular mechanisms in a variety of transgenic and genetically modified mouse lines. Like the rat model, we observed that ventral root stimulation (VRS; to evoke contraction of skeletal muscle) in an anesthetized mouse causes a depressor response and a reduction in HR. In contrast, the same stimulation in a decerebrate mouse causes a rise in MAP and HR. The responses observed in the decerebrate murine preparation are abolished by disruption of afferent innervation of the muscle via dorsal rhizotomy or by neuromuscular blockade. Moreover, we demonstrate that TRPV1 antagonism in wild type (WT) mice and Trpv1 null mice display a reduced MAP response to VRS while the response to mechanosensitive stimulation (passive stretch) remains intact. Additionally, we demonstrate that intra‐arterial infusion of capsaicin (a TRPV1 agonist) results in a dose related rise in MAP and HR that is significantly reduced by a selective and potent TRPV1 antagonist and, further, that these responses are completely abolished in Trpv1 null mice. Lastly, we observe no role of the Piezo2 receptor in mediating the EPR under physiological conditions. These data serve to validate the development of a decerebrate mouse model for the study of cardiovascular responses to exercise and further define the role of the TRPV1 receptor in mediating the EPR. This novel model will allow for extensive study of the EPR in unlimited transgenic and mutant mouse lines, thus allowing for an unprecedented exploration of the molecular mechanisms that control cardiovascular responses to exercise in health and disease.
Human Cadaver Preservation Techniques During Prolonged Dissection on Downdraft Tables in a Gross Anatomy Laboratory
Authors: Travers Benjamin , Hite Aimee , Kalthoff Rachel , Ray Olivia , Gould Douglas , Barremkala Malli
Published: April 2020

The purpose of this study is to determine the best method(s) for maintaining the integrity of cadavers during prolonged dissections on downdraft tables in the medical student anatomy course. Downdraft tables have become increasingly common in gross anatomy laboratories and are one of the most effective ways to minimize exposure to formaldehyde. Unfortunately, the improved ventilation of the cadaver during prolonged dissection increases the likelihood of the cadaver drying out. This is particularly problematic in the human gross anatomy course because the dry cadaveric tissue makes it more difficult to identify anatomical structures. Following full body dissection on downdraft tables, four cadaveric forearms were maintained in four different manners: uncovered (control), wrapped in a cloth, enclosed in a plastic bag, and wrapped in a cloth as well as enclosed in a plastic bag. Three weeks later, faculty and medical students who were blind to the preservation methods evaluated the quality of the four cadaver forearms using a Likert‐scale cadaver integrity assessment (CIA) developed by the researchers. The quality of three non‐control forearms was rated as significantly higher for all measures of the CIA than the control forearms (P < 0.05). There was insufficient evidence to conclude that there was any difference in quality between the three non‐control forearms in all but two measures of the CIA (P > 0.05). We therefore conclude that leaving the cadavers on downdraft tables for only a few weeks allows the tissue to dry out, leading to cadavers with poorly preserved anatomy. The drying out process can be mitigated in part by wrapping the limbs of the cadaver in a cloth, a bag, or both between uses. This would be especially beneficial during gross anatomy courses so that students could learn human anatomy from a cadaver with well‐preserved anatomical structures.
Gross Cross‐Sectional Anatomy as a Tool to Develop Imaging Proficiency in Medical Students
Authors: Hite Aimee , Travers Benjamin , Kalthoff Rachel , Ray Olivia , Khan Abdul-Majid , Barremkala Malli
Published: April 2020

Many first‐year medical students find imaging studies to be an abstract and difficult topic to learn. Understanding and interpreting imaging studies such as Magnetic Resonance (MR), Computed Tomography (CT) and ultrasound requires proficient knowledge of cross‐sectional anatomy of the human body. Our study aims to evaluate the use of an online module, which utilizes cadaveric gross cross‐sectional anatomical photographs of the thigh and correlates such viewpoints with the respective MR, CT and ultrasound images. Medical students at Oakland University William Beaumont School of Medicine were given access to the online module. We designed this module to consist of a pre‐module quiz, the module itself, and a post‐module quiz. The module contains cadaveric cross‐sectional photos of the thigh at three different levels as well as corresponding imaging studies of the thigh. Each eight‐question pre‐module and post‐module quiz contains two MR, two CT, two ultrasound, and two cross‐sectional anatomy questions. Comparison of students’ pre‐ and post‐ module scores as well as student scores on exam questions relevant to the module content will be used to evaluate whether the module has successfully aided students in learning the various imaging modalities. A survey will be sent to students to seek their feedback on the module in order to improve it for future medical students. This research uses a novel approach by correlating anatomical photographs with the imaging modalities; other studies exclude the ultrasound or use of clay models rather than imaging. Medical students have received the online module, and we are awaiting the results of this study, which could contribute to the future of medical education and the ease of learning MR, CT, and ultrasound via cross‐sectional anatomy.
Cytochrome P450‐Derived Epoxy Lipids of N‐3 PUFAs Protect the Heart From Ischemia‐Reperfusion Injury by Regulating Mitochondrial Sirtuin 3
Authors: Darwesh Ahmed M. , Altamimi Tariq R. , Jamieson K. Lockhart , Bassiouni Wesam , Zhang Hao , Oudit Gavin Y. , Lopaschuk Gary D. , Seubert John M.
Published: April 2020

Objective

Cytochrome P450 epoxygenases can metabolize dietary n‐3 polyunsaturated fatty acids (n‐3 PUFAs) to epoxy lipids, which have known cardioprotective effects; however, it remains unclear how this cardioprotection occurs. Our recent data demonstrate that cytochrome P450 epoxygenase‐derived metabolites of the n‐3 PUFA docosahexaenoic acid (DHA), 19,20‐epoxydocosapentaenoic acid (19,20‐EDP), exhibit protective effects toward cardiac mitochondria. Maintaining mitochondrial quality is essential to limit myocardial damage following ischemia‐reperfusion (IR) injury. The mitochondrial deacetylase sirtuin 3 (SIRT3) plays a pivotal role in the maintenance of mitochondrial function and cardiac energy metabolism. We hypothesize that the cardioprotective mechanism(s) of 19,20‐EDP against IR injury involve(s) stimulating mitochondrial SIRT3.

Methods

Langendorff and isolated working heart perfusions were performed in male and female C57BL/6 mice to assess changes in cardiac function and energy metabolism in response to IR injury. Hearts were perfused with either vehicle, 19,20‐EDP (1 μM) and/or the SIRT3 inhibitor nicotinamide (30 μM) for a 20 min baseline perfusion, following which hearts were subjected to 30 min of global no flow ischemia and 40 min of reperfusion. Glucose and palmitate oxidation rates were measured to detect alterations in cardiac energy metabolism. Mitochondrial fractions were assessed for changes in SIRT3 activity and total lysine acetylation levels. Mitochondrial SIRT3 downstream targets were assessed in human ventricular tissues obtained from individuals with ischemic heart disease (IHD) included in the Human Explanted Heart Program and compared to non‐failing control hearts (NFC) collected from unused transplant donors through the Human Organ Procurement and Exchange Program at the University of Alberta. In addition, mitochondrial respiration was assessed in cardiac fibers isolated from the human tissues.

Results

Hearts perfused with 19,20‐EDP demonstrated improved post‐ischemic functional recovery, glucose oxidation and cardiac efficiency compared to control groups. The cardioprotective effects were associated with enhanced mitochondrial SIRT3 activity and reduced global protein acetylation. We observed increased acetylation of the mitochondrial antioxidant superoxide dismutase 2 in tissues obtained from IHD human myocardium suggesting decreased antioxidant capacity. Treatment with 19,20‐EDP also markedly improved mitochondrial respiration in permeabilized human fibers obtained from IHD hearts. Importantly, the cardioprotective effects of 19,20‐EDP was abolished by the SIRT3 inhibitor nicotinamide.

Conclusion

We demonstrate that 19,20‐EDP‐mediated cardioprotective mechanisms against IR injury involve preservation of mitochondrial SIRT3 activity, which results in improved cardiac efficiency.
Metabolic and Contractile Pathophysiology Following Volumetric Muscle Loss Injury
Authors: Basten Alec M. , Raymond-Pope Christiana J. , Dalske Kyle A. , Call Jarrod A. , Greising Sarah M.
Published: April 2020

Volumetric muscle loss (VML) injury, due to abrupt and significant loss of tissue, exceeds the endogenous regenerative ability of the skeletal muscle, leading to long‐term functional impairment and disability. There is a well‐established physiologic relationship between skeletal muscle mass and metabolism, but the implications of VML injury on this relationship are widely unexplored. Recent investigations have identified oxidative and mitochondrial dysfunction associated with VML injury; however, the local metabolic myofiber response is not completely understood. We hypothesized that significant metabolic and contractile limitations would be observed in the remaining myofibers following VML injury, suggestive of motor unit reorganization. To evaluate the metabolic properties following VML, adult C57BL/6 male mice (n=16) underwent a sham procedure or multi‐muscle full thickness VML injury to the gastrocnemius, soleus, and plantaris muscles (21.1±0.7mg). Two months post‐VML, terminal histologic evaluation of capillarity, mitochondrial content, fibrotic deposition, and myosin heavy chain distribution was completed. Additionally, maximal contractile function and force‐frequency response of the posterior muscle compartment were evaluated. Maximum passive torque at 20 degrees dorsiflexion was significantly greater in the VML group (5.5±0.22 mN·m) compared to sham (2.3±0.13 mN·m). Maximum isometric torque was significantly less in VML (334±28 mN·m) compared to sham (557±66 mN·m). VML injury resulted in slower torque production and elevated twitch:tetani ratio, perhaps indicating a slower muscle fiber phenotype in the tissue remaining. Collectively, this work suggests that motor unit reorganization may alter muscle fiber phenotype following VML injury, likely limiting long‐term muscle functionality.

Support or Funding Information

Supported by W81XWH‐18‐1‐0710 & T32AR050938.
Thalidomide protects against cerebral ischemia reperfusion injury: Role of pERK1/2‐NFκB signaling
Authors: Nassar Noha N. , Mohamed Reham A. , El-Yamany Mohammed F. , Abdel Rahman Abdel A. , Al-Shorbagy Muhammad Y.
Published: April 2020

While thalidomide (TLM) protects against neuronal injury and behavioral deficits caused by cerebral ischemia reperfusion (IR), the mechanisms of TLM neuroprotective effect remain unknown. Here, we tested the hypothesis that suppression of the phospho‐extracellular signal regulated kinase (pERK1/2)‐ nuclear factor kappa (NFκ)‐B signaling, subsequent to tumor necrosis factor‐α (TNF‐α) inhibition, underlies TLM mediated neuroprotection. Male Wistar rats subjected to occlusion of both common carotid arteries for 45 min followed by declipping and allowing reperfusion for 24 h exhibited histopathological damage, memory impairment, motor incoordination and hyperactivity. Unilateral intra‐hippocampal TLM ameliorated these behavioral deficits along with the following ex vivo hippocampal effects: (i) abrogation of the IR‐evoked elevations in hippocampal TNF‐α, pERK1/2, NFκB, MPO, BDNF, iNOS contents and (ii) partial restoration of the reduced anti‐inflammatory cytokine IL‐10 and pS852 nNOS. These neurochemical effects, which were replicated by the pERK1/2 inhibitor PD98059, likely mediate the reductions in c‐Fos and caspase‐3 levels and the ensuing anti‐apoptotic effect of TLM in the IR model. These results substantiate a crucial anti‐inflammatory role for pERK1/2 inhibition in the salutary neuronal and behavioral effects of TLM in a model of IR injury.
The Impact of High Dietary Sodium on Cerebral Blood Flow Regulation
Authors: Migdal Kamila U. , Robinson Austin T. , Watso Joseph C. , Babcock Matthew C. , Serrador Jorge R. , Farquhar William B.
Published: April 2020

High salt consumption is linked to increased stroke risk. In rodents, high dietary sodium has been shown to impair cerebral blood flow regulation. In humans, cerebrovascular reactivity to carbon dioxide (CO2) is a validated method for assessing the cerebrovasculature where a reduction in cerebrovascular reactivity is associated with cerebrovascular disease. Additionally, the ability for the brain to maintain cerebral blood flow despite changes in arterial blood pressure is termed cerebral autoregulation (CA). Dysfunctional CA is also predictive of strokes and all‐cause mortality. However, the effects of a high dietary sodium consumption on cerebrovascular reactivity and dynamic CA in healthy, young adults is unknown.

Purpose

We tested the hypothesis that ten days of high dietary sodium feeding would impair cerebral blood flow regulation in healthy young adults.

Methods

Eight adults (6M/2F; age: 29 ± 11yrs; BMI: 26±3 kg/m2, Mean‐±SD) participated in a controlled feeding study that consisted of 10 days of either low‐ (LS: 1 g sodium/day), medium‐ (MS: 2.3 g sodium/day), or high‐sodium (HS: 7 g sodium/day) diets, in randomized order separated by at least a month. Urinary sodium excretion was determined to ensure compliance on diets. Transcranial Doppler of the right middle cerebral artery (MCA) velocity was assessed while participants lay supine. On the tenth day of the diet, cerebrovascular reactivity to high CO2 (via 8% CO2; hypercapnia) and low CO2 (via hyperventilation; hypocapnia) were assessed. Dynamic CA between spontaneous changes in mean arterial pressure to MCA velocity using a transfer function analysis was also measured. Data were analyzed using one‐way repeated measures ANOVA. Tukey post hoc comparisons were used when appropriate.

Results

Urinary sodium excretion (LS= 64±56, MS= 82±19, HS=250±213 mmol/24h, P=0.04) increased in a step‐wise manner. Brachial blood pressures were not different (systolic blood pressure= P=0.45; diastolic blood pressure: P=0.17). Cerebrovascular reactivity to hypercapnia (LS= 2.4±0.6, MS= 1.7±0.5, HS=2.5±0.7 %/mmHg, P=0.09) and to hypocapnia (LS= 1.8±0.6, MS= 1.9±0.6, HS=2.0±0.7 %/mmHg, P=0.72) were not different. Dynamic CA was also not different in the very low frequency (phase: LS= 71±23, MS= 72±38, HS=80±39°, P=0.69) and low frequency band (phase: LS= 42±14, MS= 45±11, HS=47±14°, P=0.62).

Conclusion

Despite the diets, these preliminary data suggest that high dietary sodium may not impair cerebral blood flow regulation in healthy young adults.

Support or Funding Information

Acknowledgements

NIH Grant 1RO1HL128388
Novel Tumor Suppressor PMM1 Suppresses Oral Cancer Growth through Activation Unfolding Protein Response Activator EGR1
Authors: Li Li Jie , Chan Ming-Hsien , Chang Yu-Chan , Chang Wei-Min , Huang Chi-Ying , Hsiao Michael
Published: April 2020

Protein glycosylation is the common post‐translational modification in the mammalian cells. More than half proteins have their own glycosylation which increasing the protein diversity and complexity or maintaining protein stability. N‐glycosylation is a quality control marker of protein folding. Aberrant n‐glycosylation usually cause unfolding protein response and endoplasmic reticulum stress. Here, we find phosphomannomutase 1 (PMM1), which converts initiation molecule in n‐glycosylation to mannose‐1‐phosphate, serves as a tumor suppressor gene in oral squamous cell carcinoma (OSCC). PMM1 peptide sequence and enzymatic substrate are highly similar to PMM2. However, we found PMM1 might have an opposite cancer progression effect in public prognosis analysis. Ectopic wildtype PMM1 expression suppressed in vitro cancer cell proliferation, but not in the catalytic domain death PMM1‐R150H clone. Furthermore, wildtype PMM1 also suppressed the in vivo OSCC cell growth in the Nod‐SCID immunodeficient mice. Transcriptome analysis by cDNA microarray and ingenuity pathway analysis revealed that PMM1 stimulated early growth response protein 1 (EGR1) pathway activation. As we known, EGR1 is a tumor suppressor and the master activator in unfolding protein response. Our study suggests that PMM1 is a gatekeeper in cellular protein folding and OSCC tumor suppressor through regulating EGR1 expression. This study provides new insights into an important mechanism for n‐glycosylation and protein folding in oral carcinoma, which may be helpful for biomarker and therapeutic development in the future.
The P2Y2 Nucleotide Receptor Mediates Tissue Factor Expression in Human Monocytes
Authors: Peng Qianman , Shen Jianzhong
Published: April 2020

Recently we reported that in human coronary artery endothelial cells, activation of the P2Y2 receptor induces up‐regulation of tissue factor (TF), a key initiator of the coagulation cascade. However, it has been shown by others that monocyte TF is more important than endothelial TF in terms of provoking pro‐thrombotic state. Thus, we aimed to study whether human monocytes express P2Y2 receptor and its role in control of TF expression. RT‐PCR and receptor activity assays revealed that among the eight P2Y nucleotide receptors, the P2Y2 subtype was selectively and functionally expressed in human monocytic THP‐1 cells. Consistent with this, stimulation of the THP‐1 cells by ATP or UTP at micromolar concentration dramatically increased TF protein expression measured by Western blotting, which was abolished by AR‐C118925XX, a selective P2Y2 receptor antagonist, suggesting a key role for P2Y2 receptor. In addition, UTP or ATP treatment induced a rapid accumulation of TF mRNA, which was maximal at 2h and preceded with an increased TF pre‐mRNA, indicating enhanced TF gene transcription. However, we also observed a significant increase in TF mRNA stability after UTP and ATP treatment. Furthermore, activation of the monocyte P2Y2 receptor by a low dose of UTP or ATP (1μM) significantly activated the MAPK pathways including ERK1/2, JNK, and p38, along with their downstream transcription factors including c‐Jun, c‐Fos and ATF‐2, whereas blocking the ERK1/2, JNK or p38 pathways respectively, all significantly suppressed P2Y2 receptor‐mediated TF expression. These findings demonstrate for the first time that the P2Y2 receptor mediates TF expression in human monocytes through new mechanisms involving ERK1/2, JNK and p38, and that both transcriptional and posttranscriptional mechanisms contribute to a pro‐thrombotic status in monocytes through the P2Y2‐TF axis, highlighting monocyte P2Y2 receptor may be a new drug target for the prevention and/or treatment of relevant thrombotic diseases.

Support or Funding Information

Supported by NIH funding 1R01HL125279‐01A1 (JS)
Measuring the Kinetics of G‐protein‐coupled Receptor Signaling and Biased Agonism.
Authors: Hughes Thomas Edward , Tewson Paul , Hoare Sam
Published: April 2020

Quantifying the time course of G‐protein‐coupled receptor signaling can reveal biological properties of GPCR function such as receptor desensitization, pathway selectivity, and spatiotemporally compartmentalized signaling. Applying signaling kinetics to modern receptor research and drug discovery requires assay modalities that can monitor signaling responses over time, and data analysis methods to extract the kinetic drug parameters. Here we describe a suite of fluorescent biosensors for monitoring multiple pathways of GPCR signaling (Gs, Gi, Gq and arrestin). These biosensors are genetically‐encoded and amenable to kinetic analysis, with continuous “real time” responses detected on plate readers or imaging systems. A kinetic data analysis framework is applied to the time course data using familiar equations on commonly‐used curve‐fitting software (e.g. GraphPad Prism). The analysis yields the initial rate of signaling (kTau), a biologically‐meaningful parameter of ligand efficacy at generating the signal. Biased agonism assessment is simplified using this approach, and demonstrated using arrestin recruitment and second messenger signaling via the AT1 angiotensin and GLP‐1 receptors. Bias is quantified as the ratio of the initial rates of signaling reported by each assay. This platform provides a straightforward method that investigators can use to quantify GPCR receptor pharmacology and biased signaling with the application of kinetic analysis to understanding receptor function and to the development of new therapeutics.

Support or Funding Information

This work was supported by grants from the National Institutes of HealthR44 GM125390‐02 and R44 DA050357‐01
Effects of Continuous vs Interval Aerobic Exercise on Cognitive and Vascular Function of Juvenile Rats
Authors: DE MORAES CAMILA , Rebelo Macário Arosti , Garcia Nádia Fagundes , Padovan Cláudia Maria
Published: April 2020

Physical exercise is well‐known as an effective approach to improve cardiovascular function as a response to the cardiac overload and arterial shear stress that occurs during physical effort. In recent years physical exercise has been show to be effective to improve cognitive function, particularly memory, in individuals with neurodegenerative diseases but less is known about this effect in health individuals. Furthermore, an emerging issue on exercise science is to determine the optimal dose of exercise in favor of these benefits. The aim was to evaluate the effects of continuous versus interval aerobic exercise training on spatial learning and spatial memory, anxiety‐like behavior and vascular reactivity of juvenile rats. Male Sprague Dawley rats (10‐week‐old) were divided into three groups: sedentary (SD), continuous training (C‐tr), and interval training (I‐tr). The continuous (running at 60% during 40 minutes) and interval training (3 x 2 min of running at 70% with 4 min running at 35%) were performed 3 times/wk during 6 weeks. Elevated plus maze test (EPM) was carried out on Monday of the sixth week and Barnes maze task was initiated on Friday of the same week. Contraction response of carotid rings was determined by tension increase, from baseline, using cumulative concentration‐response curve to phenylephrine (Phe 1nM‐100μM) in intact rings. Endothelial function was measured either by cumulative concentration‐response curve to acetylcholine (ACh 10nM‐100μM) or measuring endothelium‐dependent increase in tension to L‐NAME (100 μM) in rings contracted by Phe (2 μM). Exercise regimens did not influence spatial learning and memory as primary escape latency was not modified. Open arm exploration of EPM was 42% higher in C‐tr indicating lower anxiety‐like behavior. Regarding vascular function, Phe potency was lower in C‐tr and maximal response was higher in I‐tr. However, both trained groups had L‐NAME‐induced tension increased by half compared to SD group. Neither maximal response nor potency of ACh was modified. In conclusion, spatial learning and spatial memory were not affected by both exercise training protocols. However, C‐tr produced better outcomes regarding anxiety‐like symptoms. Endothelial function was improved in a similar way, and then I‐tr can be considered time‐effective. These results could be useful to support the selection of exercise training protocol in animal model. It is of particular importance as physical exercise parameters (training regimen, effort intensity and duration, weekly frequency) produces specific behavioral and physiological adaptation.

Support or Funding Information

FAPESP Grant # 2017/27042‐6