bluuebunny/crossref_metadata_2025 huggingface dataset:
GPR183 and GPR19 Activation Improves Pancreatic Alpha Cell‐Induced Glucose Homeostasis
Authors: Rossiter Jackie L. , Yosten Gina L.C. , Kolar Grant R.
Published: April 2020

Diabetes mellitus (DM) is characterised by pancreatic islet failure and dysregulation. The pancreatic islet is comprised of three primary cell types: alpha, beta and delta cells. Pancreatic beta cell destruction is responsible for the onset of classical symptoms of DM consequent to loss of endogenous insulin production and has been the subject of decades of research effort. The mechanisms underlying alpha cell dysfunction are less well understood, but no less important. Alpha cell dysfunction contributes to counter regulatory failure and predisposes to severe hypoglycemia, a disabling and potentially fatal diabetic complication. Thus, it is critical to identify new targets for the treatment of alpha cell dysfunction. Using a PCR‐based screen of AlphaTC1‐9 cells, we identified G‐protein coupled receptors 183 and 19 as highly expressed in alpha cells. G‐protein coupled receptor 183 (GPR183, also known as EB12) is the receptor for the potent GPR183 agonist 7alpha 25‐dihydroxycholesterol. GPR183 expression is increased in human pancreatic beta cells and GPR183 activation by 7alpha 25‐dihydroxycholesterol promotes both insulin transcription and release in these beta cells. G‐protein coupled receptor 19 (GPR19) is the receptor for adropin, a small peptide which plays roles in metabolism and energy homeostasis. Circulating adropin acts on the liver to enhance Insulin receptor substrate (IRS) sensitivity and reduce hepatic glucose release. Adropin levels are reduced in obesity, and adropin administration to diet‐induced‐obese mice reduces insulin resistance and lowers fasting blood glucose. The effect of adropin on pancreatic alpha cell function had not been examined prior to this study. We hypothesize that GPR183 and GPR19 act in the pancreatic alpha cell to facilitate glucose homeostasis. To test this hypothesis, Alpha TC1‐9 cells were treated with GPR183 agonist, 7alpha 25‐dihydroxycholesterol, at 1nM, 10nM, and 100nM, or GPR19 agonist, adropin, at 1nM, 3nM, or 9nM. AlphaTC1‐9 were treated with agonists unstimulated, stimulated for 1 hour at 25mM glucose (HG) and treated at 5.5mM glucose (NG), or stimulated for 1 hour at 25mM glucose and treated at 2.2mM glucose (LG). Treatment with the GPR183 agonist 7alpha 25‐dihydroxycholesterol increased proglucagon transcript and reduced GPR183 and GPPR19 transcript in alphaTC1‐9 cells. Treatment with adropin had no effect on proglucagon transcript and robustly increased GPR183 transcript. These results suggest GPR183 and GPR19 as novel therapeutic targets to reduce the incidence of hypoglycemia in DM.

Support or Funding Information

NIDDK
Aortic Banding and Low‐Intensity Exercise Training do not Alter Autophagy in a Yucatan Mini‐Swine Model of Pressure Overload‐Induced Heart Failure
Authors: Silva Kleiton Augusto Santos , Thorne Pamela , Ivey Jan , Emter Craig A.
Published: April 2020

The autophagy‐lysosome system is an essential intracellular proteolytic process mediating recycling of dysfunctional proteins and organelles. This process is important to cellular renovation, and activation of autophagy by exercise training (ET) is considered essential to the maintenance of cellular vitality. However, the implications of chronic low‐intensity ET on autophagic function in an experimental setting of heart failure (HF) remains unclear. The purpose of this study was to determine if autophagy contributes to cardiovascular dysfunction that is treatable by ET in a pre‐clinical model of pressure overload‐induced HF. We hypothesized ET would prevent aortic banding‐induced decreases in autophagy in the heart, coronary vasculature, and skeletal muscle. Male Yucatan mini‐swine (29–32 kg; 8 mo‐old) were assigned to the following groups (6–7 pigs/groups): control (CON), heart failure (HF), heart failure low‐intensity continuous ET (HF‐CT), and heart failure low‐intensity interval ET (HF‐IT). Chronic pressure overload was induced by an aortic band placed on the ascending aorta proximal to the brachiocephalic artery. Two months post‐surgery and after the development of pathological hypertrophic remodeling, animals began treadmill ET 3 times/week at 55 minutes/session for 17 weeks. Autophagic markers measured by protein level of LC3B (a marker of autophagosome formation), p62 (autophagosome cargo receptor) and Beclin 1 (regulation of autophagy initiation), were assessed in the left ventricle (LV), right ventricle (RV), left circumflex artery (LCX), right coronary artery (RCA), deltoid, and long head of the triceps (TLH). We found no changes in autophagic markers (LC3B‐I, LC3B‐II, LC3B‐II/I ratio, p62, and Beclin 1) in LV, RV, LCX, RCA, deltoid, and TLH when comparing HF group with CON group (p>0.05). Similarly, ET of any intensity did not alter autophagic markers in LV, RV, LCX, RCA, deltoid, and TLH compared to CON and HF groups (p>0.05). Contrary to our hypothesis, protein levels of a number of different well‐established markers of autophagy were not decreased in the HF group. Further, our results do not demonstrate any change in this important cellular recycling mechanism following chronic low‐intensity ET. Although activation of autophagy is considered a positive adaptation of ET in a setting of physiological health, these data suggest autophagy may not be a mechanism by which ET of an intensity tolerable to a heart failure patient improves cardiovascular and skeletal muscle health in a pre‐clinical model of pressure overload‐induced HF.

Support or Funding Information

Craig A. Emter – NIH/NHLBI R01 HL112998, and Kleiton A. S. Silva – AHA: 18POST33960472
Transmural Variation of Collagen Orientation and Structure Distributions in the Right Pulmonary Arterial Wall
Authors: Pursell Erica , Valdez-Jasso Daniela
Published: April 2020

Pulmonary arterial hypertension (PAH) is a disease characterized by elevated blood pressure (mean pulmonary arterial pressure above 25 mmHg. Remodeling of pulmonary arteries due to this elevated blood pressure compromises their normal physiological function. Our previous work showed that the right pulmonary artery significantly stiffens circumferentially in PAH and collagen fibers become engaged and significantly aligned in the medial layer (Pursell et al 2016). To better understand the changes in tissue mechanical response, here we explore the distributions of collagen orientation and tortuosity through the vessel wall.

Methods

To induce PAH, four male Sprague‐Dawley rats were injected with 60 mg/kg monocrotaline. Once PAH was established (4 weeks post‐injection), pulmonary arteries were harvested, and collagen imaged via multiphoton microscopy. A custom MATLAB code was then used to determine collagen fiber orientation and tortuosity.

Results

MPM images of collagen fibers from the intima, media, and adventitia of normotensive and hypertensive rat RPAs were traced with our custom MATLAB code. In the hypertensive animals, fiber direction and tortuosity varied more between vascular wall layers than those of normotensive animals.

Conclusions

While our previous study only examined the medial layer, this study determined that fiber distribution varies throughout the vessel wall. This will significantly affect the mechanics or behavior of pulmonary vessels. Therefore, future studies will need to incorporate collagen structures from multiple vessel layers to better understand the relationship between vascular remodeling and mechanics.
Exosome‐associated ELAVL1 adversely affects cardiac macrophage‐fibroblast crosstalk in diabetes
Authors: Krishnamurthy Prasanna , Govindappa Prem , Patil Mallikarjun
Published: April 2020

Background

Macrophage dysfunction, inflammation and excessive fibroblast activation has been implicated in the pathogenesis of diabetes‐associated heart disease. However, the paracrine mechanisms are not completely understood. We sought to unravel the influence of diabetes on exosome (Exo) transfer of Human antigen R (HuR or ELAVL1, an mRNA stabilizing protein) and its implications on cardiac fibrosis and dysfunction.

Methods and Results

We evaluated the invitro and invivo effects of macrophage cellular‐ and exosome‐HuR or its deficiency on paracrine mechanisms of diabetic milieu‐induced inflammatory and fibrosis‐related signaling. Human failing hearts show higher HuR levels as compared to non‐failing hearts. Diabetic milieu (db/db mice) show increase in HuR expression in cardiac‐ and cultured bone marrow derived‐macrophages (BMMac). High glucose (HG, 30mM) stimulates HuR nuclear‐to‐cytoplasmic translocation and transfer of HuR mRNA and protein into exosomes. PKC‐delta inhibition blocks HG‐induced HuR translocation. Exosome from macrophages exposed to diabetic milieu (HG or db/db mice) significantly increases proinflammatory and profibrogenic responses in fibroblast (invitro) or cardiac fibrosis in mice. Exo‐HuR deficiency (HuR knockdown in macrophage) abrogates the above effects. In diabetic mice, macrophage depletion followed by reconstitution with HuR‐deficient‐BMMac decreases ANG II‐induced cardiac fibrosis and improves LV dysfunction as compared to BMMacWT administered mice.

Conclusions

To the best of our knowledge, this is the first study to demonstrate that diabetes‐mediated increase in BMMac‐HuR expression, its cytoplasmic translocation and exosome transfer activates fibrogenesis signaling invitro and invivo, and HuR deficiency attenuates these deleterious effects. The data suggests that HuR might be targeted to alleviate macrophage dysfunction and pathological fibrosis in diabetes mellitus.

Support or Funding Information

National Institutes of Health (NIH) grants HL116729 (to P.K.), HL138023 (to P.K. and J.Z.), and American Heart Association Grant‐in‐aid GRNT 25860041 (to P.K.) and Transformational Project Award 19TPA34850100 (to P.K.).
Identifying the Substrate Interactions of KDAC7
Authors: Mohammed Hajjia S. , Nichols Kyara A. , Toro Tasha B. , Watt Terry J.
Published: April 2020

Protein post‐translational modifications are chemical adaptations that take place in the human body to control behavior of proteins. The acetylation of lysine is one type of protein post‐translational modification. Lysine deacetylases (KDACs or HDACs), are enzymes that help regulate proteins by reversing this modification. It has been shown that KDAC7 is essential in many biological processes, but KDAC7 has no identified substrate proteins. Therefore, better understanding of KDAC7 substrate preferences could lead to the improved treatment of diseases. Using a gain‐of‐function variant of KDAC7 that significantly increases enzyme activity, we have identified several possible substrates using peptides derived from identified acetylated proteins. Molecular dynamics simulations suggest possible important interactions between these peptides and the enzyme. We are also examining the effect of the second, non‐catalytic domain of KDAC7 on substrate preferences and overall activity. Possible substrates identified by these experiments provide leads for cell‐based assays to determine in vivo substrates and/or binding partners. This work provides a greater understanding of how KDAC7 interacts with potential substrate proteins and the biological activity of the enzyme.

Support or Funding Information

NIH TL4GM118968, UL1GM118967, 5G12MD007595, and R15GM129682; NSF CHE 1625993 and MCB 1817358; U. S. Army Research Laboratory and the U. S. Army Research Office W911NF1810450; and the Louisiana Cancer Research Consortium.
Interactive, Team‐Based Learning Environment Enhances Students Learning and Confidence
Authors: DiFrancesca Heidi
Published: April 2020

Many students enter a class lacking appropriate self‐confidence, which can negatively impact their performance in the course. Active learning environments lend to a milieu whereby strategies that allow students to fully participate in the course even when understanding is low are used, which could provide opportunities for confidence to grow among these students. Additionally, research has shown that active‐learning strategies can improve both student’s exam performance and their satisfaction with the course. A flipped teaching model is an interactive pedagogical approach that focuses on learner‐centered instruction by employing learning strategies in the classroom. Coupled with team‐based methodology, such a learner‐centered model can facilitate collaboration‐based discussion and small group work that ask students to engage in higher order thinking, possibility leading to a gain in confidence that translates into an improvement in performance. While evidence that supports the effectiveness of a learner‐centered approach on the benefits of student learning is well established, a growing body of research has shown this approach to have a positive impact on student’s self‐confidence and its relationship to grades. In this preliminary study, students enrolled in a team‐based, flipped undergraduate Human Anatomy and Physiology course were asked to anonymously self‐assess their level of confidence in learning the course material both before the course and after it. 54% of students (n=28) reported either highest or high increases in their confidence from the start of the course to the end of it, whereas, 15% reported their confidence levels as neutral (no change) and 2% of students reported low confidence. Additionally, students were asked to assess the impact team‐based quizzes and active learning activities had on their confidence in learning the course material. 56% of students of students enrolled in the course noted an increase in confidence as a result of the team‐based quizzes, and 52.4% reported that the in‐class active learning activities positively boosted their confidence in learning the material. 4% indicated that team‐based quizzes had a no impact on confidence. Not one student indicated that the in‐class activities did not increase their confidence in some way. Taken together, these preliminary results suggest that an interactive, team‐based learning approach in a Human Anatomy and Physiology course can have a positive impact on confidence and should be further explored.
Non‐coding RNA modulates cardiovascular adverse effects of anthracycline chemotherapeutic agent
Authors: Krishnamurthy Prasanna , Henderson John , Patil Mallikarjun
Published: April 2020

Introduction

Non‐coding RNA regulate numerous pathophysiological processes involved in cardiac health and disease. microRNA‐377 dysregulation has been implicated in cardiac dysfunction and remodeling.

Objectives

We evaluated its role in chemotherapy agent (doxorubicin, DOX)‐induced cardiotoxicity, specifically in DOX‐induced cardiomyocyte (CMs) stress and endothelial dysfunction.

Methods

miR‐377 expression and its targets were evaluated in human and mouse ischemic‐myocardial tissue by qRT‐PCR and RNA‐sequencing. We determined miR‐377 effect on Dox‐induced cardiomyocyte cell death, oxidative stress gene and cardiac pathology.

Results

Human cardiac biopsies from HF patients show significant increase in miR‐377 expression in comparison to non‐failing hearts. Cardiac ischemia‐reperfusion (I/R) injury in mice increases myocardial miR‐377 expression. Furthermore, single cell suspension of mouse ischemic heart and qPCR analysis show a robust increase (~13 fold) in miR‐377 expression specifically in the cardiomyocyte. Following DOX‐induced cellular stress, miR‐377 expression in CMs was elevated, and inhibition of miR‐377 attenuates DOX‐induced cell death and inflammatory response in CMs in vitro. RNA‐sequencing data suggests miR‐377 regulates numerous genes involved in cell survival and growth in cardiomyocytes. Furthermore, transfection of miR‐377 mimics in HUVECs significantly inhibits VEGF induced migration and vascular tube formation. In addition, proteome profile of human CD34+ cells transfected with miR‐377 mimics show significant decrease in numerous proangiogenic proteins as compared to nonspecific control transfected cells. Interestingly, anti‐miR‐377 injection in mice after doxorubicin administration shows increase in left ventricular wall thickness and reduction in LV chamber size.

Conclusions

These findings suggest that doxorubicin‐induced cardiotoxicity is associated with an increase in miR‐377 expression in the myocardium and that miR‐377 overexpression is detrimental to cardiomyocyte viability and endothelial cell function. Furthermore, in vitro, inhibition of miR‐377 was protective against DOX‐induced apoptosis, and attenuated the expression of anti‐oxidant genes. Therefore, targeting miR‐377 might protect against DOX‐induced cardiotoxicity.

Support or Funding Information

National Institutes of Health (NIH) grants HL116729 (to P.K.), HL138023 (to P.K. and J.Z.), and American Heart Association Grant‐in‐aid GRNT 25860041 (to P.K.) and Transformational Project Award 19TPA34850100 (to P.K.)
Greenhouse Gas Concentrations Driven by Microbial Community Structure in Boston Groundwater Wells
Authors: Momyer Victoria , Al-Haj Alia , Angell John , Fulweiler Robinson W.
Published: April 2020

Methane is a powerful greenhouse gas with a global warming potential 34 times that of carbon dioxide. Recent work has demonstrated high methane concentrations in groundwater wells. While previous research has focused on anthropogenic sources of methane in groundwater wells, it is also possible that methane is produced in situ via archaea. Here we determine the source(s) of the high methane concentrations in groundwater wells in Boston, MA. We also assess the groundwater well methanogen and methanotroph community. To do so, we collected gas, water, and DNA samples from nine groundwater wells throughout the city between 2017 and 2019. Methane, carbon dioxide, and nitrous oxide concentrations were determined using gas chromatography. DNA samples were extracted, replicated with archaeal and bacterial 16S primers, and sequenced using Illumina Mi‐Seq High Output DNA Sequencing. We found that 27.9% of our measurements could be attributed to natural gas leaks near the groundwater wells (>2.5% methane). In groundwater wells with no natural gas leaks, methane concentrations were negatively correlated with dissolved oxygen concentrations (r = −0.5480; p = 0.001416) and positively correlated with salinity (r = 0.5466; p = 0.001465). Additional data on the groundwater well microbial community is forthcoming and will also be presented. Overall, this study provides insight into how various environmental conditions such as salinity, dissolved oxygen, and distance to seawater impacts methanogen and methanotroph populations.

Support or Funding Information

Support and/or Funding provided by Boston University Undergraduate Research Opportunities Program, Boston University Kilachand Honors College, and the American Society for Biochemistry and Molecular Biology.
STAT3 is essential for proper limb skeletal development
Authors: Rigueur Diana
Published: April 2020

The Interleukin‐6/STAT3 (IL‐6/STAT3) signaling pathway has been well investigated in the development of the immune system and in disease. From autoimmune disorders, cancers, rheumatoid, and osteoarthritis, STAT3 gain‐of‐function and loss‐of‐function studies have shown that in humans, its regulation is imperative for control of disease. However, the precise role that STAT3 and its downstream effectors play in the formation, maintenance, and disease of the skeleton, remain understudied. Our recent work shows that STAT3 is imperative for multiple aspects of limb skeletal development. Through whole‐mount skeletal preparations, X‐ray micrographs, and histology, conditional loss of STAT3 in limb, via the Prx1‐Cre driver, show that STAT3 mediates proper cartilage, bone, tendon, and ligament formation. Moreover, loss of STAT3 results in severe bending and twisting of the fore and hind limbs, as well as abnormal tendon and ligament attachments. Furthermore, loss of STAT3 shows forelimb regional specificity of ectopic bone formation. Additional investigation of STAT3 mediated downstream effectors can identify a mechanism involved in proper cartilage and bone formation, as well as tendon and ligament‐to‐bone integration. Fundamental understanding of the role STAT3‐mediated signaling plays in skeletal formation may identify future therapeutics for regenerative medicine in tendon/ligament bone repair.

Support or Funding Information

NIH R01 80094
Kinetic Characterization of Escherichia coli Mutant Methylenetetrahydrofolate Reductase Flavoenzymes Leu277Ala and Leu277Ile
Authors: Hua Junmin , Tran Tin , Chien Jason , Trimmer Elizabeth E
Published: April 2020

The flavoprotein E. coli methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10‐methylenetetrahydrofolate (CH2‐H4folate) to 5‐methyltetrahydrofolate (CH3‐H4folate) utilizing NADH as the source of reducing equivalents. Leu277, whose expression is highly conserved in MTHFRs of different species, is one of four hydrophobic amino acids that surround the p‐aminobenzoate (PABA) ring of folate. Leu277 is also in close proximity to the ribose ring of NADH (3.529 Å) and presumably contributes to the stability of the highly folded structure of this substrate
[1]. Therefore, we hypothesize that Leu277 enhances folate and/or NADH substrate‐MTHFR binding affinity. To test our predictions, we genetically engineered Leu277Ala MTHFR and Leu277Ile MTHFR mutants as potential non‐functional and functional analogues to the wild‐type enzyme. In the steady‐state NADH‐menadione oxidoreductase assay, where the NADH half‐reaction is rate limiting, we show that Leu277Ala mutant increases Km of NADH by 7‐fold while decreasing kcat by 2‐fold. The Leu277Ile mutant increases Km of NADH by 3‐fold, but has comparable kcat to wild‐type MTHFR in this assay. We also report that, in the steady‐state CH3‐H4folate‐menadione oxidoreductase assay, kcat for Leu277Ala mutant is decreased by 2‐fold, and Km of this mutant is similar to the wild‐type enzyme. In this assay, Leu277Ile mutant decreases kcat of CH3‐H4folate by 4‐fold and Km by 3‐fold. In conclusion, Leu277 contributes significantly to NADH‐MTHFR binding, but has trifling effect on catalysis of both half reactions.
Developing an efficient ocular delivery system for the hydrogen sulfide releasing compound, diallyl trisulfide
Authors: Singh Neetu , Opere Catherine , Singh Somnath
Published: April 2020

Purpose

Although hydrogen sulfide (H2S) exhibits ocular hypotensive and retinal neuroprotective actions (Ohia et al., JOPT, 34:61,2018), its ocular delivery is challenging. Diallyl trisulfide (DATS) is a lipophilic, H2S‐releasing compound that rapidly degrades under aqueous conditions. Therefore, we sought to prepare a novel solid lipid nanoparticle (SLN) of DATS for ocular delivery.

Methods

DATS content was determined by HPLC method using standard solutions (3.12–100 ug/ml). The HPLC parameters consisted of C18 column (Zorbax, 150 mm × 4.6 mm, 5 μm) isocratic elution with methanol and water (85:15). Following method validation, the SLN loaded with DATS were prepared using hot emulsification process, characterized and influence of process parameters on particle size were evaluated.

Results

A well resolved DATS peak was detected at 2.8 mins. The method was validated for linearity (R2 > 0.999); precision (<10%) and accuracy (>90 %). Increasing the concentration of the poloxamer 188 from 0.2% to 1% (w/v) decreased the particle size from 296.7 to 90.46nm. Increasing lipid concentration from 1% to 3% (w/v) increased particle size from 65±5 to 103.4 ± 6 nm. Without the surfactant, glyceryl behenate (GBH) elicited higher particle size (330.8 ± 8.5 nm) than glyceryl monostearate (GMS) (254.4 ± 5.5 nm). Presence of surfactant (poloxamer 1%, soy lecithin 0.2%) reduced particle size to 238.6 ± 8 nm and 85 ± 4.5 nm for GBH and GMS, respectively. Thus, GBH was selected for SLNs and particle size, polydispersity index, zeta potential, and drug loading were 124 ± 2.67 nm, 0.189 ± 0.005, −23.57 ± 3.22mV and 1.5%, respectively. FTIR studies indicated complete encapsulation of DATS.

Conclusion

An accurate method for quantification of DATS was developed and validated. A biocompatible system for delivery of DATS was obtained with a particle size less than 200 nm. Studies are in progress to determine release pattern in ocular tissues.

Support or Funding Information

Creighton University‐SPAHP, Pharmaceutical Sciences Graduate Program
Methocinnamox (MCAM) Reverses and Provides Extended Protection from Fentanyl‐Induced Respiratory Depression in Awake Unrestrained Rats
Authors: Jimenez Victor M. , Maguire David R. , Gerak Lisa R. , France Charles P.
Published: April 2020

Opioid use disorder affects over 2 million Americans with an increasing number of deaths due to overdose from the synthetic opioid fentanyl and its analogs. Opioid toxicity is due, in large part, to the suppression of respiratory function. The FDA‐approved opioid receptor antagonist naloxone (e.g. Narcan®) is used currently to reverse opioid‐induced respiratory depression; however, its short duration of action limits its clinical utility. Many abused opioids have a duration of action longer than naloxone and reemergence of respiratory depression is common after rescue with naloxone. Methocinnamox (MCAM) is a selective, pseudoirreversible mu opioid receptor antagonist with a long duration of action that may reverse and provide long‐term protection from the respiratory‐depressant effects of fentanyl. Previous research has shown that MCAM reverses and prevents the respiratory‐depressant effects of heroin in nonhuman primates. However, the effects of MCAM on opioid agonist‐induced respiratory depression has not been examined in other species. Moreover, its effectiveness in antagonizing the respiratory depressant effects of intravenous (i.v.) opioids, including fentanyl, has not been examined. This study compared the ability of naloxone and MCAM to reverse and protect from fentanyl‐induced respiratory depression in rats. On different occasions, male Sprague‐Dawley rats (N = 8) received 0.01, 0.032, 0.056, 0.1, or 0.178 mg/kg fentanyl i.v. and ventilatory parameters were recorded using a whole‐body plethysmograph. Fentanyl decreased respiration in a dose‐ and time‐dependent manner. Compared with respiration under control conditions, these doses of fentanyl decreased minute volume by 20–65% with 0.178 mg/kg of fentanyl producing the largest and longest decrease in minute volume. Within 1 minute after i.v. administration, naloxone (10 mg/kg) and MCAM (10 mg/kg) reversed fully the respiratory depressant effects (minute volume) of 0.178 mg/kg fentanyl. The day after antagonist administration, MCAM, but not naloxone, attenuated fully the respiratory depressant effects of 0.178 mg/kg fentanyl. This study extends to another species (rats), another route of administration (i.v.), and another mu opioid receptor agonist (fentanyl) the ability of MCAM to reverse and provide extended protection from the respiratory depressant effects of opioids. Prolonged antagonism by MCAM may provide a useful treatment option for the current opioid crisis, including toxicity from fentanyl and related highly potent analogs.

Support or Funding Information

Supported by NIH (R01DA005018 and R01DA048417) and the Welch Foundation (AQ‐0039).
An approach to measure the effect of bridging protein size on DNA loop probability
Authors: Tader Brooke , Tse David , Becker Nicole , Maher Louis Jim
Published: April 2020

Using DNA ligase‐catalyzed cyclization assays, the in vitro persistence length of DNA has been determined to be about 150 base pairs (bp). In living E. coli bacteria, where DNA loops regulate the lac operon, DNA looping appears to be much more probable than expected from in vitro data. Why? A possible explanation for this paradox is that DNA loops in bacteria are bridged by proteins, reducing the amount of required costly DNA bending. Current in vitro methods to measure DNA flexibility are tedious and artificial, in that they require DNA cyclization, not just DNA looping. No high‐throughput method has been described to measure DNA flexibility for loops involving bridging proteins of different sizes. We are developing a high‐throughput method to measure the probability of DNA looping when bridged by a fusion protein that includes PCV2 and micrococcal nuclease domains. A bead‐tethered 1000‐bp DNA molecule of a known sequence is conjugated to this fusion protein through the PCV2 domain, and looping allows self‐cleavage such that the distribution of cleavage sites revealed by next generation sequencing reflects the energetics of DNA looping. We hypothesize that this distribution will be affected as a function of bridging protein size. Measuring how the size of bridging proteins affects tight DNA loop probability enables improved understanding of genetic switches.
Fresh‐frozen cadaveric dissection for medical students using specimens from surgical CME workshops
Authors: Ehrhardt John Dennis , Woodhouse Palina , Gomez Ferdinand
Published: April 2020

Introduction

Many medical schools have reduced, modified, or eliminated cadaveric dissection from their curricula. While the digital anatomy paradigm shift allows educators to reinforce concepts in an integrated, longitudinal manner, some students, especially those pursuing surgical specialties, remain interested in cadaver exposure. These students often practice manual dexterity by suturing on poultry and bovine parts, despite its limited ability to simulate human skin and connective tissues. Fresh‐frozen cadaver represents the highest quality specimen for appreciating anatomy and performing dissection. Unfortunately, student access is often prohibited by cost and lack of embalming limits prolonged use.

Aim

We sought to augment second‐year medical student anatomy knowledge and teach fundamental surgical principles through an elective experience using fresh‐frozen human cadaver.

Methods

Surgeons attend continued medical education (CME) workshops with fresh‐frozen specimens to learn advanced operative approaches. Specimens are typically discarded afterwards. Our school’s surgery interest group coordinated with faculty to create a two‐day session focused on head and neck dissection and suturing with fresh‐frozen cadavers leftover from a temporomandibular joint surgery CME workshop. This is a pilot study of an extracurricular supplement at our medical school and was not considered for IRB review.

Results

This session increased the educational value of donated human specimens. Students highly regarded the event for reinforcing challenging aspects of regional head and neck anatomy. They gained a higher appreciation for tissue planes than afforded by traditional formalin‐preserved cadaver. Dissection and suturing was comparable to what students can expect to encounter in the operating room. All twelve participating students advocated for increasing the size and frequency of these sessions. Formal data collection instruments were not utilized because this was a qualitative pilot experience.

Conclusions

Fresh‐frozen cadaver can be re‐used by students after surgical workshops for dissection and suturing to ethically gain the most educational value out of donated human specimens. These elective sessions can be dispersed throughout an integrated preclinical curriculum, each focused on a different anatomic region. The tactile experience of working with fresh‐frozen specimens is superior to formalin‐preserved tissues and enhances the education of students interested in the surgical profession.
2020 Diameter of the Common Carotid Artery correlates with Age of Death in a multi‐campus, multi‐year study.
Authors: Bee Mary Tracy , Garmo Atalie , Chaney Adrienne , Barton Scott Thomas
Published: April 2020

Over the past six years, in a multi‐campus and multi‐year study, we have investigated the relationship of the size of the common carotid artery versus age of death. The common carotid is a paired artery, vitally conducting blood to the head and neck region. With a sample size over 130 human cadavers, we have identified a significant and positive correlation in the diameter of the common carotid arteries versus age of death (p=0.013). Human cadaver ages ranged from 24 to 102 years old. As the diameter of the common carotid artery has been associated with the occurrence and severity of strokes, this finding may provide insight into circulatory‐related procedures in the cervical region or may be indicative of a related genetic predisposition to a higher vascular flow.
Protective Effect of Diminazene Aceturate in Acid‐Induced Airway Obstruction
Authors: Liao Yan-Shin , Kuan Shin-Ping , Guevara Maria Valentina , Collins Emily , Atanasova Kalina , Dadural Joshua , Vogt Kevin , Schurmann Veronica , Reznikov Leah R.
Published: April 2020

Mild airway acidification occurs in several airway diseases, including cystic fibrosis and asthma. However, the role of airway acidification in airway pathology is controversial. Here, we studied mucus secretion in piglets forty‐eight hours following an intra‐airway acid challenge. Airway submucosal glands of acid‐challenged piglets exhibited faulty submucosal gland secretion, with a retainment of mucin 5B (MUC5B) in the submucosal gland and decreased concentrations of MUC5B in lung lavage fluid following cholinergic stimulation. Concomitantly, intrapulmonary airways were obstructed with glycoprotein rich material under both basal and methacholine‐stimulated conditions. Blocking detection of acid with the diminazene aceturate, a small molecule that inhibits the acid‐sensing ion channel (ASIC), partially restored submucosal gland function and mitigated acid‐induced airway obstruction. These findings suggest that transient airway acidification significantly impacts mucus secretion and highlight diminazene aceturate as an agent beneficial in alleviating some mucus defects.

Support or Funding Information

HL119560, 1OT2OD023854
Role of O‐GlcNAcylation in SEC24C function
Authors: Lamb Caitlin , Bisnett Brittany , Cox Nathan J. , Smith Tim , Boyce Michael
Published: April 2020

Coat protein complex II (COPII) mediates forward protein and lipid cargo trafficking from the endoplasmic reticulum via five protein components required for in vitro vesicle formation: SAR1, SEC13, SEC23, SEC24, and SEC31. While the structure and function of COPII proteins are well understood, we do not fully understand how COPII is regulated in response to environmental and cellular stimuli. We and others have found that O‐linked‐beta‐N‐acetylglucosamine (O‐GlcNAc), a dynamic single‐sugar modification added to serines and threonines of intracellular proteins, decorates many human COPII components, including all four SEC24 paralogs. Additionally, others have shown that COPII, and specifically SEC24, may play a role in the formation of the autophagosome for autophagy, a process in which the cell degrades its own components. However, the regulation of SEC24’s canonical and autophagic functions remains unclear. To determine the function of SEC24 O‐GlcNAcylation, we mapped O‐GlcNAc sites on SEC24C using mass spectrometry and created unglycosylatable serine or threonine to alanine mutations at each O‐GlcNAc site. We then used CRISPR‐Cas9 to create SEC24C−/− HEK 293T and HeLa cells, and reintroduced unglycosylatable SEC24C mutants. By comparing SEC24C−/− to control HeLa cells, we discovered a baseline difference between levels of autophagy. Furthermore, SEC24C−/− 293T cells may be more sensitive to ER stress than control cells are. With these differences between SEC24C −/− and control cells, we will next determine whether the differences persist when comparing the unglycosylatable mutants to wild type SEC24C, using Western blots and immunoprecipitation. This study will enhance our understanding of the role of O‐GlcNAcylation on SEC24C function and the interplay between canonical COPII function and autophagy.

Support or Funding Information

Research on this project in the Boyce Lab is supported by NIH grant 1R01GM117473.
Development of Functional, Contractile 3‐D Human Muscle Constructs for Studies of the Effects of Microgravity on Muscle Mass and Function.
Authors: Jones Samantha W. , Janvier Adam J. , Shigdar Shahjahan M. , Henstock James R. , McArdle Chris , Hoettges Kai , McArdle Anne , Jackson Malcolm J.
Published: April 2020

Major demographic changes are affecting modern societies, leading to rapidly increasing numbers of older people with relatively poor health and quality of life. The mechanisms underlying age‐related loss of muscle have not been fully evaluated, but we have previously demonstrated that muscle from ageing animals and humans show attenuated adaptations to exercise that compromises their ability to maintain muscle mass and function. We have evaluated key mechanisms underlying adaptations of skeletal muscle to exercise and demonstrated that the exercising muscle of young or adult animals and humans generates reactive oxygen species (ROS) that stimulate activation of specific transcription factors leading to increased generation of a number of protective and cytoprotective proteins. Such responses do not occur in muscle from older animals and man in response to exercise. Astronauts and animals exposed to microgravity also lose skeletal muscle mass and their muscles are also relatively unresponsive to aerobic or resistance training in microgravity. Within a call from the UK Space Agency for an upcoming national mission on board the International Space Station (ISS), we will utilise in vitro studies of 3‐D skeletal muscle constructs to determine whether an analogous failure of muscle adaptations to contractile activity occurs in muscle fibres exposed to microgravity as that which occurs in older people on earth. The aim of the current study was to develop appropriate 3‐D constructs which remained viable and contractile following field electrical stimulation in specifically designed, sealed hardware for space flight for up to 7 days. Initial experiments examined a variety of immortalised and primary murine myocytes which were found unsuitable due to lack of excitability and high oxygen consumption. Subsequent experiments have utilised immortalised human myocytes obtained from Dr. Vincent Mouly (Paris, France) which have been fabricated on 3D printed scaffolds encapsulated in a fibrin hydrogel solution (4×106 cells/construct) and polymerised at 37°C. Once matured the constructs were checked for contractility and the medium was replaced with Leibrovitz‐L15 medium (supplemented with 2 mg/mL 6‐aminocaproic acid) to minimise oxygen consumption while retaining viability. These constructs retain viability and contractility for up to 7 days and display significant adaptations to short periods of contractile activity in normal gravity. Specific hardware to maintain the constructs and permit electrical stimulation of contractions has been designed by Kayser Space Ltd., (Harwell, UK), and the experiment will fly to the ISS in 2021.

Support or Funding Information

Supported by the UK Space Agency
Ecm16 protein confers resistance against the DNA intercalator antibiotic echinomycin
Authors: Gade Priyanka R. , Erlandson Amanda , Mathews Irimpan I. , Chen Xi , Mera Paola , Kim Chu-Young
Published: April 2020

Echinomycin is a nonribosomal peptide antibiotic that acts by intercalating double‐stranded DNA. The echinomycin biosynthetic gene cluster of Streptomyces lasaliensis contains a gene of unverified function, ecm16. We show that expression of Ecm16 in the echinomycin‐sensitive E. coli K12 renders cells resistant to echinomycin. Additionally, we have determined the X‐ray crystal structure of Ecm16 at 2.0 Å resolution. Interestingly, the three‐dimensional structure of Ecm16 resembles that of UvrA, the DNA damage sensor protein from the prokaryotic nucleotide excision repair pathway. Ecm16, like UvrA, contains two nucleotide binding domains on a single polypeptide chain, and it can bind double‐stranded DNA. However, our results show that neither UvrA nor Ecm16 can complement each other’s function in vivo.

Support or Funding Information

The University of Texas System STARs Award
Evaluation of Hindlimb Arteriolar Vasodilation Evoked by Dynamic Muscle Contraction in Goto‐Kakizaki Rats Using in vivo X‐ray Microangiography
Authors: Sonobe Takashi , Tsuchimochi Hirotsugu , Pearson James T.
Published: April 2020

Vascular endothelial dysfunction is a well established comorbidity in diabetes mellitus. In peripheral resistance arteries, it has been reported that endothelium‐dependent hyperpolarization (EDH), which is an important factor to induce vasodilation in small arteries, is downregulated in diabetic animals. We hypothesized that downregulation of EDH inhibits exercise‐induced vasodilation in diabetic patients and causes exercise intolerance. To test this hypothesis, the objective of this study was to evaluate peripheral vascular function in response to muscle contraction and the contribution of EDH to vasodilation in anesthetized diabetic rats. Type 2 diabetic Goto‐Kakizaki (GK) rat and healthy Wistar rat were anesthetized by urethane + α‐chloralose, and then mechanically ventilated. Carotid artery and jugular vein were cannulated for monitoring blood pressure and administering pharmaceutical agents. By means of X‐ray angiography, the hindlimb vasculature was visualized by infusing iodine contrast agent via the catheter inserted into the femoral artery. Angiography was performed before and after rhythmic muscle contraction (0.5 s tetanic contraction + 0.5 s relaxation, repeated 30 times) evoked by sciatic nerve stimulation (40 Hz, 0.1 ms duration). Femoral blood flow was simultaneously monitored by a transit‐time ultrasonic flowmeter. To investigate the contribution of EDH to the changes in arterial diameter and vessel recruitment, angiography was repeated after an intravenous administration of small conductance and big conductance potassium channel inhibitors (50 μg/kg apamin+charybdotoxin), which are known to suppress the EDH‐dependent vasodilation.

We observed increases in arterial diameter (~100% increase) in most of hindlimb arteries after the muscle contraction except for the superficial artery and the saphenous artery. We also observed recruitment of small arteries (diameter less than 100 μm), which were not visualized at baseline condition. These vasodilative effects on the hindlimb arteries were similar in GK rat compared to Wistar rat. Increase in femoral blood flow at peak and decrease in vascular resistance at the nadir were also similar between the groups. Inhibition of potassium channels did not affect baseline diameter in arteries and the changes in vasodilation after muscle contraction in either Wistar or GK rats. In conclusion, effects of acute muscle contraction on the changes in arterial diameter and recruitment were preserved in the GK rat. EDH may not contribute to induce the peak response in arterial diameter after muscle contraction.

Support or Funding Information

This work was supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan [18K10882].
Myeloid‐Specific STAT4 Deletion Improves Glucose Homeostasis in Diet‐Induced Obesity
Authors: Hossain Sakib , D’silva Melinee , Nadler Jerry L.
Published: April 2020

The signal transducer and activator of transcription 4 (STAT4) is activated in response to IL‐12 and stimulates expression of genes necessary for T helper 1 cell differentiation. Recent evidence has suggested that STAT4 is involved in an inflammatory cascade leading to atherosclerosis and insulin resistance in mouse models. However, the role of STAT4 in myeloid cell functions has not been studied. IL‐12 and STAT4 is expressed in the pancreatic beta cell and therefore changes in activity could lead to altered glucose homeostasis as typically seen in type 2 diabetes (T2D). For this reason, elucidation of cell‐specific roles of STAT4 on glucose homeostasis is critical for treatment and prevention of T2D. Previous studies have shown that global STAT4 deletion significantly improves insulin signaling and glucose tolerance in high fat fed C57Bl6 mice. This study was undertaken to determine the role of myeloid specific STAT4 deletions on glucose homeostasis and islet function. We developed a novel mouse model with targeted deletion of STAT4 in myeloid cells. Here we show that high fat fed C57Bl6 mice with macrophage specific STAT4 deletion (STAT4 LysmCre) display improved glucose metabolism compared to control mice. After 16‐weeks, control mice gained an average of 24.5 grams while the STAT4 LysmCre cohort gained an average of 20 grams, indicating only moderate differences in weight gain. However, STAT4 LysmCre mice showed an improved glucose tolerance compared to control mice [AUCGlucose 68780±2820 vs. 56025±6687 (mg/dl)*min]. These results may suggest that STAT4 signaling in myeloid cells plays a key role in glucose homeostasis during high fat feeding. Further studies in progress will explore the role of myeloid STAT4 deletion on pancreatic beta cell function.

Support or Funding Information

This research is being funded by NIH grant 5R01HL142129‐02
Hidden cardiotoxicity of drugs ‐ paradigm change in cardiac safety testing in ischemic and comorbid conditions
Authors: Ferdinandy Peter
Published: April 2020

Unexpected cardiac adverse events are one of the leading causes of interruption of clinical trials and drug withdrawals. It has been shown that cardiovascular risk factors and comorbidities (such as aging, metabolic diseases, etc) and their medications (e.g. nitrates, antidiabetic drugs, statins, etc) may interfere with cardiac ischemic tolerance and molecular signaling of endogenous cardioprotection. Indeed certain drugs may exert adverse events on the diseased heart that is hidden in the healthy myocardium. Hidden cardiotoxic effects of drugs may occur due to (i) enhancement of unwanted signaling due to ischemia/reperfusion injury and/or the presence of risk factors and/or (ii) inhibition of cardioprotective signaling pathways, both of which may lead to ischemia‐related cell death and pro‐arrhythmic events. This led to novel concept of “hidden cardiotoxicity”, i.e. cardiotoxicity seen only in the diseased heart, i.e. ischemia/reperfusion injury and/or its major comorbidities (Ferdinandy et al, Eur Heart J, 2019). Hidden cardiotoxicity cannot be revealed by the routinely used cardiac safety testing methods in “healthy” test systems, moreover, the mechanism of hidden cardiotoxocity is largely unknown. Therefore, here we summarize the current knowledge on hidden cardiotoxicity and urge the need for development of novel cardiac safety testing platforms for early detection of yet “hidden” cardiotoxicity. We also show an example of rofecoxib a COX2 inhibitor with hidden cardiotoxic properties.

Support or Funding Information

This study was supported by the National Research, Development and Innovation Office of Hungary (NKFIH; NVKP‐16‐1‐2016‐0017 National Heart Program). The research was also financed by the Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary, within the framework of the Therapeutic development thematic programme of the Semmelweis University