bluuebunny/crossref_metadata_2025 huggingface dataset:
Graduate Education Trends in the Anatomical Sciences: Are anatomy PhDs nearing extinction or adapting to change?
Authors: Kaza Niroop , Wilson Adam B. , Singpurwala Darius J. , Brooks William S.
Published: April 2020

Introduction

Human anatomy is the cornerstone of health sciences education, yet literature over the past 50 years has demonstrated a severe and continued shortage of anatomy educators. Many have postulated that the factors responsible for the dwindling numbers of anatomists are multiple.

Aim

This study (1) assessed trends in doctorates awarded in anatomy and related fields within the U.S. over the past 48 years and (2) evaluated the modern graduate education‐training paradigm in the anatomical sciences.

Methods

Retrospective data relative to the anatomical sciences were compiled from the National Science Foundation (NSF) Survey of Earned Doctorates (SED), an annual census of research doctorates within the U.S. The number of doctorates in anatomy and related fields and the total number of doctorates by sex and race/ethnicity were plotted for trend analysis. Additionally, the number of currently active PhD anatomy training programs within U.S. allopathic & osteopathic medical schools were assessed. Curricula and major characteristics of each active program where evaluated through website searches and phone interviews with program directors. The IRB at UAB did not qualify this study as human subjects research.

Results

Since 1969, the number of PhDs awarded in Anatomy has declined, on average, by 3.3 graduates per year (Max = 163, 1982; Min = 9, 2017). Conversely, doctorates in Cell Biology, Developmental Biology, and Neuroscience have observed marked growth over the past 40 years at rates of 8.0, 4.3, and 31.3 graduates per year, respectively. Males have historically dominated the anatomical fields, but more females received doctorates than males from 2009–2016. The proportion of PhDs in the anatomical sciences awarded to underrepresented minorities (URMs) has steadily increased with an all‐time high of 11.1% in 2015, but still lags behind Science & Engineering (14.1%). The proportion of graduates entering employment directly after graduation declined from a high of 55% in 1969 to the current rate of 21% and is inversely proportional to the number of graduates entering additional training positions, which is 78% currently. As of August 2019, 21 active doctoral programs in anatomy were identified from among 187 institutions affiliated with a medical school. PhD programs were categorized as either Anatomy Education (N=8), Classic Anatomy (N=8), or Anthropology/Evolutionary Anatomy (N=5) programs based on coursework in anatomical science disciplines and education/teaching‐based curricula.

Discussion & Conclusion

Declining numbers of anatomy PhD graduates and misalignment between training expectations and job market needs has contributed significantly to the shortage of anatomists. A concerted effort by all stakeholders to increase the diversity and number of anatomy educators is necessary to ensure the future health of the profession.
Biophysical Folding Sensors for the Detection of Misfolded Proteins by Hsp70
Authors: Karunnayake Chamithi , Page Richard C.
Published: April 2020

Chaperoned ubiquitination complexes play a defining role in responding to chemical and pathological stresses and an understanding of how these complexes are regulated on a molecular level offers the opportunity to identify new avenues of treatment for a wide range of diseases. Hsp70 participates in chaperoned ubiquitination by recruiting and attempting to refold misfolded protein “clients” in an ATP‐dependent process. Misfolded clients interact with a binding cleft within the substrate binding domain (SBD) formed by a beta‐sheet subdomain (SBD‐beta) and helical lid subdomain. Understanding how Hsp70 recruits clients and positions clients relative to the E3 ligase CHIP is critical for understanding the core interactions between CHIP and Hsp70 that dictate the outcome of chaperoned ubiquitination. Herein we describe a set of folding sensors representing a spectrum of folded and unfolded states. When utilized in ubiquitination reactions this system provides a tunable folded/unfolded client with an Hsp70‐SBD binding site that provides control over the distance from the model unfolded client to the E2~Ub active site bound to the E3 ligase CHIP, thereby providing a measure of how folding state dictates degree of ubiquitination.

Support or Funding Information

NIGMS R35 GM128595
Mitochondrial Protein Hyperacetylation in Rodents with Doxorubicin‐Induced Cardiac Dysfunction
Authors: Tomczyk Mateusz M. , Cheung Kyle G. , Xiang Bo , Agarwal Prasoon , Kereliuk Stephanie M. , Wilkins John A. , Dolinsky Vernon W.
Published: April 2020

Objective

Doxorubicin (DOX) is an effective chemotherapeutic but has dose‐dependent cardiotoxic effects that limits its use in pediatric patients. Previous studies in our lab showed that DOX decreases expression of the mitochondrial lysine deacetylase SIRT3 and mitochondrial phospholipid cardiolipin (CL) in the mouse heart. We hypothesize that DOX impairs cardiac function as a consequence of reduced SIRT3 expression resulting in increased acetylation of mitochondrial proteins involved in cardiac energy and oxidative stress homeostasis.

Methods

C57BL6 mice were given DOX (8.0mg/kg body weight) or saline control injections for 4 weeks. Transthoracic echocardiography was performed on all mice (n=10 per group) and parameters of cardiac structure, systolic and diastolic function were measured. Cardiac mitochondria were isolated from saline and DOX mice and an anti‐acetylated lysine antibody was used to enrich for tryptic digested peptides containing acetylated lysines followed by mass spectroscopy analysis (n=6).

Results

DOX treated mice exhibit decreased left ventricular posterior wall thickness (P<0.05), increased intraventricular relaxation time and reduced ejection fraction, compared to controls (P<0.05). Quantitative PCR of cardiolipin biosynthesis genes revealed Ptpmt1 and Crls1 gene expression was reduced by half with DOX compared to controls (p<0.01). In DOX treated mice, we observed an enrichment of cardiac mitochondrial acetylated peptides of proteins involved in metabolic, CL remodelling processes and oxidative stress resistance (eg. ATP5F1A, TFEα, SOD2, P<0.05, >2‐fold increase). MitoSOX staining of human induced pluripotent stem cell derived cardiomyocytes revealed an increase (2.5‐fold, p<0.001) in reactive oxygen species production with DOX treatment which was attenuated with adenoviral SIRT3 overexpression.

Conclusion

Alterations to the mitochondrial acetylome may be responsible for DOX‐induced cardiac dysfunction in mice. SIRT3 overexpression attenuates production of reactive oxygen species in human derived cardiomyocytes and SIRT3 may prevent cardiotoxic effects of DOX.
Self‐powered bioluminescence in a marine tube worm
Authors: De Meulenaere Evelien , Puzzanghera Christina , Deheyn Dimitri D.
Published: April 2020

An increasing number of luminous organisms are being found in the ocean, presenting opportunities to discover new biochemical reactions associated with light production, and thus new bioreporter systems for biotechnology and biomedicine. The marine parchment tube worm Chaetopterus sp. is of particular interest because it secretes a luminous mucus that glows for a long time (hours, possibly days), although in seawater and isolated from any continuous metabolic support. This contrasts with the short flashes, or the bacterial, metabolically driven glow that are more typical for naturally observed bioluminescence. We have recently found that the light production in this mucus is activated by Fe2+ and that the long‐lasting glow may therefore be supported by the proven presence of the iron storage protein ferritin.

We have also observed a temporary increase in the bioluminescence after exposure of the mucus to blue light specifically, which is capable of triggering Fe2+ release from ferritin through a photo‐reduction process. Since the mucus bioluminesces in the blue region of visible light, we are investigating whether it could be powering its own light production.

Support or Funding Information

This research is supported by the Air Force Office of Scientific Research (grant # FA955017 0189 to D. D. Deheyn).
Neutrophil Elastase Facilitates Cancer Cell Motility via Induction of Akt Signaling Pathway
Authors: Ardi Veronica C. , Deryugina Elena I. , Quigley James P.
Published: April 2020

Cancer cells create a microenvironment favoring growth of the primary tumor and cancer dissemination. We and others have demonstrated that immune cells recruited to a primary tumor actively contribute to pro‐tumorigenic and pro‐metastatic conditions. Neutrophils, one of the first inflammatory leukocytes infiltrating a developing tumor, facilitate intratumoral angiogenesis and tumor cell dissemination via newly formed blood vessels. Upon arrival to the site of primary tumor, the recruited neutrophils release their potent enzymes, including neutrophil MMP‐9, a structurally unique metalloproteinase strongly inducing angiogenesis. Herein, we demonstrate that a neutrophil‐derived serine protease, neutrophil elastase (NE), enhances cancer cell motility, which is essential for efficient cancer cell dissemination from primary tumors to secondary sites. We have shown that the treatment of tumor cells with purified NE significantly increased their migration in a chemotactic Transwell assay. Our data show significant (by 2‐fold) increase in migration rates of NE‐treated over non‐treated control for both epidermoid HEp3 and prostate PC3 carcinoma cells. We found no direct effect of NE treatment on cell proliferation, thus excluding cell division from the causes of NE‐mediated increase in rates of cell migration. However, we found that NE treatment induced activation of Akt, a protein kinase that regulates many important cell functions. We have demonstrated that NE‐mediated activation of Akt was sensitive to the specific NE inhibitor, α1PI, implicating the catalytic activity of NE in the enhancement of cell migration. Furthermore, NE‐induced cell migration was completely abrogated if tumor cells were pre‐treated with inhibitors of Akt signaling, Dasatinib and Wortmannin, before NE treatment. Together, our findings indicate that neutrophil‐specific protease, NE, is a potent inducer of tumor cell migration, and that Akt‐activation signaling constitutes an essential mechanism for NE‐enhancing effects on tumor cell migration.
Investigation of Cytochrome P450 2J2 Ligand Binding Modes
Authors: Motl Nicole , Scott Emily E
Published: April 2020

Human cytochrome P450 2J2 (CYP2J2) plays a multifaceted role in cardioprotection and xenobiotic metabolism. CYP2J2 oxidizes endogenous polyunsaturated fatty acids P450s, including arachidonic acid and linoleic acid, to various epoxyeicosatrienoic acids (EETs). These EETs are implicated in vasodilation and protection against hypoxia‐reperfusion, but also promote tumor growth and metastasis, where inhibition may be a clinical advantage. Recently, it has been reported that CYP2J2 is inhibited by anti‐hypertensive drugs including manidipine, azelnidipine, and telmisartan. In its drug metabolism role, CYP2J2 metabolizes numerous xenobiotics including anti‐histamine and anti‐cancer agents. While kinetic and metabolic parameters for many of these endogenous and xenobiotic compounds have been reported, it remains to be elucidated how such a wide variety of ligands are accommodated by the CYP2J2 active site. This study determined the binding modes of a catalog of CYP2J2 ligands, including substrates, inhibitors, xenobiotics, and a broad panel of azole containing compounds for the purpose of better understanding their interactions with CYP2J2. The binding modes and affinities were determined by monitoring the spectral shift of the P450 heme Soret absorbance, which is indicative of perturbation of the heme environment. Such spectral shift indicated varying responses: displacement of the heme‐coordinated water (a type‐I binding mode), compound coordination to the heme iron (a type‐II binding mode), or no spectral shift. As the latter result can be consistent with no binding or a binding in the active site in a way that does not perturb the heme center, these latter compounds were investigated via a luminescence‐based inhibition assay to verify their interaction with CYP2J2. Overall, this study aides in improving our understating of different ligand scaffolds that are compatible with CYP2J2 binding to support the development of therapeutics and inhibitors of CYP2J2.

Support or Funding Information

National Institutes of Health Grant R37 GM076343
An RNA‐Seq technology that assesses the complete transcriptome from miRNA to mRNA and exhibits exemplary performance on FFPE tissue samples
Authors: Shulewitz Mark , Okino Steven , Kong Michelle , Patel Pranav , Bibillo Arek , Ma Jason , Wang Yan
Published: April 2020

RNA‐Seq is a powerful technique that measures the presence and abundance of RNAs in a cellular sample. There are two general RNA‐Seq approaches. Standard RNA‐Seq processes long RNAs, typically larger than 200 bases. Small RNA‐Seq assesses RNAs shorter than 200 bases and often targets miRNAs in the 20–26 base range. Neither of these technologies captures all RNAs in a sample and allow for interrogation of the entire transcriptome. Here we present a new RNA‐Seq technology that leverages an engineered reverse transcriptase that enables the analysis of RNAs of all sizes from microRNAs (miRNAs) to messenger RNAs (mRNAs). In addition, because the new RNA‐Seq approach processes short RNAs very efficiently, it has exemplary performance analyzing highly degraded RNA samples. We observe over 30‐fold improvement in processing FFPE RNA samples compared to other RNA‐Seq kits as well as better gene detection sensitivity and accuracy. We envision that this “Complete RNA‐Seq” technology will enable researchers to develop new insights into the complex behavior and interconnection of the entire transcriptome, analyze standard and challenging RNA samples and facilitate the discovery of new biomarkers.
Acute Intermittent Hypoxia Results in Baroreflex Resetting to Higher Blood Pressures in Young Men but Not Young Women
Authors: Limberg Jacqueline K. , Jacob Dain W. , Baker Sarah E. , Ott Elizabeth P. , Ivie Clayton L. , Harper Jennifer L. , Acevedo Camila Manrique
Published: April 2020

Background

Repetitive hypoxic apneas, similar to what is observed in sleep apnea, result in resetting of the sympathetic baroreflex to higher blood pressures (BP) in humans. This resetting of the baroreflex is associated with increased BP in preclinical models of sleep apnea (chronic intermittent hypoxia, CIH); however, the majority of our understanding comes from men. There are data to suggest female rats exposed to CIH do not develop high BP. With this, we sought to examine potential sex‐related differences in the effect of intermittent hypoxia (IH) on the sympathetic baroreflex. We hypothesized young men would exhibit resetting of the baroreflex to higher BP following IH, with no change in young women.

Methods

BP and muscle sympathetic nerve activity (MSNA) were assessed in 17 men (30±1 yrs) and 10 women (28±1 yrs) during quiet normoxic rest prior to and following 30‐min of experimental IH [30‐s hypoxic exposures (0.05 FiO2) alternating with 90‐s room air breathing] resulting in 15 hypoxic events (91.7±0.5 % SpO2). Sympathetic baroreflex sensitivity was evaluated using the modified Oxford technique before and after 30‐min of IH.

Results

Diastolic BP increased in men following IH (71±2 to 74±2 mmHg; p<0.01) with no change in women (75±3 to 75±3 mmHg; p=0.97). Sympathetic baroreflex sensitivity did not change following IH in either group (Men: −1.8±0.2 to −1.9±0.3; Women: −1.6±0.3 to −2.1± 0.5 bursts/100 heart beats/mmHg; Main effect of IH, p=0.14; Interaction of group and IH, p=0.28).

Conclusion

Acute IH resets the sympathetic baroreflex to higher BP in young men, with no change in young women. Baroreflex sensitivity slopes were unchanged in either group following IH. Our results support sex differences in the effect of IH on sympathetic control of BP and show that young women are protected from the BP‐raising effects of IH. These data enhance our understanding of sex‐specific mechanisms that may contribute to the development of hypertension in sleep apnea.

Support or Funding Information

NIH HL130339, Mayo Clinic Center for Biomedical Discovery
Protein splicing and tryptophan fluorescence of salt‐dependent inteins from Haloquadratum walsbyi
Authors: Yalala Vaishnavi , Chavez Angel , Lynch Abigeal K. , Mills Kenneth V.
Published: April 2020

Protein splicing is a self‐catalyzed reaction by which an intein, an intervening polypeptide, catalyzes its own removal from exteins, the flanking polypeptides, as well as ligation of the exteins. We are studying the inteins that interrupt a cell division control protein in the halophilic archaea Haloquadratum walsbyi. Halophiles like H. walsbyi are able to survive in water with high concentrations of sodium chloride. H. walsbyi has four inteins that interrupt the cdc21 gene, and we have studied the two inteins (Hwa cdc21A and Hwa cdc21D) that lack an intervening homing endonuclease domain. We are studying the splicing activity under various salt concentrations for the inteins both separately and when expressed as part of a single fusion protein. Hwa cdc21A splices optimally at 2.5 M NaCl, while Hwa cdc21D splices optimally at 1.5 M NaCl. We also are using native Trp fluorescence to study the influence of salt concentration on the proper folding of the inteins.

Support or Funding Information

This work was supported by NSF grant MCB‐1517138, a Henry Dreyfus Teacher‐Scholar Award, and NIH Grant 1R15GM132817‐01.
Effects of activity and selection on mouse femoral strength
Authors: Ona Jack , Castaneda Melanie , Garland Theodore , Horner Angela
Published: April 2020

Bone is a hard tissue providing mechanical support to vertebrates and is influenced by both genetic and environmental factors. Stress from mechanical loading causes micro‐fractures in bones, triggering dynamic bone remodeling by shifting the balance of absorption and formation to net formation. Prolonged inactivity can lead to bone dystrophy, which reduces bone strength. In order to assess how genetic and environmental factors can independently affect the structure of bone, we used mice from lines that have been selectively bred for high levels of voluntary wheel running (High Runner, or HR lines) that had access to cage wheels throughout their lives. Femora from HR and control mice were dissected and defleshed in order to perform material strength testing on the bone. A custom‐made rig was designed to hold the femora in place as a materials testing system (MTS) was used to perform a three‐point bending test at the mid‐diaphysis of each individual femur. We recorded morphometric data prior to testing and the modulus (stiffness) and failure stress were calculated from the MTS tests. HR mice have thicker femora, but are lighter in density, than control mice. HR mice with wheel access had longer than average femora, but stiffness and failure strength was similar among all groups.
Are you copying me? Leveraging Expert Visual Scan Path to Transmit Visual Literacy In Novice Histology Students
Authors: Hirt Lisa , Leonard Carly , Lee Lisa M.J.
Published: April 2020

Histology requires visual literacy, the recognition and interpretation of tissue organization in the body, but this is a challenging skill for novice students to acquire. Developing pattern recognition requires expert guidance and exposure to diverse visual objects, which is limited by minimal contact hours in health sciences curricula. Using eye tracking, visual literacy is interpreted through visual scan paths. Novice students demonstrate improved visual scan paths and recognition of simple tissues after feed forward training (FFT) of an expert’s scan path, but this has yet to be tested in more complex tissues. The aim of the current IRB approved (#19‐1820) study is to apply FFT to complex histological tissues. Evidence suggests a high mental rotation test (MRT) score, ability to cognitively rotate and manipulate 2D objects, correlates with accelerated learning of visual objects. To account to MRT differences, this study evenly distributed participants into two groups, based on their MRT scores; while sex was evenly distributed. Both groups were a part of a two‐phase study. In phase one, group A received FFT on a series of 3 gastrointestinal (GI) organ micrographs showing: areas where the expert’s eyes fixated, pathway the eyes traveled across the micrograph (scan path), without focusing on distractors (saccades), and the total distance their eyes traveled across the image (total scan path distance). Group B viewed the same training micrographs, but FFT was omitted. Both groups were tested on visual identification of the 3 GI organs while their eye movements were captured by an eye‐tracking device, Gazepoint. Phase two was identical to phase one, except, students were exposed to the lymphatic system and group B received the FFT. 3,150 data points (N = 25) were collected and analyzed with MATLAB and SPSS. Neither group had a significant difference in scan path distance (A: p = 0.96; B: p = 0.83), or average fixation numbers (A: p = 0.25; B: p = 0.70) when analyzed individually. At phase two, both groups answered significantly faster (A: p < 0.01; B: p = 0.04) and their mean saccade length reduced (A: p = 0.01; B: p = 0.02). One‐way ANOVA on phase one data revealed female participants in both groups exhibiting less proficiency in visual search patterns with a significantly higher average fixation number (p < 0.01) and total scan path distance (p < 0.001) compared to their male counterparts. A linear regression revealed no correlation between the MRT and tissue identification accuracy (r = 0.35, p = 0.15), suggesting a high MRT may not be related to success in histology. Our data suggests watching an expert’s scan path improves visual literacy, and, for the first time, that there is a sex difference in histological search strategies. Future studies should examine if amount of time examining tissue specific areas of interest predict accuracy in testing.
Misoprostol attenuates hypoxia‐induced neonatal cardiomyocyte proliferation through Bnip3 and perinuclear calcium signaling
Authors: Gordon Joseph W. , Martens Matthew D. , Field Jared T. , Chapman Donald , Day Chelsea , Keijzer Richard , West Adrian R. , Ivanco Tammy L.
Published: April 2020

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. Although the molecular mechanisms are still unknown, metabolic stressors resulting from neonatal hypoxia drive aberrant glycolysis and cardiomyocyte proliferation, which may be initially adaptive, but ultimately can program the heart for early life pathological remodeling. Recent evidence suggests the prostaglandin E1 analogue misoprostol is cytoprotective in the hypoxia‐exposed neonatal heart by promoting the alternative splicing of Bnip3, resulting in the generation of an isoform lacking the third exon (Bnip3ΔExon3) known as small Nip (sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary ventricular neonatal cardiomyocytes and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In cultured myocytes, molecular markers and fluorescent indicators of proliferation, such as cylcin‐D1 and PHH3, were significantly elevated with hypoxia exposure and were attenuated with misoprostol treatment (p<0.01). Moreover, hypoxia reduced mitochondrial respiration and increased cytosolic production of NADH, which were restored by misoprostol treatment. In addition, both sNip and misoprostol promoted cardiomyocyte maturation by increasing nuclear calcium accumulation, and drove the nuclear localization of NFATc3 (p<0.01). Gene expression studies identified that misoprostol and sNip reduced the expression of the proliferative MEF2C‐myocardin‐BMP10 pathway, while favoring expression of the cardiac maturation factors BMP2 and MEF2A (p<0.01). Furthermore, an siRNA targeting sNip restored hypoxia‐induced proliferation in misoprostol‐treated cells. Finally, we observed a significant increase in left ventricular nuclei number and PHH3 staining in hypoxia‐exposed rat pups, which were also attenuated with misoprostol treatment (p<0.05). These findings suggest a novel mechanism by which misoprostol promotes Bnip3 alternative splicing and myocyte maturation, thereby blocking hypoxia‐induced neonatal cardiomyocyte proliferation.

Support or Funding Information

This work is supported by the Natural Science and Engineering Research Council of Canada, the Heart and Stroke Foundation of Canada, and the Children’s Hospital Research Institute of Manitoba.
Structural studies of human Fis1 reveals a dynamic region important for Drp1 recruitment and mitochondrial fission
Authors: Hill Blake , Enger John , Harwig Megan , Ihenacho Ugochukwu , Widlansky Michael
Published: April 2020

Fission protein 1 (Fis1) and dynamin‐related protein 1 (Drp1) were initially described as being evolutionarily conserved for mitochondrial fission, yet the role of Fis1 in human fission is unclear and disputed by many. In budding yeast where Fis1 helps to recruit Drp1 from the cytoplasm to mitochondria for fission, an N‐terminal “arm” of Fis1 is required for function. The yeast Fis1 arm interacts intramolecularly with a conserved surface that governs in vitro interactions with yeast Drp1. In human Fis1, NMR and x‐ray structures show a different arm conformation and its importance for human Drp1 recruitment is unknown. Here, we use MD simulations and comparisons to experimental NMR chemical shifts to show the human Fis1 arm can adopt an intramolecular conformation akin to that observed with the yeast molecule. This finding is further supported through intrinsic tryptophan fluorescence and NMR experiments on human Fis1 with and without the arm. Using NMR, the human Fis1 arm is also observed to be sensitive to environmental changes. The importance of these findings are revealed in cellular studies where the removal of the hFis1 arm reduces Drp1 recruitment and mitochondrial fission suggesting an important role for Fis1 in human mitochondrial fission.

Support or Funding Information

This project was supported by the following National Institutes of Health grants: R01GM067180 (to RBH), and R01HL128240 (to MEW).
Role of Transient Receptor Potential Ankyrin‐1 (TRPA1) in Endothelial‐Specific Effects of Diverse Aldehydes
Authors: Conklin Daniel , Jin Lexiao
Published: April 2020

Introduction

Structurally diverse aldehydes are found in abundance in the environment including in foods, beverages and tobacco‐derived aerosols. Some aldehydes are associated with cardiovascular disease risk especially in cigarettes smokers, however, the detailed mechanisms by which aldehydes influence cardiovascular health and disease are incompletely known.

Objective

Because reactive aldehydes, in general, are considered instigators of endothelium dysfunction, we explored the direct effects of a diverse set of aldehydes in isolated murine blood vessels, i.e., aorta and superior mesenteric artery (SMA).

Methods

Aldehydes: acetaldehyde (AA); formaldehyde (FA); cinnamaldehyde (CA); crotonaldehyde (CR); and a TRPA1 agonist, allyl isothiocyanate (AITC, mustard oil), were tested for vascular effects in isolated murine aorta and SMA using isometric myography.

Results

All aldehydes induced endothelium‐dependent relaxation in SMA. AA (1–100 mM), FA (30–1,500 μM), CA (30–300 μM) and CR (1–100 μM) strongly and reversibly relaxed (>75% relaxation) agonist‐induced contractions. High levels of FA (1.2–1.6 mM) and CR (>300 μM) irreversibly altered vascular function in aorta and SMA. To identify the mechanism of aldehyde‐induced relaxations, isolated blood vessels were pre‐contracted with phenylephrine (PE), U46,619 (thromboxane A2 analog), or 60 mM K+ (High K) and then exposed to AA, FA, CA, CR or AITC with intact or mechanically‐impaired endothelium (air‐perfused) as well as in the absence or presence of: 1) Nω‐Nitro‐L‐arginine methyl ester hydrochloride (L‐NAME); 2) an inhibitor of guanylyl cyclase (ODQ); and, 3) a TRPA1 antagonist (A967079; 1 μM). The sensitivity (EC50) but not the efficacy (% decreased tension) of FA‐ and AA‐induced relaxations was dependent on the contractile agonist (i.e., PE < U46,619 ≤ High K) indicating multiple mechanisms. In PE pre‐contracted SMA, vasorelaxations at the lowest levels of AA, FA, CA, CR and AITC were inhibited significantly by mechanically‐disrupted endothelium, L‐NAME, ODQ, and by TRPA1 antagonist except for AA, which was TRPA1 independent. TRPA1 was co‐localized with isolectin immunofluorescent staining in SMA endothelium but not in aorta.

Conclusion

Because low levels (physiological) of several aldehydes (FA, CA, CR) induce reversible and efficacious vasorelaxations via TRPA1 channels, we propose that endogenous and exogenous sources of these aldehydes may significantly impact gut (mesenteric) blood flow to augment postprandial hyperemia in aid of digestion and nutrient uptake.

Support or Funding Information

Research funding from the National Institutes of Health: GM127607, HL122676, U54HL120163.
Daily Intermittent Fasting in Mice Enhances Morphine Induced Anti‐Nociception while Mitigating Multiple Side Effects
Authors: Duron David I. , Streicher John
Published: April 2020

The opioid epidemic has continued to plague the United States with high levels of abuse and poor quality of life for chronic pain patients requiring continuous use of opioids. Many potential solutions have been proposed to mitigate this epidemic including new medications to reduce the abuse and side effect profiles of opioids; however, new medications are plagued by low efficacy, abuse potential, and toxicities. These limitations suggest that non‐pharmacological interventions could be more efficacious with low side effect profiles. Intermittent fasting (IF) is a time restriction diet which has recently grown in popularity over the past decade in the prevention and mitigation of a variety of pathological states. Numerous animal and human studies have shown the benefits of IF in these disease states, but not in opioid treatment paradigms. We thus subjected male and female CD1 mice to 18‐hour fasting intervals followed by 6‐hour feed periods with standard chow for 1 week. Mice which underwent this diet displayed an enhanced anti‐nociceptive response to systemic morphine both in efficacy and duration using thermal tail flick and post‐operative paw incision pain models. While showing enhanced anti‐nociception, IF mice also demonstrated a reduction in the development of anti‐nociceptive tolerance using the thermal tail flick assay paired with daily systemic morphine treatment. We also found a reduction in the development of opioid induced constipation using both fecal mass and fecal pellet count in IF mice. Seeking a mechanism for our behavioral findings, we performed 35S‐GTPγS assays to assess the functionality of mu opioid receptors within the spinal cord and periaqueductal grey (PAG) tissues from IF mice with and without chronic morphine treatment. The resulting DAMGO concentration‐response curves demonstrated an increased efficacy of mu opioid receptors within the spinal cord in IF mice and an absence of the development of tolerance within the PAG in IF mice, suggesting site‐specific receptor mechanisms for the behavioral effects we observed. These data suggest that a daily IF diet may improve the therapeutic index of acute and chronic opioid therapies for pain patients in the clinic, both in improved analgesia and reduced tolerance and constipation.
The Role of Glutamate in Maintaining Muscle Spindle Afferent Excitability in Adult Mice
Authors: Ortiz Serena , Kim Enoch , Than Kimberly , Bustos Cebrina , Chu Sarah , Klier Nikola , Occiano Alyssa , Salazar Alexandra , Villegas Natanya , Wilkinson Katherine A.
Published: April 2020

Group Ia and II muscle spindle afferents are sensory neurons critical to detecting muscle movement and length, which is important in proprioception and motor control. Glutamate is released by synaptic‐like vesicles (SLVs) at muscle spindle afferent nerve endings and exogenous glutamate increases sensory neuron firing rate during stretch (Bewick, et al., 2005). However, the activity of individual muscle spindle afferents was not measured, allowing for the possibility that this increased firing also included other sensory neurons known to have glutamate receptors, like nociceptors. Here we test the hypothesis that glutamate increases the firing rate of individual muscle spindle afferents. We isolated the extensor digitorum longus muscle and the sciatic nerve from adult mice (8–12 wks old) and recorded the activity of individual muscle spindle afferents during exogenous glutamate treatment. The muscle‐nerve preparation was perfused in an oxygenated synthetic interstitial fluid tissue bath, an extracellular recording electrode was placed on the nerve to measure the activity of individual muscle spindle afferents, and a battery of 4s ramp‐and‐hold stretches were performed on the muscle. Glutamate addition led to a significant increase in muscle spindle afferent firing rate during stretch (1mM; n=12, 18.8%). We also decreased glutamate release by blocking the vesicular glutamate transporter 1 (VGLUT1) with the inhibitor xanthurenic acid (XA). We found a significant decrease in muscle spindle afferent firing rates (3mM; n=17, −39%), with 5 afferents exhibiting a complete absence of firing. However, we noticed heterogeneity in muscle spindle afferent responses to glutamate and XA treatment, with some afferents not responding at all to the drug treatments. To confirm our pharmacological findings, we obtained a transgenic mouse line lacking a single copy of vesicular glutamate transporter 1 (VGLUT1; B6.129X1‐Sic17a7tm1Edw/MmcD; Fremeau, 2004). We observed two muscle spindle afferents responding normally to stretch, while another two failed to sustain firing at the end of stretch, something that is only rarely seen in wild type animals. This heterogeneity may be due to differences in VGLUT1 protein levels in individual afferents. We are currently increasing our sample size, but our preliminary results support our hypothesis that glutamate is critical to maintaining muscle spindle afferent firing during maintained stretch. These studies will lead to a better understanding of how muscle spindle afferent stretch sensitivity is regulated.

Support or Funding Information

This work was supported by NIH SC3 GM127195 (KAW) and NIH R25 GM071381 (SO and NV).
Mechanisms of Toll‐like Receptor (TLR) 4 Mediated Pro‐inflammatory Cytokine Production in Human Pulmonary Microvascular Endothelial Cells
Authors: Nelin Leif Daniel , Pool Caitlyn , Jin Yi , Liu Yusen
Published: April 2020

Endothelial cell apoptosis is an early event in acute lung injury (ALI) leading to acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia (BPD). An early pathogenic feature of ALI is inflammation‐induced endothelial cell death. We have previously shown that cytomix (lipopolysaccharide (LPS), interleukin (IL)‐1β, interferon‐γ, and tumor necrosis factor‐α), but not LPS alone, results in robust cell death in human pulmonary microvascular endothelial cells (hPMVEC). We hypothesized that the inflammatory cascade associated with cytomix treatment in hPMVEC would result in the release of HMGB1, a damage‐associated molecular pattern (DAMP) that interacts with TLR4, and thereby enhances TLR4 mediated production of pro‐inflammatory cytokines. Treatment of hPMVEC with cytomix for 24 hours resulted in a robust induction (~80‐fold, p<0.005) of HMGB1 protein levels in the media compared to controls. While, there was only a small and statistically non‐significant increase in the HMGB1 protein levels in the cells following cytomix treatment compared with controls. To assess the role of TLR4 in the inflammatory response of hPMVEC following cytomix treatment, we utilized TAK‐242, a TLR4 antagonist. Cytomix treatment of hPMVEC resulted in nearly 5‐fold greater cleaved caspase‐1 protein levels than in control hPMVEC. This cytomix‐induced increase in cleaved caspase‐1 was prevented by the addition of TAK‐242 to the media. Cytomix treatment of hPMVEC also resulted in a robust induction of both pro‐IL‐1β and cleaved IL‐1β protein levels compared to controls. However, the cytomix‐induced pro‐IL‐1β and cleaved IL‐1β protein levels were significantly attenuated when the hPMVEC were also treated with TAK‐242. Our results demonstrate that treatment with cytomix results in substantial release of HMGB1. Furthermore, cytomix treatment results in robust production of the TLR4‐mediated pro‐inflammatory cytokine IL‐1β. We speculate that the release of damage‐associated molecular patterns (DAMPs), such as HMGB1, may be crucial in the innate immune response of hPMVECs. These findings may have implications for the pathogenesis of ALI in inflammatory lung diseases.
The Role of Metadherin in Inflammatory Breast Cancer Progression
Authors: Ortiz-Soto Gabriela , Suárez-Arroyo Ivette J. , Martínez-Montemayor Michelle M.
Published: April 2020

Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer where patients have a 43% increased risk of death compared to women with non‐IBC. The lethality of IBC originates from its nature of invading the vascular and lymphatic systems, absence of a typical tumor mass, and the generation of tumor emboli. The arising challenges in IBC include the accurate and early diagnosis and the development of effective targeted therapies. A previous study investigated potential biomarkers involved in tumor spheroid integrity by characterizing IBC’s cell surface proteome. Plasma membrane proteins were identified via “stable isotope labeling with amino acids in cell culture” (SILAC) with subsequent mass spectrometry (MS)‐based quantitative proteomics analysis using the IBC cell line SUM‐149 vs. non‐ cancerous mammary epithelial cells, MCF10A. SILAC results showed overexpression of Metadherin (MTDH) in SUM‐149 cells (>2.0 fold change). Subsequently, we validated the overexpression of MTDH in SUM‐149 IBC cells, IBC tissues and in the emboli. MTDH is a cell adhesion molecule overexpressed in many cancer types, including breast cancer. MTDH promotes cancer progression by modulating various signaling pathways including NF‐kB, which is related to cell survival, proliferation, invasion and metastasis. Therefore, the aim of this study is to assess the functional role of MTDH in IBC progression. To elucidate the mechanism of action of MTDH in colony formation and tumor spheroid we silenced MTDH (CRISPR or shRNA) SUM‐149 and SUM‐190 IBC cells. We assessed the expression of MTDH by immunoblotting and immunofluorescence where we observed reduced MDTH levels. The number of colonies formed in SUM‐149 MTDH silenced cells was ~40% lower when compared to non‐silenced cells. Tumor spheroids of SUM‐149 and SUM‐190 MTDH silenced cells were smaller than non‐silenced cells. Aditionally, MTDH silencing results in a decrease in the phosphorylation of the p65 subunit of NF‐kB while no changes are observed in total protein abundance in SUM‐149 cells. We can conclude from our preliminary results that MTDH serves as a potential IBC target for IBC progression.

Support or Funding Information

This work was supported by NIH NIGMS #GM111171 (MMM), SGRP 2017‐00143 (MMM and GOS), NIMHD #MD007583 (MMM), GM103475 (UPR MMM), Title‐V‐PPOHA #P031M105050, Title‐V‐Cooperative #P031S130068 from the U.S. Dept. of Education and Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health #P20GM103475 (GOS).
Eicosapentaenoic Acid Increases Browning Markers in Subcutaneous Adipose Tissue and Primary Adipocytes from Wild Type and UCP‐1 Deficient Mice
Authors: Ramalingam Latha , Jayarathne Shasika , Pahlavani Mandana , Scoggin Shane , Moustaid-Moussa Naima
Published: April 2020

Objective

Uncoupling protein ‐1 (UCP‐1) plays a critical role in brown adipose tissue (BAT) thermogenesis as well as in white adipose tissue (WAT) browning, which primarily occurs in subcutaneous adipose tissue (SAT). We have previously reported that fish oil‐enriched with eicosapentaenoic acid (EPA) reduced obesity, and increased UCP‐1 in BAT of B6 mice fed high fat diets (HF). Furthermore, we and others demonstrated that these protective metabolic effects of fish oil or EPA in BAT were independent of UCP‐1. Our goal here is to determine if EPA exerts anti‐obesity effects in SAT, by increasing browning and thermogenic markers and whether these effects are mediated by UCP‐1.

Methods

Wild type (WT) and UCP‐1 knock‐out (KO) male mice starting at 6 weeks of age were maintained at thermoneutral environment and fed HF with or without 36g/kg of EPA‐enriched fish oil (800 mg/g) for 14 weeks. The thermoneutral environment was used to induce obesity in UCP‐1 KO mice. SAT was collected for histological, gene and protein analyses. Moreover, SAT was collected from chow diet‐fed WT and KO mice at ambient environment to prepare stroma vascular cells, which were differentiated into adipocytes, then treated with 100uM EPA for 48 hours and harvested for mRNA and protein analyses. Lastly, extracellular flux analyzer was used to measure the mitochondrial capacity and oxygen consumption in differentiated SAT adipocytes. Differences among groups were analyzed using one way ANOVA with significance at p<0.05..

Results

WT mice supplemented with EPA had similar body weight to WT mice fed HF. However, UCP1 KO mice fed HF had higher body weight than all other groups (p<0.05). Interestingly, WT and KO mice fed EPA had smaller SAT adipocytes compared to their respective HF control groups. mRNA levels of fibroblast growth factor‐21 (FGF‐21) and Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC1α) which are oxidative markers were not altered in WT groups. However, UCP‐1 KO mice fed had HF or EPA had lower levels of FGF21 and PGC1α compared to l HF WT mice p<0.05, with no differences between the KO genotype. EPA significantly increased gene expression of UCP‐2, UCP‐3 and FGF21 in differentiated SAT adipocytes from both WT and KO compared to control. KO mice fed HF had lower levels of browning markers such as transmembrane protein 26 (TMEM26) and T BOX ‐1 (TBX) in SAT compared to WT mice fed HF, while EPA rescued these only in KO mice, but not in WT mice. Lastly, only the spare respiratory capacity was improved with EPA in both WT and KO primary adipocytes. Additional mechanistic studies are currently underway to further dissect EPA effects on different fat depots in WT vs KO mice.

Support or Funding Information

Funding Sources

NIH/NCCIH grant #R15AT008879‐01A1 (N.M.M.)
Expression of Aquaporins AaAQP2 and AaAQP6 in the Osmoregulatory Organs of Mosquito Larvae (Aedes aegypti)
Authors: Guardian Elia Edith Grieco , Donini Andrew
Published: April 2020

The juvenile stages of the yellow fever mosquito, Aedes aegypti, are found in semi‐permanent water storage containers, septic tanks, and natural coastal rock pools. Changing climates and anthropogenic pollutants affect the ionic (salt) composition of these mosquito breeding sites which can influence the spread and distribution of vector populations. These habitats impose different challenges to the salt and water balance of the mosquitoes which is accomplished by regulation of salt and water transport across epithelial barriers. Aquaporins (AQPs) are transmembrane proteins first discovered as selective water channels and can be classified by their transport capabilities to other small solutes. Six aquaporin isoforms have been identified in A. aegypti mosquitoes, AaAQP1‐6, and here we investigated the protein localization and abundance levels of AaAQP2 and AaAQP6 in the osmoregulatory organs of the aquatic larvae reared in either ion‐poor water (IPW; reverse‐osmosis water), artificial freshwater (FW), or brackish water (BW; 30% seawater). Using custom antibodies with immunohistochemistry and western blotting it was found that AaAQP2‐like and AaAQP6‐like immunofluorescence was primarily intracellular in the Malpighian tubules (MTs) and gastric caeca (GC). Rearing salinity did not affect protein abundance of AaAQP2 in MTs, GC and hindgut which appeared as a putative dimer of ~ 45 to 50 kDa; however, in the hindgut of BW reared larvae an additional band of ~ 30 kDa was apparent which may represent the monomer. Western blots for AaAQP6 are currently being optimized. Results suggest that AaAQP2 and AaAQP6 are required in these osmoregulatory organs regardless of the salinity of the external environment during development. Furthermore, AaAQP2 may have additional functions in the hindgut of larvae that develop in higher salt environments.

Support or Funding Information

NSERC ‐ DISCOVERY RGPIN‐2018‐05841
Expression and purification of N‐terminally acetylated Ssa1 chaperone from Saccharomyces cerevisiae
Authors: Griffith Alijah A. , Holmes William
Published: April 2020

N‐terminal acetylation leads to proteomic diversity by constituting a cellular acetylome wherein proteins exhibit an alternate function and dynamic, allowing cells to react specifically to environmental stressors or cellular perturbation. N‐terminal acetylation is catalyzed by a collection of N‐terminal acetyltransferases (NATs), which transfer acetyl moieties from acetyl‐coenzyme A to α‐amino groups at specific N‐terminal residues. One theoretical target of N‐terminal acetylation is Ssa1, an Hsp70 class chaperone endogenous to Saccharomyces cerevisiae. Ssa1 works in tandem with its co‐chaperone Sis1 to restore the native conformations of aberrantly‐folded proteins through a transient ATP‐driven mechanism. The consequences of N‐terminal acetylation on Hsp70 function remain to be fully characterized. To further probe the functional and mechanistic changes associated with the N‐terminal acetylation of Hsp70, we developed an efficient, user‐friendly, one‐step purification of Ssa1 using previously constructed Protein‐A fusion yeast strains. Our results indicate that our method isolates high yields of pure acetylated and unacetylated Ssa1 without compromising enzymatic activity, allowing for future evaluation of the effects of N‐terminal acetylation on Hsp70 chaperone function and dynamic.

Support or Funding Information

Research supported by RI‐INBRE (#P20GM103430) from NIGMS, NIH.