bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Three‐Dimensional Drawing Activity to Learn the Dural Venous Sinuses
Authors: Shaia Jacqueline K. , Elzie Carrie
Published: April 2020

Background

The use of drawing as a pedagogical tool in anatomy has been shown to be effective at increasing knowledge and engaging students. Creating three‐dimensional drawings or models can be especially beneficial as students struggle to translate two‐dimensional images into three‐dimensional structures in order to comprehend the dynamic aspects of functional anatomy.

Objective

We developed a three‐dimensional drawing activity of the dural venous sinuses to increase student knowledge of anatomical structures and improve their understanding of the spatial orientation and drainage system.

Methods

Students were given a pre‐test, a lecture, put in pairs, given markers and white swim caps, asked to draw the sinuses on their partner with anatomical accuracy, and completed a post‐test. All pre and post‐tests were evaluated using a T‐test. Effectiveness of the activity was gauged using a modified Likert Scale and was evaluated based on mean and median values.

Results

Results from the post‐test revealed a significant increase in knowledge related to structure identification and drainage (p = 0.017). Further, students found drawing to be an effective way to learn anatomical structures and strongly agreed that translating a two‐dimensional picture into a three‐dimensional drawing improved understanding of anatomical orientation.

Conclusion

Overall, this was a quick, effective activity utilizing three‐dimensional drawing to solidify the understanding of the dural venous sinuses. This educational tool required minimal time, direction and supplies, making it an ideal intervention for both small groups and large lab settings in any anatomy or neuroanatomy course.
Identification and Characterization of the Regulation of N‐Methyl‐D‐Aspartate Receptor by the Protein Tyrosine Phosphatase Ptpmeg
Authors: Robertson Quayd , Hill Carlin , Hoss Grace , Murcia Luis , Ree Brandy
Published: April 2020

Cell death in neurodegenerative diseases such as Alzheimer’s or after traumatic brain injury is associated with over stimulation of the N‐methyl‐D‐aspartate receptor (NMDAR). NMDAR is a glutamate‐gated Ca2+ channel that normally plays an important role in memory formation, but whose hyperactivation in pathological conditions leads to cell death. The NMDA receptor is a known target of tyrosine phosphorylation in mammals and Drosophila. Ptpmeg, a protein tyrosine phosphatase found in Drosophila, is highly expressed in the brain. Here it is responsible for the proper formation and maintenance of axonal projections in the mushroom body, the center for olfactory learning and memory. Ptpmeg has two vertebrate homologs, PTPN3 and PTPN4. Like the Drosophila counterpart, PTPN4 is expressed in neurons. PTPN4 physically associates with a subunit of the NMDAR, NMDAR2B, in both cultured cells and in the brain. Because PTPN4 is expressed in the neurons where it associates with a protein known to play a role in neuron death, NMDAR, this leads to potential implications of PTPN4 having a role in neuron death, and thus neurodegenerative diseases. However, analysis of the molecular and phenotypic impact of this interaction is lacking. Drosophila are a convenient and inexpensive way to address this knowledge gap. Our initial findings revealed that Ptpmeg physically interacts with dNR1 in neural tissue. We subsequently confirmed that a loss of Ptpmeg results in a statistically significant increase in the phosphorylated form of the dNR1 subunit of NMDAR. Of note, absence of Ptpmeg produced a defect in programmed cell death in non‐neural tissue. Collectively, these data suggest a role for Ptpmeg in governing NMDAR activity associated with neuronal death during neurodegeneration.

Support or Funding Information

Arkansas College of Osteopathic Medicine
Extracellular Matrix Remodeling with Ageing Associates with Cardiac Functional and Structural Changes in a Sex‐Specific Manner
Authors: de Castro Brás Lisandra , Grilo Gabriel A. , Shaver Patti R. , Stoffel Hamilton J. , Morrow Caleb Anthony , Johnson Octavious T. , Iyer Rugmani Padmanabhan
Published: April 2020

Age‐related remodeling of the heart causes structural and functional changes in the left ventricle (LV) that are associated with a high index of morbidities and mortality worldwide. Some cardiac pathologies in the elderly population vary between genders revealing that cardiac remodeling during aging may be sex‐dependent. Herein, we analyzed the effects of cardiac aging in male and female C57Bl/6 mice in four age groups, 3, 6, 12, and 18 month old (n=6–12 animals/sex/age), to elucidate which age‐related characteristics of LV remodeling are sex‐specific. We focused particularly in parameters associated with age‐dependent remodeling of the LV extracellular matrix (ECM) that are involved in collagen metabolism. LV function and anatomical structure were assessed both by conventional echocardiography and strain analysis. We then measured ECM proteins that directly affect LV contractility and remodeling. All data were analyzed across ages and between sexes and were directly linked to LV functional changes. Echocardiography confirmed an age‐dependent decrease in chamber volumes and LV internal diameters, indicative of concentric remodeling. As in humans, animals displayed preserved ejection fraction with age. Notably, changes to chamber dimensions and volumes were temporally distinct between sexes. Complementary to the traditional echocardiography, speckle tracking echocardiography (STE) revealed that circumferential strain rate declined in 18 month old females, compared to younger animals, but not in males, suggesting STE as an earlier indicator for changes in cardiac function between sexes. Age‐dependent collagen deposition and expression in the endocardium did not differ between sexes; however, other factors involved in collagen metabolism were sex‐specific. Specifically, while decorin, osteopontin, Cthrc1, and Ddr1 expression were age‐dependent but sex‐independent, periostin, lysyl oxidase, and Mrc2 displayed age‐dependent and sex‐specific differences. Moreover, our data also suggest that with age males and females have distinct TGFβ signaling pathways. Overall, our results give evidence of sex‐specific molecular changes during physiological cardiac remodeling that associate with age‐dependent structural and functional dysfunction. These data highlight the importance of including sex‐differences analysis when studying cardiac aging.

Support or Funding Information

We acknowledge funding support from the East Carolina University and the American Heart Association [grant numbers 18AIREA33960311 and 19PRE34450066].
Obesity‐associated Hypoxia Contributes to Aberrant Methylation of Genes Implicated in Inflammation and vascular Function
Authors: Ali Mohamed M. , Naquiallah Dina , Hassan Chandra , Masrur Mario , Bianco Francesco M. , Frederick Patrice , Cristoforo Giulianotti , Gangemi Antonio , Phillips Shane A. , Mahmoud Abeer M.
Published: April 2020

Introduction

Obesity is a major risk factor for cardiovascular disease. We previously demonstrated an impaired vascular function in obese adults (OB). We now hypothesize a role of obesity‐associated hypoxia in disturbing the methylation/expression of genes involved in inflammation/vascular function. We also propose a mediating role of the hypoxia‐inducible factor, HIF1α and the DNA hydroxymethylase, TET1.

Methods

We obtained subcutaneous and visceral adipose tissue (AT) biopsies from bariatric patients (n=60; age: 36±7 yrs; BMI: 50.7±8.7 kg/m2) and non‐obese (NOB) adults having elective surgeries (n=30; age: 36±2 yrs; BMI: 25.8±1 kg/m2). AT‐isolated arterioles were tested for vasoreactivity in response to pressure gradients of Δ10‐Δ100 cmH2O. Arteriolar nitric oxide (NO) and reactive oxygen species (ROS) were measured. Protein expression of HIF1α and TET1 and methylation/expression of leptin, IL1β, IL6, IL8, IL17, CXCL5, TNF‐α, and IFNγ were measured in the AT.

Results

Flow‐induced dilation (FID) was 40–50% higher in NOB than OB adults across all pressure gradients (p<0.05). NO production was higher, and ROS generation was lower in OB arterioles compared to NOB. HIF1α and TET1 proteins were 2–4‐fold higher in OB compared to NOB adults and correlated negatively with arteriolar FID, NO, and brachial artery FID (r=0.82, 0.64, 0.91, respectively; p<0.001) and positively with ROS (r=−0.71, p<0.01). Global hydroxymethylation and adipocytokine promoter hypomethylation and increased expression were observed in OB compared to NOB.

Conclusion

Our results suggest that vascular dysfunction in OB adults may be attributed to aberrant DNA methylation and increased expression of adipocytokines. This conclusion was also supported by invitro mechanistic studies.

Support or Funding Information

This research was funded by the National Heart, Lung, and Blood Institute (NHLBI), grants K99HL140049 and 4 R00 HL140049‐03 (AM) and HL095701 (S.P.)
Confirmation of Allosteric Switches in Human Sirtuin 1 Using a Sirtuin Activating Compound
Authors: Nguyen Thi-Tina N. , Amaya Carina Araceli , Huynh Angelina , Lustig Brooke , Wang Ningkun
Published: April 2020

Sirtuin 1 (SIRT1) is a protein deacetylase that requires cosubstrate NAD+ for its enzymatic function. A favorable target for drug development, SIRT1 is involved in stress response, metabolism, and age‐related diseases. Sirtuin activating compounds (STACs) are molecules that bind to a domain in SIRT1’s N‐terminus called the SBD (STAC Binding Domain), and change SIRT1 activity. In particular, resveratrol, an allosteric modulator of SIRT1, is one of the best‐studied STACs and is used in our experiment. This change in SIRT1 activity is due to allostery— a phenomenon where a modulator binds distal to the active site of an enzyme. Presently, the mechanism of how the N‐terminus interacts with the catalytic core to change enzyme activity is poorly understood. We hypothesized that SIRT1 contains an allosteric switch within the STAC binding domain that is the key to allosteric regulation of SIRT1. The allosteric switch is postulated to be a region of multiple residues that undergo a change in secondary structure upon allosteric interactions. The objective of this study is to predict and confirm a novel allosteric switch region in human SIRT1 (hSIRT1) using enzymatic assays with resveratrol and various peptide substrates. Using computational methods, SIRT1 residues 186–189 (TFVQ) and 190–193 (QHLM) were found to be highly ordered and thus predicted to be possible switch regions within the SBD. Mutating all four residues to serines would increase the disorder propensity of this region, which is expected to abolish the switch‐like behavior. To test this, we have cloned and purified these two serine mutations of SIRT1: SIRT1(186–189S) and SIRT1(190–193S). We performed enzyme‐coupled assays in triplicates to measure the steady‐state kinetics of these mutants to test for allosteric response to resveratrol and to ensure that they remained catalytically active. We obtained the Michaelis‐Menten parameters for the wildtype SIRT1 and the two mutated SIRT1 constructs, using Ac‐p53W as the peptide substrate. Wildtype SIRT1 showed an increase in catalytic efficiency after the addition of resveratrol. For both mutated constructs, the catalytic efficiency after adding resveratrol decreased, mostly due to an increase in KM, which is the opposite of what happens with wildtype SIRT1. This suggests that the mutations affected the allosteric network. Identifying an allosteric switch will provide insight on the mechanism of STACs with the wildtype enzyme, adding valuable information on allosteric regulation and the potential of developing drugs for age‐related diseases, stress responses, and metabolism.

Support or Funding Information

SJSU Undergraduate Research Grant, California State University Program for Education and Research in Biotechnology Travel Grant Award, and NIH Grant 1SC2GM122000.
Intestinal alkaline phosphatase is a diagnostic biomarker for necrotizing enterocolitis in preterm infants
Authors: Buckley Rebecca , Heath Maya , Gerber Zeromeh , Davis Porcha , Linneman Laura , Gong Qingqing , Barkemeyer Brian , Fang Zhide , Good Misty , Penn Duna , Kim Sunyoung
Published: April 2020

Necrotizing enterocolitis (NEC) in preterm infants is an often fatal, gastrointestinal emergency. There is a need for a robust NEC biomarker that can be adopted in pathology labs and that is not confounded by sepsis. Use of such a NC biomarker could improve bedside management, lower morbidity and mortality, and permit patient selection in clinical trials for therapeutic candidates. We evaluated if aberrant intestinal alkaline phosphatase (iAP) biochemistry in infant stool is a molecular biomarker for necrotizing enterocolitis and not correlated to sepsis in the absence of NEC. There were 136 premature infants enrolled in three hospitals in this observational and prospective study. Median birthweight (IQR) was 1050 g (790‐350 g) and median gestational age was 28.4 weeks (26‐0.9 wks). Twenty‐five infants were diagnosed with severe NEC, 19 were suspected of having NEC, and 91 were non‐NEC infants. Twenty‐six patients were diagnosed with late‐onset sepsis and 14 had other non‐GI infections. Infant stool samples were collected between 24–40+ weeks postconceptual age (PCA). Enrolled infants were subject to abdominal radiography at physician and hospital site discretion. Enzyme activity and relative abundance of iAP were measured using fluorometric detection and immunoassays, respectively. Post‐measurement, biochemical data were evaluated against clinical entries from infants’ hospital stay. Fecal iAP content was 99±19, 123±41, and 5±3 (median±SE) for severe, suspected, and non‐NEC samples, respectively. Enzyme activity was 183±108, 355±180, and 613±218 kU/g (median±SE) for severe, suspected, and non‐NEC samples, respectively. ROC area under the curve (AUC) values were 0.96, 0.97, 0.54, and 0.58 for relative iAP content measurements at time of severe NEC, suspected NEC, sepsis, and other non‐GI infections; AUC values for iAP activity were 0.60, 0.76, 0.52 and 0.57, respectively. High amounts of iAP protein in stool and low iAP enzyme activity are reliable and specific biomarkers for NEC: iAP biochemistry uniquely has clinical utility in distinguishing NEC from sepsis.

Support or Funding Information

This work was supported by the National Institutes of Health (R41HD095779 to RB and SK; R01GM097350 to SK; K08DK101608, R03DK111473 and R01DK118568 to MG), National Science Foundation (IIP‐1713220 and IIP‐1547932 to RB and SK); March of Dimes (5‐FY17‐79 to MG), Louisiana Board of Regents (LEQSF‐RD‐D‐07 to SK), LSU LIFT2 (HSCNO‐2017‐LIFT‐006 to SK), Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital (MG), Department of Pediatrics at Washington University School of Medicine, St. Louis (MG), LSU School of Medicine (BB, SK, and MH), and LSU Health Foundation (MH, ZG, and SK).
Molecular Mechanisms of Skeletal Muscle Atrophy in Cerebral Ischemic Stroke
Authors: Mohamed Junaith , Khan Mohammad , Ferrandi Peter , Paez Hector , Pitzer Christopher , Stephen Alway
Published: April 2020

Stroke is the leading cause of death and the top cause of adult disability in patients world‐wide. Skeletal muscle is the main effector organ accounting for disability in stroke patients due to severe muscle atrophy and related weakness. However, the molecular signaling mechanisms that cause post‐stroke muscle atrophy remains elusive. To determine the mechanisms, we used a preclinical model of stroke in which we induced 60 minutes middle cerebral artery occlusion (MCAO) followed by 3 days reperfusion. We found that stroke caused a significant brain lesion and motor deficit. Stroke induced severe skeletal muscle atrophy in the paretic tibialis anterior muscle compared to the corresponding tibialis anterior muscle of sham mice, as evidenced by reduced muscle mass and FCA (fiber cross‐sectional area). Our transcriptome analysis showed that stroke altered mRNA expression profile in skeletal muscle. Some of the genes associated with catabolic pathways are highly dysregulated in post‐stroke muscle. We also found that stroke dramatically repressed the protein deacetylase Sirtuin1 (SirT1) gene expression and its function in skeletal muscle. Interestingly, restoring SirT1 function in post‐stroke muscle notably reversed muscle atrophy, in part, through regulating the key players of autophagy and ubiquitin proteasomal pathway. Collectively, our data uncovered a potential mechanism that causes cerebral ischemic stroke‐induced muscle atrophy and identified SirT1 as a critical regulator of post‐stroke muscle mass.
Low‐Tech Gamification and TBL Strategies to Teach Physiolgy
Authors: Tejada-Simon Maria Victoria , Silverthorn Dee
Published: April 2020

The concept of gamification in education refers to the process of adding active game elements into a course to encourage participation. Games help develop skills and confidence in healthcare professionals. Herein, we are aiming to engage the students in a game experience that will uncover the topic in question in a scaffolding manner. Specifically, we assisted first year Pharmacy students in understanding the autonomic nervous system’s (ANS) structure and function using an interactive game coupled to a team‐based learning session, devoid of any technology. We used a flipped/TBL classroom design in a 2‐hour interactive problem‐solving supplemental instruction session (SI). Prior to class, the students completed pre‐class activities in form of diagrams, crossword puzzles and fill‐in the blank to engage them with anatomical terminology. During the SI, 8 students/group (128 students total) worked collaboratively. The class met in an active learning designed room to facilitate small group interaction. The organization of the SI followed a constructive alignment: (1) Pre‐class activities: self‐paced interactive learning and reading; (2) Assessment of foundational learning before SI session with group activity, pair‐share/team‐based learning (TBL), 1–3 minutes microlecture; (3) Student group activity with discussion: game, diagram, fill in the blanks, match activities; (4) Group share: white board sharing “quiz” among the groups. To evaluate the success of this approach we used a grading rubric for pre‐class activities, post‐lecture assessments, measure classroom activity using GORP, student evaluation/survey, faculty peer evaluation.

Support or Funding Information

Promoting Active Learning and Mentoring Network (PALM); College of Pharmacy Faculty Development ‐ University of Houston
Overexpression of SIRT3FL Improves Cardiac SERCA2a Function Following High Fat‐High Sucrose Feeding in Mice
Authors: Oldfield Christopher J. , Moffatt Teri L. , O'Hara Kimberley A. , Xiang Bo , Dolinsky Vernon W. , Duhamel Todd A.
Published: April 2020

Background

Diabetes induces a distinct form of cardiomyopathy, termed diabetic cardiomyopathy (DCM). A key pathophysiological mechanism of DCM is reduced level and activity of sarco(endo)plasmic reticulum Ca2+ ATPase 2a (SERCA2a). Recently, it has been discovered that the function of SERCA2a is negatively regulated by the post‐translational modification, acetylation. The sirtuin (SIRT) isoform, SIRT3 full‐length (SIRT3FL), deacetylates protein substrates, though it is unknown if SIRT3FL deacetylates SERCA2a. Therefore, the objective of this study was to determine if the overexpression of SIRT3FL alters cardiac SERCA2a acetylation and function in a mouse model of diabetes, induced by high fat‐high sucrose (HFHS) feeding.

Methods and Results

Adult (12 weeks of age) male C57BL/6J Wild Type (WT) mice and adult male MCK‐mSIRT3‐M1‐Flag transgenic (SIRT3FLTG) mice on a C57BL/6J background, who overexpress the SIRT3FL isoform in skeletal and cardiac muscle, were randomized into groups fed either a standard lab diet (Control) or a HFHS diet for 4‐months. After 4‐months, HFHS‐fed mice exhibited increased body weight, left ventricle (LV) weight, LV weight relative to body weight, and liver weight, compared to control fed‐mice. LV systolic parameters were unchanged by HFHS feeding, while the A and E wave forms of HFHS‐fed mice were slowed, compared to control‐fed mice. HFHS‐fed mice exhibited LV thickening, compared to control‐fed mice. Glucose tolerance was impaired in HFHS‐fed mice, compared to control‐fed mice. Diet did not alter SIRT3 protein content. Likewise, HFHS feeding did not alter the level or function of cardiac SERCA2a. LV SERCA2a protein content was not altered by genotype. However, SIRT3FLTG mice, regardless of diet, had 21% greater maximal SERCA activity (Vmax) and an 11% greater Hill coefficient, compared to WT mice. The Ca50, which is the Ca2+concentration that elicits 50% of Vmax, was 24% lower in SIRT3FLTG‐HFHS‐fed mice, compared to WT‐HFHS‐fed mice, indicating an increase in Ca2+ sensitivity.

Conclusion

HFHS feeding induced a diabetic phenotype in WT and SIRT3FLTG mice but level and function of cardiac SERCA2a was unchanged. However, overexpression of the SIRT3FL isoform improved cardiac SERCA2a function, regardless of HFHS feeding.

Support or Funding Information

This work was supported by a grant‐in‐aid from the Heart and Stroke Foundation of Canada
Testosterone Reverses the Urethra Hypercontractility in Ovariectomized Rat: Role of Voltage‐Operated Calcium Channel
Authors: Bonilla Sandra Milena , Antunes Edson
Published: April 2020

Introduction and Aim

Postmenopausal women exhibit overactive bladder (OAB), as evidenced by urgency, frequency and noctúria (1). Ovariectomized (OVX) rats display OAB and urethral hypercontractility that is normalized by in vivo testosterone replacement (2). Androgen receptors (AR) have been found in urinary bladder, suggesting they play an important role in the urinary continence (3,4). However, the mechanism of action of testosterone in urethra of female rats has not been elucidated. L‐type voltage‐operated calcium channels (VOCC) are implicated in urinary contraction during the voiding phase (4). We hypothesized here that testosterone via VOCC activation regulates the female urethra. This study aimed therefore to evaluate the effects of testosterone and its modulation by VOCC on urethra contractility in OVX rats.

Materials and methods

Two‐month old female Sprague Dawley rats were submitted to bilateral ovariectomy, whereas Sham‐operated rat were manipulated but the ovaries were left intact. After 4‐month OVX, the urethra was removed, and concentration‐response curves to calcium chloride (CaCl2; 10 μM – 300 mM) were performed in the presence of testosterone (100 nM; 30 min), vehicle (alcohol; 0.5 μL) and/or the VOCC inhibitor nifedipine (3 nM; 30 min).

Results

Bilateral OVX significantly increased the urethra contraction to compared with Sham group (P < 0.05). Testosterone (100 nM prevented CaCl2‐induced urethral hypercontractility in OVX group. Pre‐incubation with nifedipine (3 nM) also reduced the contractile response to extracellular calcium in OVX group. In Sham group, testosterone and/or nifedipine had no effects.

Conclusion

Our data suggests that testosterone via non‐classical genomic pathway acts to inhibit VOCC to normalize the urethral hypercontractility in OVX rats.

All animal procedures were approved by the Ethical Committee on Animals Use CEUA/UNICAMP (CEUA; No. 4421‐1, 5250‐1).

Support or Funding Information

FINANCIAL SUPPORT: FAPESP (2017/26564‐9).
Upregulation of miR‐210‐5p in Systemic Lupus Erythematosus Impairs Silent Clearance of Dead Cell Remnants
Authors: Wu Yi-Hsuan , Chan Yen-Fan , Hsieh Hsi-Lung , Hwang Tsong-Long
Published: April 2020

Deficiency of dead cell clearance plays a significant contributing factor in the progression of Systemic Lupus Erythematosus (SLE). While several studies support macrophage‐mediated innate immune response for SLE development, the mechanism about “silent” clearance by polarized monocyte/macrophage populations in SLE still needs to be elucidated. By analyzing miRNA level, the expression of miR‐210‐5p from peripheral blood mononuclear cells (PBMCs) in patients with active SLE (n=17) was higher than that in healthy controls (n=16). We further used macrophage‐like THP‐1 as cell models to delineate the status of miR‐210‐5p upregulation on silent clearance and immune response. By miR‐210‐5p mimic transfection, differentiation and phagocytosis analysis, miR‐210‐5p‐overexpression increased accumulation of secondary necrotic cells (SNECs) in THP‐1 cells. ROS production of miR‐210‐5p‐overexpressed macrophage‐like THP‐1 cells was decreased as compared with that in negative controls upon secondary necrotic cells (SNECs) or H2O2 treatment by DCF‐DA staining. Impaired SNECs clearance in miR‐210‐5p‐overexpressed THP‐1 cells was mediated by a decrease in superoxide production through NOX pathway. These findings support the notion that increased miR‐210‐5p in SLE is, in part, due to decrease the NOX‐mediated redox signaling, which affects impaired dead cell clearance and immune response.
Inhibition of actin‐related protein (Arp) 2/3 complex blocks vasopressin‐induced AQP2 membrane accumulation
Authors: Liu Chen-Chung Steven , Cheung Pui Wen , Bouley Richard , Brown Dennis
Published: April 2020

Aquaporin 2 (AQP2) is a water channel protein located primarily on principal cells of kidney collecting ducts, and is crucial for regulating body water homeostasis. Regulation of AQP2 trafficking is subject to hormonal control, mainly via the canonical vasopressin (VP) signaling pathway which stimulates AQP2 membrane accumulation. Active actin cytoskeleton remodeling also plays an important role in AQP2 trafficking, but the mechanism is incompletely understood. Using immunohistochemistry and confocal microscopy, we discovered that actin‐related protein (Arp) 2/3 complex, an actin nucleator, was highly expressed in inner medullary principal cells where it colocalized with AQP2. Using an Arp2/3 complex inhibitor, CK‐666, we found that VP‐induced AQP2 membrane accumulation was inhibited both in rat kidneys and LLC‐AQP2 cells in vitro. Instead of distributing throughout the cytoplasm, AQP2 in cells treated with CK‐666 was concentrated in vesicles forming a perinuclear patch, which was also positive for Rab‐11 (a recycling endosome marker) and clathrin (a trans Golgi Network (TGN) marker). Similar perinuclear AQP2 patches appear in cells incubated at 20°C (cold block), which allows endocytosis to continue, but prevents protein exit from the TGN. By rewarming the cells to 37°C, these perinuclear patches dissipate, and AQP2 quickly redistributes throughout the cytoplasm (cold block release). However, we found that in cells exposed to a 20°C cold block and treated with CK‐666, AQP2 patches failed to dissipate upon rewarming, suggesting that CK‐666 blocked release of AQP2 from the TGN in the exocytotic pathway. This effect of CK‐666 was independent of VP signaling, and did not alter the VP‐induced phosphorylation state of AQP2 at residues serine‐256, S269 and S261. In conclusion, inhibition of the Arp2/3 complex blocks VP‐induced AQP2 plasma membrane accumulation by blocking AQP2 exocytosis at the level of the TGN and the recycling endosome, but did not affect VP signaling pathway. This result suggests that actin filament nucleation and growth via Arp2/3 activity is essential for AQP2 recycling and trafficking.

Support or Funding Information

CCL is supported by NIH training grant 5T32DK007540. PWC is supported by NIH grant DK115901. DB is supported by NIH grant DK096586
Occupational Stress Among Doctors ‐ Role of Duty Schedule and De‐Stressing Techniques.
Authors: Khan Mohammed Moizuddin
Published: April 2020

Introduction

Job or occupational stress is something we all face as employees or employers and we all handle it differently. It is a mismatch between the individual capabilities and organizational demands. Presently various challenges are faced by doctors making medical profession a tough job leading to occupational stress. Doctors are involved in long and irregular working hours which is resulting in varying levels of exhaustion and ultimately occupational stress. Emergencies or night duties are an inseparable part of medical profession so its desirable to find best possible ways to manage occupational stress.

Aim & Objectives

To evaluate the role of emergencies and night duties in causing occupational stress among doctors. To study the effect of de‐stressing techniques used by doctors to cope up with occupational stress.

Material & Methods

This is a cross sectional Observational study conducted in Indian doctors. Structured pro‐forma was used to collect demographic data, professional work pattern, exercising habits etc. To assess the psychological occupational stress a standardized professional psychological stress test questioner was used.

Results

It was seen that the doctors who attended more than 6 calls/week the percentage of occupational stress was found to be on higher than who attended less numbers. 28.89% had moderate stress while 13.33% and 6.67% were having stress or major stress. There was statistically significant association between emergency call/week and psychological occupational stress score. Physical exercise, plying sports, de‐stressing techniques like listening to music, net surfing were significant in coping with psychological occupational stress.

Conclusion

Occupational stress among doctors can be managed effectively by adopting a combination of measures which include modification in working schedules, regular psychological stress monitoring at institutional level and incorporating healthy lifestyle practices like physical exercise, sports etc. at personal level.

Support or Funding Information

College of Medicine, Dar Al uloom University, Riyadh
TMED2 is Required for Trophoblast Migration, Differentiation, and Proliferation
Authors: Aber Rachel , Kaiser Caroline , Yuan Libin , Jerome-Majewska Loydie
Published: April 2020

Essential cargo receptors, specifically members of the Transmembrane emp24 domain (TMED) protein family, are implicated in numerous cellular morphogenesis. The localization and levels of TMED proteins are tightly controlled to regulate cell survival, migration, and proliferation. TMED proteins have an N‐terminal coiled‐coil region essential for interactions with TMED partners and a Golgi dynamics (GOLD) domain required for heterodimerization and cargo recognition. Although the structure of TMED2 is known, the function of TMED2 in placental development remains poorly understood. Mouse embryos homozygous for a null mutation in the signal sequence of TMED2 died at midgestation from failure to form a placental labyrinth layer. Moreover, ex vivo cultures of pre‐attachment chorion and allantois suggest that cell‐type‐specific transport of fibronectin and VCAM1, by TMED2, is required for chorionic‐allantoic cell communication and for trophoblast differentiation. Thus, we hypothesize that events necessary for labyrinth layer formation such as trophoblast migration and syncytiotrophoblast fusion, require functional TMED2. To test the role of TMED2 in cell migration and survival, we conducted scratch and viability assays in HEK293 cells overexpressing wild‐type and mutant TMED2. We also tested the effects of mutating residues in the GOLD domain of TMED2, E48 and K91, that are predicted to be involved in heterodimerization with TMED10. We found that HEK293 cells overexpressing wild‐type TMED2 displayed reduced cell migration and increased ER stress. Similarly, overexpression of mutant TMED2‐E48Q, resulted in a comparable reduction in cell migration. However, cells overexpressing TMED2‐K91R, showed increased cell migration and viability compared to TMED2 and TMED2‐E48Q cells. To more precisely investigate the absence of a labyrinth layer in TMED2 deficient embryos, we conducted proliferation and fusion studies in two human choriocarcinoma cell lines: JEG‐3 and BeWo. Overexpression of wild type TMED2 in JEG‐3 cells was sufficient for syncytiotrophoblast fusion; however, knockdown of TMED2 in BeWo cells inhibited fusion, supporting a requirement for TMED2 in this process. Moreover, proliferation and metabolic activity assays revealed that knockdown of TMED2 in BeWo cells increased cell proliferation and decreased cell migration. Overall, our data suggest that levels of TMED2 must be tightly regulated for cell proliferation and viability. In addition, we found that the K91 residue of TMED2, which is predicted to help mediate heterodimerization with TMED10 is important for TMED2 function. We propose that the abnormalities displayed by embryos and cells deficient in or overexpressing TMED2 reflect the inadequate transport and localization of fibronectin and likely more unidentified proteins critical for regulating proliferation and migration of trophoblast cells.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada
Assessment of Novel Rexinoids for Therapeutic Potential in Combating Alzheimer's Disease
Authors: Pedroza Morgan Arielle , Mallick Sanchita , Marshall Pamella , Wagner Carl , Jurutka Peter
Published: April 2020

Bexarotene is an FDA approved anti‐cancer agent, but more recently this drug has been assessed for its potential as a therapeutic for dementia. Bexarotene may reduce amyloid beta (α□) plaques that are thought to play a role in Alzheimer’s disease pathophysiology, although clinical use of bexarotene causes unwanted side‐effects including hyperlipidemia and hypothyroidism. Bexarotene drives retinoid‐X‐receptor (RXR) homodimerization on the retinoid‐X‐response element (RXRE), as well as facilitating heterodimers with liver‐X‐receptors (LXR) and peroxisome proliferator activated receptor gamma (PPARγ). These RXR‐containing dimers are hypothesized to induce apolipoprotein (ApoE) in glial cells to control lipid and cholesterol homeostasis in the brain. We tested the ability of bexarotene and a panel of novel rexinoids to drive RXR dimerization using dual luciferase reporter assays to assess transcriptional efficiency of these compounds. We employed both RXRE‐mediated reporter assays, as well as a mammalian 2 hybrid system (M2H) to probe the ability of our novel rexinoids to activate RXR signaling. These compounds were also tested as activators of LXRE‐directed transcription, and as inducers of ApoE mRNA in U87 glial cells. Results from these multiple assay systems reveal that our panel of RXR analogs contain compounds with diverse activities, some of which bind to RXR with greater affinity than others, and in some cases can stimulate ApoE expression to a greater extent than bexarotene. These results suggest that chemical modifications to the parent bexarotene chemical scaffold may yield novel analogs with an amplified ability to activate RXR and possibly combat Alzheimer’s disease via a reduction in amyloid beta (α□) plaques.

Support or Funding Information

Coins For Alzheimer’s Research Trust
Advanced Activity‐Based Probes to Monitor the Proteasome in Live Cells
Authors: Trader Darci Jones , Zerfas Breanna
Published: April 2020

The probes currently available to monitor the activity of the proteasome need to be used at high micromolar concentrations to obtain significant signal and can be hydrolyzed non‐specifically by other cellular proteases. We have been using an LC/MS‐based screening method to discover peptide scaffolds that are cleaved more efficiently by the proteasome than other enzymes in human serum. Our new probes incorporate a Rhodamine 110 fluorophore and a peptoid moiety to enhance cell permeability. These new probes can be used in live cells to detect real‐time activity of various isoforms of the proteasome.

Support or Funding Information

NIH R21GM131206
Antihypertensive effect of methanol leaf extract of Anacardium occidentale against L‐Nitro Arginine Methyl Ester (L‐NAME)‐induced hypertension in male Wistar rats
Authors: Saba Adebowale Benard , Adebayo Adedeji Kolawole , Oyagbemi Ademola Adetokunbo , Omobowale Temidayo Olutayo , Ola-Davies Olufunke Eunice , Adedapo Adeolu Alex , Oguntibeju Oluwafemi Omoniyi , Yakubu Momoh Audu
Published: April 2020

The Anacardium occidentale plant; commonly known as Cashew has been widely reported for its efficacy in the treatment of various ailments such as hypertension. The efficacy of the plant has been attributed to its phytochemical constituents. Hypertension was induced in rats via oral administration of L‐Nitro Arginine Methyl Ester (L‐NAME); a non‐selective nitric oxide synthase inhibitor. To examine the antihypertensive properties of the Anacardium occidentale extract, twenty‐five (25) male Wistar rats were divided into 5 groups of 5 animals each and were grouped as follows: Group A (Distilled water), Group B (L‐NAME 40 mg/kg), Group C (100 mg/kg extract, 40 mg/kg L‐NAME), Group D (200 mg/kg extract, 40 mg/kg L‐NAME), Group E (10 mg/kg of Lisinopril, 40 mg/kg L‐NAME) orally for a duration of 4 weeks. Blood pressure parameters, markers of oxidative stress and antioxidant enzymes were determined. Immunohistochemistry of the angiotensin converting enzyme, mineralocorticoid receptor, angiotensin II type 1 receptor, and kidney injury molecule‐1 (Kim‐1) expressions were also determined. Treatment of hypertensive rats with Anacardium occidentale methanol leaf extract normalised systolic, diastolic and mean arterial blood pressure compared to hypertensive untreated rats. There was significant improvement in serum nitric oxide bioavailability of hypertensive rats treated with Anacardium occidentale methanol leaf extract in comparison to hypertensive untreated rats. Furthermore, there was a significant reduction in markers of oxidative stress and concurrent increase in the levels of renal antioxidant enzymes of hypertensive treated rats. Immunohistochemistry revealed significant reduction in the renal expressions of angiotensin converting enzyme, mineralocorticoid receptor, angiotensin II type 1 receptor, and kidney injury molecule‐1 of hypertensive treated rats. The results of this study suggest that Anacardium occidentale methanol leaf extract holds promise for the management of hypertension through the inhibition of angiotensin converting enzyme as its novel mechanism of action
Hyperhomocysteinemia: Instigating the Mechanism of Periodontal Disease
Authors: Stanisic Dragana , George Akash K. , Molnar Jack , Mallonee Carissa Jo , Homme Rubens P. , Smolenkova Irina , Tyagi Suresh C. , Singh Mahavir
Published: April 2020

Periodontal disease is one of the most common conditions resulting from poor oral hygiene and is characterized by a destructive process in the periodontium which essentially include gingiva, alveolar mucosa, cementum, periodontal ligament, and the alveolar bone. Notably, the destructive event in the alveolar bone has been linked to homocysteine (Hcy) metabolism however; it has not yet been fully investigated. Therefore; the implication of Hcy towards initiation, progression, and maintenance of the periodontal disease remains incompletely understood. Our research has led to the discovery that hyperhomocysteinemia (HHcy) causes changes in the periodontal tissues as well as it leads to an increase in the angiogenesis‐related proteins, potentially indicating a faster onset of periodontal disease in the susceptible individuals. To study periodontal tissue status and angiogenesis‐related protein levels, we compared wild‐type C57BL/6J (WT) and CBS+/− (HHcy) mice strains. The distance between cementoenamel junction, and the alveolar bone crest (CEJ‐BC) were measured histomorphometrically using histological preparations (Hematoxylin and Eosin; H&E, and the Masson’s Trichrome staining). Serum angiogenesis‐related levels namely the angiogenin, angiopoietin‐1, endostatin/collagen XVIII, FGF acid, IGFBP‐1, IGFBP‐2, MMP‐9 (pro and active forms), PDGF‐AA, PDGF‐AB/PDGF‐BB, pentraxin‐3 were measured using the Proteome ProfilerTM Array Kit specific for the mouse. The data suggest an increase in the distance between cementoenamel junction, and the alveolar bone crest. Levels of the angiogenesis‐related proteins in CBS+/− mouse strain were also significantly increased in comparison to the WT control mice. Interestingly, some of these angiogenesis‐related proteins have been shown to be upregulated during the periodontal disease. Our findings thus underscore the importance of Hcy metabolism towards the loss of periodontal tissue homeostasis, and the causation of the periodontal disease indicating that regulating Hcy metabolism can help prevent periodontal tissue damage, and the periodontal disease in people who suffer from chronic metabolic disorders such as hyperhomocysteinemia.

Support or Funding Information

Funding from the National Institutes of Health (NIH): HL139047, AR071789, HL74815, HL107640, HL139047, and DK116591.
Photo‐SNAP‐Tag, a Light‐Regulated SNAP‐Tag Chemical Labeling System
Authors: Cleveland Joseph , Tucker Chandra
Published: April 2020

Methods that allow labelling and tracking of proteins during dynamic cellular events have been instrumental for understanding their function. Traditional methods for labeling proteins include fusion to fluorescent proteins or self‐labeling chemical tagging systems such as SNAP‐Tag or Halo‐Tag. These latter approaches allow bright fluorophores or other chemical moieties to be attached to a protein of interest though a small fusion tag. In this work, we sought to improve the versatility of self‐labeling chemical‐tagging systems by regulating their activity with light. We used light‐inducible dimerizers to reconstitute a split SNAP‐Tag (modified human O6‐alkylguanine‐DNA‐alkyltransferase, hAGT) protein, allowing tight light‐dependent control of chemical labeling. In addition, we generated a split SNAP‐Tag version that can efficiently self‐assemble, showing high labeling efficacy. We envision these tools will extend the versatility and utility of the SNAP‐Tag chemical system for protein labeling applications.
Galectin‐8 Regulates Primary Cilia Assembly and Contacts Development
Authors: Syu JhanJhang , Jou Tzuu-Shuh
Published: April 2020

Primary cilium is a significant sensory organelle protruding from cellular surface to sense and thereby transduce environmental clues. A critical aspect of cilium morphology is defined by its length and contact formation between neighboring cilia. Galectins are a group of β‐galactoside‐binding animal lectins found both in the cytosol, and secreted in the extracellular space through unconventional pathway. Galectin‐8 (Gal‐8) is a protein with 2 tandem‐repeat carbohydrate recognizing domains (CRDs), and a high affinity of its N‐terminal CRD for sialylated or sulfated glycoconjugates which distinguishes it from other galectins. We found that knockdown of endogenous Gal‐8 inhibited primary ciliogenesis in polarized Madin‐Darby canine kidney (MDCK) cells, and both endogenous and epitope‐tagged Gal‐8s were noted at the surface of primary cilia. Furthermore, application of purified recombinant Gal‐8 protein at sub‐micromolar concentration to the apical, but not to the basolateral domain, resulted in growth and adhesion of PC in polarized MDCK cells within minutes. To identify the putative binding partner at the cilia surface for Gal‐8, we exploited a strategy applying surface biotinylation and surface pull‐down followed by proteomic assay. With these approaches, we found out several potential candidates and would examine their roles in ciliogenesis by engineering gene‐editing mutant cell lines for future study.
HUMAN ANATOMY TUTORING PROMOTES IMPROVEMENT OF THE STUDENT'S TEACHING AND LEARNING PROCESS
Authors: Forte Guilherme Aguiar , de Paula Uchôa Francisco Ewerton , Nunes Bruna Marques Barreto , Sanders João Victor Souza , Barreto João Erivan Façanha , Cerqueira Gilberto Santos
Published: April 2020

The academic tutoring activity is understood as an educational support exercise officially instituted in Brazilian universities. The teaching‐assistant, while supporting the teaching‐learning process of fellow students who contribute to the increase in their interest in a given area, becomes the main beneficiary with the practice of tutoring. The tutoring of the Human Anatomy discipline consists of teaching, research and extension activities, thus representing an indispensable strategy for facilitating the construction and mastery of anatomical knowledge. Based on this premise, the objective of this work was to describe the role of human anatomy tutoring as a tool for improving the teaching and learning process and extending the medical course. During a year of tutoring, teaching activities were carried out using cadavers and anatomical pieces, extension activities for public and private schools, technical training activities, anatomical competitions and the teaching of anatomical dissection. It was observed that the tutoring of human anatomy in the School of Medicine of the Federal University of Ceará represents a complementary way of learning anatomy. This activity deepened the anatomical knowledge of the teaching‐assistants mainly due to the activity of dissection and assistance in practical undergraduate classes. Anatomy tutoring provided the motor development of cadaver handling skills that are important for surgical technique, developed ethical competence and respect for the relationship with death. In addition, the tutoring contributes to the continuous renovation of anatomical pieces and to the activity of visiting the anatomy laboratory by the community. It was found that the tutoring of anatomy is an important tool for the dissemination of anatomical knowledge and improvement of the teaching and learning process in the undergraduate and community in general.
Chemical approaches towards regulated gene therapy in astrocytes
Authors: Cervasio Danielle A. , Pizzuto Isabella R. , Gunawardhana Nipuni , Preston Alyssa N. , Tan Kevin C. , Laughlin Scott T.
Published: April 2020

Astrocytes are glial cells that tile the entire central nervous system. Within the brain, they control metabolism, blood flow, and water flux, as well as dynamic processes like synaptic formation, maintenance, and pruning. Targeting astrocytes using gene therapies could be an attractive therapeutic option for seizure, epilepsy, Parkinson’s or Alzheimer’s disease, where there exists an imbalance in ion, neurotransmitter, or protein clearance— processes all modulated by astrocytes. We have recently developed chemical tags that ferry small molecule cargo into astrocytes. Here, we adapt these astrocyte targeting tags to control transcription, with the ultimate goal of using them for regulated gene therapy. Essentially, we utilize a modified transcription activator, doxycycline, to turn on the tetracycline‐inducible gene expression (Tet‐On) system— obtaining temporal control over transcription in astrocytes. Astrocyte specificity is attained by attaching a permanently positive astrocyte targeting moiety via organic chemistry to the small molecule drug doxycycline, which retains its Tet‐On activating capabilities. Our current probes include doxycycline and 9‐cyanodoxycyline variants with appended targeting moieties to improve astrocyte targeting and reduce cell permeability, decreasing off‐target affects. Our first probe, doxycycline methyl pyridinium (doxyMP) can successfully activate transcription in primary astrocytes under the strong, mammalian cytomegalovirus (CMV) promoter, eliminating the need for a cell‐specific promoter. In classical gene therapies, constitutive systems suffer from off‐target effects or variable gene expression of cell‐specific promoters. Using an astrocyte‐targeted transcription activator, we eliminate the need for these variably expressed, cell‐specific promoters, while obtaining specific and robust gene expression in astrocytes. Despite the continued development of gene therapies, there are few that utilize regulated gene expression systems. We hope to employ these molecules therapeutically where current constitutive gene therapies for Parkinson’s or glioblastoma, for example, could benefit from regulated gene expression.