bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental Programming of Glomerular Podocyte Endowment
Authors: Bertram John , Cullen-McEwen Luise , Gazzard Sarah , Bertram michael , van der Wolde James , Walton Sarah , Moritz Karen , Haruhara Kotaro , Goncalves Gessica
Published: April 2020

A range of perturbations to the maternal environment result in low nephron endowment in offspring, which is often linked to increased blood pressure and renal pathology in later life. Given that nephrogenesis in mammals ends shortly before or after birth, this nephron deficit is permanent. To date, very few studies have examined the morphology of nephrons in offspring born with low nephron endowment. Given the link between podocyte loss and glomerulosclerosis in adults, we examined whether maternal perturbations also result in low podocyte endowment in offspring, thereby rendering offspring at increased risk of adult disease.

Three models of maternal perturbation were investigated: (1) gestational hypoxia in mice; (2) maternal low protein diet (LPD) in mice followed by postnatal hyperglycaemia; and (3) maternal low protein diet in rats followed by high fat feeding. Podocytes were counted at postnatal day 21 in whole glomeruli contained in tissue slices immunostained for specific podocyte markers, cleared and imaged with confocal microscopy.

In all three models, total podocyte number per glomerulus was reduced in settings of maternal perturbation. In male mice exposed to hypoxia, podocyte endowment was 15% lower than controls (p<0.01), but no effect was observed in females; in male and female mouse LPD offspring, podocyte endowment was 15% (p<0.001) and 9% (p<0.05) lower than controls; and in male LPD rats (females were not studied), podocyte endowment was 15% (p<0.0001) lower than controls.

This is the first report of developmental programming of low podocyte endowment. We have identified low podocyte endowment in two species, three models and both sexes. We are currently investigating whether this podocyte deficit is permanent, and the effects on renal function in later life.
Interactive, Illustrative Video to Improve Student Retention and Comprehension of Sympathetic Autonomic Pathways and Effects
Authors: Alspaugh Nathan , Van Winkle Katherine
Published: April 2020

The autonomic nervous system is always a dreaded, but critical lecture for medical and health professional students, due to its seemingly complex pathways and convoluted delivery. In the context of our current technology‐centric student culture, we need new ways to deliver information effectively and succinctly to students and professionals alike without over‐simplifying content. We developed a video that creates an easy to follow schematic of the sympathetic nervous system. However, in attempt to address the diversity of student learning styles, we expanded upon our original audiovisual approach to a multi‐method design. Videos are a passive activity, but the best learning outcomes seem to derive from incorporating a myriad of learning strategies. Therefore in addition to the video we included a printable outline of the drawings to allow students to actively follow along as they listen to the video. This makes it a visual, auditory and kinetic learning experience, which we believe will improve students’ comprehension and retention. Data on its efficacy and reception will be collected both qualitatively and quantitatively in the next few months. This will be achieved through the distribution of pre and post‐viewing surveys, assessing students’ confidence as well as quantitative quizzes that will test changes in subject comprehension. The first video will represent our pilot project to establish a video series starting with autonomics and then addressing other difficult concepts in anatomy at the medical professional education level.
Determining the Effects of Legionella pneumophila SidE Effector Proteins on Host Vesicular Trafficking
Authors: Favichia Michelle , Allgood Samual C. , Neunuebel M. Ramona
Published: April 2020

Legionella pneumophila is an opportunistic intracellular pathogen responsible for Legionnaires’ disease in humans. Upon infection, L. pneumophila forms the Legionella‐containing vacuole (LCV), shielding itself from the host immune response. From the LCV, L. pneumophila uses a specialized secretion system to translocate over 350 effector proteins into the host cell. Effectors target and manipulate host pathways in order to optimize the host’s cellular environment to support L. pneumophila replication
(1). One important cellular process targeted during infection is endocytic recycling. Recycling pathways are generally categorized as slow or fast, depending on the time it takes for endocytosed cargo to return to the plasma membrane. Rab11 is a key regulator of the slow endocytic recycling pathway
(2) and it moves along microtubules partly through its interaction with Rab11‐FIP1A. It was previously shown that mutation of Rab11‐FIP1A disrupts transferrin recycling
(3). In the context of infection by L. pneumophila, our group has shown that transferrin recycling in human macrophages is inhibited early during infection. We hypothesize that the L. pneumophila effectors protein family SidE is at least partly responsible for this defect. Pull‐down assays revealed that interaction between Rab11 and Rab11‐FIP1A is disrupted in the presence of SdeC. In transiently transfected CHO cells, GFP‐Rab11‐FIP1A is found on vesicles in the perinuclear region. However, when GFP‐Rab11‐FIP1A and mCherry‐SdeC are co‐expressed in CHO cells, we observed that GFP‐Rab11‐FIP1A changed its localization to several enlarged structures. We are investigating whether SdeC’s ubiquitination activity is responsible for disrupting interaction of Rab11 with Rab11‐FIP1A. These results will help establish the effects of ubiquitination by SidE effector proteins on the slow recycling pathway.

Support or Funding Information
Fibroblast Inflammasomes Promote Inflammation and Fibrosis in the Breast Tumor Microenvironment
Authors: Postma Makenzie
Published: April 2020

Clinically, both incidence and mortality of breast cancer remain significant, despite progress in our understanding of cancer, and advances in treatments. Fibrosis is an independent prognosis marker of breast cancer, and cancer‐associated fibroblasts promote cancer growth. Whether inflammasome activities of fibroblasts critically influence inflammation and fibrosis remains unclear. Thus, we determined whether fibroblast inflammasome activation altered the secretions of pro‐inflammatory cytokines and fibrosis associated proteins. We assessed (1) the expression of the inflammasomes’ proteins and (2) the secretions of the pro‐inflammatory cytokines IL‐1ß and IL‐18 along with collagen I secretions by L929 fibroblasts in vitro. The effects of conditioned media collected from the aggressive 4T1 mammary tumor cells were also assessed. Our data indicate that L929 fibroblasts express the proteins associated with functional NLRP3 inflammasomes. Furthermore, following incubation with LPS, a classical inflammasome activator, L929 fibroblasts secreted significantly more IL‐1ß, matrix metalloproteinase 14, and collagen I. The effects of the NLRP3 inflammasome inhibitor MCC950 on L929 fibroblast secretions of pro‐inflammatory cytokines and pro‐fibrotic extracellular matrix proteins are also presented. These results (1) confirm the activity of inflammasome in fibroblasts, and (2) highlight the role of fibroblast inflammasomes in the generation of the pro‐inflammatory and pro‐fibrotic microenvironment in breast cancer.

Support or Funding Information

Makenzie Postma is an Honors student enrolled in the Honors in Biological Sciences Program at UNC Charlotte. This research is supported by the University of North Carolina at Charlotte under the direction of Dr. Didier Dréau.
Type 1 Diabetes Results in Significant Purinergic Receptor Remodeling in Podocytes
Authors: Klemens Christine A. , Spires Denisha , Levchenko Vladislav , Palygin Oleg , Staruschenko Alexander
Published: April 2020

P2 (purinergic) receptors have been proposed to be potential viable targets for the treatment of various cardiovascular and renal disorders. Previous studies in our lab have demonstrated that the predominant purinergic signaling pathway in healthy podocytes is through P2Y1 metabotropic receptors. It was also reported that expression of purinergic receptors in different tissues, including kidney, is dynamic and might be changed under pathological conditions such as diabetic nephropathy. We hypothesized that prolonged type 1 diabetes (T1D) would result in substantial changes to podocyte purinergic calcium signaling and result in a transition from predominantly metabotropic signaling through P2Y1 to increased ionotropic signaling through P2X receptors. To do this we utilized the established T1D model of streptozocin‐injected Dahl Salt Sensitive (SS) rats, and measured intracellular calcium ([Ca2+]i) signaling in podocytes from their freshly isolated glomeruli. We determined that in comparison to non‐diabetic rats, which have a rapid elevation of [Ca2+]i levels in response to ATP (40 μM) that returns to baseline within 2 min, podocytes from T1D rats had reduced peak response amplitudes (0.53 ± 0.04 vs. 0.34±0.02, p<0.001). Furthermore, podocytes of glomeruli isolated from diabetic rats had prolonged sustained elevation of [Ca2+]i level, which was not observed in non‐diabetic rats. Additionally, we used P2 specific antagonists to determine which receptors resulted in the altered calcium response curve in T1D podocytes. We found that pharmacological inhibition of P2X7 and P2X4 (10 μM A438079 and 5‐BDBD, respectively) altered the calcium curve dynamics, but did not significantly reduce the total [Ca2+]i level over a 5 min time period, as determined by area under the curve (AUC) measurements. Similarly, inhibition of P2Y1 (1 μM MRS2500) changed the [Ca2+]i curve response, but did not reduce the AUC. Combining both P2X7 and P2X4 inhibitors reduced the AUC; however, did not completely eliminate podocyte purinergic signaling (51.42 ± 5.52 vs 30.39 ± 5.00). Combined inhibition of P2X7, P2X4, and P2Y1 resulted in the near complete elimination of [Ca2+]i release in response to ATP (AUC, 51.42 ± 5.52 vs 10.57 ± 2.56, p<0.001). These results demonstrate that T1D increases ionotropic purinergic signaling in podocytes; however, does not eliminate metabotropic signaling. These studies establish the potential for targeting both P2X4 and P2X7 for therapeutic intervention in T1D.

Support or Funding Information

National Institutes of Health grants T32 HL134643 and R35 HL135749, and Department of Veteran Affairs I01 BX004024
Using VR‐Enriched Tasks (VRETs) to Promote Active Learning in Pre‐Clinical Gross Anatomy Education
Authors: Cheung Chun Chung , Cecot Tomasz Stanislaw , Tipoe George Lim , Yang Jian
Published: April 2020

With the blooming growth of technological innovations, virtual reality (VR) is slowly being incorporated into anatomy education and has provided a brand‐new area of research interest for anatomy educators. It is paramount to make anatomy classes more active and student‐centred as that creates deeper understanding of the subject matter. The use of VR is not merely to create another atlas for students to browse in their free time. Rather, VR should be used to enhance active learning. For that, task‐based learning is believed to be an ideal approach to create an active and engaging learning environment. We aim to introduce VR into large classes of over 250 students and integrate it seamlessly into dissection classes. This pilot study looked at our students’ perception and satisfaction of a learning activity assisted with VR.

Prior to the pilot session, three VR‐enriched tasks (VRETs) were written by two anatomy teachers. All tasks required participants to work in groups of minimum two people, one using the VR device while the other giving instructions. Two other anatomy teachers, who did not participate in task writing, were invited to review and perform the tasks to verify the tasks were of adequate difficulty and written with clear instructions. During the VRETs sessions, the participants were given an introductory demonstration and a warm‐up task before proceeding to the three VRETs. Each session lasted on average 60 minutes and participants were asked to fill out a 7‐point Likert scale questionnaire (7=strongly agree and 1=strongly disagree) at the end of the session.

Two VRETs sessions were held with a total of 48 participants. 10 sets of VR devices were deployed and anatomy teachers and technicians were present to assist the participants. All participants were second year medical students at the University of Hong Kong and all completed and returned the questionnaire. The questionnaire was modified based on a recent publication on technology‐enabled active learning inventory. Overall, three participants reported to have previous VR experience. 4 items received an average score over 5.9, in which participants moderately agreed that VRETs gave them deeper insights to issues they did not think of; piqued their curiosity; act as extra support for learning anatomy and improved the overall learning experience. Participants also gave written feedback, which mainly focused on the steep learning curve of operating the VR devices.

This pilot study showed that VRETs were well‐received by participants. However, extra training time should be given to users as many commented on the operational difficulty to novice users. Further study would be conducted to look at the impact of VRETs in learning and whether it is useful in creating an active learning environment. We would also look at how VRETs can be run in conjunction to dissection classes. IRB approval Ref: HKU/HA HKW IRB, Ref: UW 19‐277.
Sex Differences in Airway Inflammation and Structural Properties in a Mouse Model of House Dust Mite Challenge
Authors: Silveyra Patricia , Fuentes Nathalie , Montes Deborah T. , Spinelli Amy , Abraham Thomas , Purushothaman Indira , Chroneos Zissis
Published: April 2020

Background

Asthma is a chronic airway disease that leads to compromised lung function and affects more than 300 million patients worldwide, leading to >180,000 deaths each year, many of which are avoidable with proper treatment and care. Despite significant advances in diagnostic and treatment of the disease, asthma continues to be a significant health problem and an economic burden. Epidemiological studies in adult populations have revealed sex differences in asthma morbidity, with higher incidence in females than males. The causes of this disparity are still unclear, but studies have shown that sex‐specific signaling pathways regulated by hormones contribute to differences in immune cell activation and airway reactivity in response to different environmental stimuli.

Methods

With the goal of understanding the mechanisms associated with the observed sex disparities in allergen‐induced asthma, we challenged both male and female adult C57BL/6J mice with a house dust mite (HDM) solution from two species, Dermatophagoides pteronyssinus and Dermatophagoides farinae. The protocol consisted of five consecutive intranasal challenges with 25μg of HDM or control (PBS) followed by two days off, repeated for five weeks. The asthma phenotype was assessed by testing airway hyperresponsiveness (AHR) to methacholine challenge, and airway inflammation was characterized by cell differential assessment of bronchoalveolar lavage fluid obtained from experimental animals. Lung tissue was harvested for inflammatory mRNA expression determinations and histology. Airway remodeling was characterized using the two‐photon imaging technique, second harmonic generation (SHG), on lung tissue sections to determine collagen content in mouse airways (Velocity, imaging quantification software).

Results

We found that HDM promoted alterations in respiratory mechanics in challenged animals, including alterations in lung function measures that led to airway hyperresponsiveness (AHR) and increased parenchymal elastance in both males and females. While HDM‐challenged male mice showed higher AHR, airway collagen & elastin content, and BALF eosinophilia than females, challenged females displayed higher BALF neutrophilia and expression of type II pro‐inflammatory cytokines than males.

Conclusion

Our results indicate sex differences in both inflammatory and immune responses, as well as in airway structural changes induced by HDM exposure in mice. Characterization of the mechanisms leading to these sex differences will provide better insight into the causes of sex disparities observed in asthma prevalence and severity.

Support or Funding Information

This work was supported by NIH grants HL133520 (PS) and HL141618 (PS) and the APS Porter Fellowship (NF)
Explorations of the Apoptotic Protease Activating Factor 1 (Apaf‐1) in its Formation of the Seven‐Spoked Apoptosome Responsible for Programmed Cell Death
Authors: Kessler Eric , Bhatia Samishti , Caviar Morgan , Cleary Jacob , Fine Sam , Fritz Sylvia , Hayne Gracie , Husted Ryan , Johnson Zoe , Kessler Jacob , Kosiborod Sam , Looney Gwen , Malik Rivka , Markley Ella , Monson Lakota , Smith Grace , Wickoren Evan
Published: April 2020

Multicellular life forms first appeared in the fossil record in a burst of evolution termed during the Cambrian period. With multicellularity came the development and specialization of cells, alongside their coordination in the formation of tissues and organs. An adult human, composed of 30–40 trillion cells, progresses through repeated choreographed rounds of growth and cell division from its microscopic begins as a single celled zygote. Some have argued that this is the most critical stage of one’s life. Equally important for development, relatively overlooked, is the intrinsic process of cell death. For example, the webbing between our fingers and toes disappears around week six of gestation. This macroscopic observation during fetal development is the result of a complex series of molecular events initiated by the apoptotic protease activating factor 1 (Apaf‐1) as it forms the holo‐enzyme the apoptosome. The Blue Valley CAPS 2020 SMART Team (Students Modeling a Research Topic), with support from the CBM at MSOE, modeled the Apaf‐1 using JMol and 3D printing technologies in order to better understand how this molecular machine carries out programmed cell death. Structurally, Apaf‐1 is composed of 1249 amino acid residues that form a C‐terminal regulatory domain, a nucleotide binding and oligomerization domain (NOD), and an N‐terminal caspase recruitment domain (CARD). The regulatory domain is composed of two WD40 b‐propellers that are responsible for binding cytochrome c, which itself is released from the inner membrane of the mitochrondria under conditions of stress. Cytochrome c bonding causes a disruption in a salt bridge between Lys192 and Asp616 leading to changes in the orientation of the propellers. The nucleotide binding and oligomerization domain is composed of a number of helical subdomains. Conformational changes in the regulatory domain stimulates nucleotide exchange, where an ADP molecule bond in Apaf‐1’s inactive and closed monomeric state is exchanged for an ATP molecule leading to a more open monomeric state and the subsequent formation of the heptameric apoptosome. The Arg265 trigger which stabilizes an aspartate triad in the inactive monomeric state becomes compromised after ATP binding leading to conformational changes that open up Apaf‐1. Once the polymeric apoptosome forms, the CARD binds and activates procaspase‐9, which itself cleaves loops in procaspases 3 and 7 which become active and responsible of the apoptosomes proteolytic function. Proteolysis ultimately leads to cell death.
Interplay of microRNA‐503 and N‐acetylcysteine in regulating hypoxia‐reoxygenation injury in cardiomyocyte H9C2 cells
Authors: He Yanjing , Cai Yin , Irwin Michael G , Xia Zhengyuan
Published: April 2020

microRNAs(miRs) are involved in many cardiovascular diseases including myocardial ischemia / reperfusion injury (IRI). Myocardial IRI can lead to oxidative stress, many miRs are regulated in response to oxidative stress. N‐acetylcysteine (NAC) –a known antioxidant, is reported to have protective effects against myocardial IRI. Previously, we and others observed that cardiac miR‐503 was down‐regulated during myocardial IRI in vivo in rodents or in cardiomyocyte H9C2 cells subjected to hypoxia and reoxygenation (H/R). This study is aimed to explore the interplay of miR503 and NAC in the regulation of hypoxia‐reoxygenation injury and possible molecular mechanisms in cardiomyocyte H9C2 cells. Here, H9C2 cells were subjected to hypoxia for 10 hours followed by reoxygenation 6 hours as simulated myocardial IRI. Results showed that cell viability was decreased and lactate dehydrogenase (LDH) activity was increased upon H/R; Pretreatment with NAC (1mM) to H9C2 cells for 30 hours can improve cell viability and reduce LDH activity in both normoxia and H/R states, while pretreatment with NAC (2mM) decreased cell viability and increased LDH activity. We also found that NAC (1mM) can down‐regulate miR‐503 expression level in H9C2 cells subjected to normoxia, it also down‐regulated miR‐503 expression level upon H/R. In order to inhibit endogenous miR‐503, H9C2 cells was transfected with Antagomir‐503, inhibition of miR‐503 can improve cell viability and reduce LDH activity upon H/R; Western blotting results showed that there was no significant difference among control, HR, HR+negative control of Antagomir‐503(NC) or HR+Antagomir‐503 groups for proapoptotic Bax protein, while anti‐apoptotic Bcl2 protein was increased after transfection of Antagomir‐503 compared with HR+NC. Target‐Scan software showed that Phosphoinositide‐3‐kinase, regulatory subunit 1(PI3K p85) is one of miR‐503 target genes, and P85 protein was decreased after H/R, but it was increased significantly after administration of Antagomir‐503, its down‐stream protein phospho‐AKT (Thr450) was also significantly increased. However, Antagomir‐503 and NAC (1mM) used in combination to H9C2 cells upon H/R, did not provide an additional advantage in protecting cell injury. It is concluded that administration of NAC (1mM) or Antagomir‐503 alone can alleviate cell injury, while the combination of NAC (1mM) and Antagomir‐503 has no synergistic cytoprotective effects, the possible molecular mechanisms need to be further explored.

Support or Funding Information

Supported by Hong Kong RGC(17158616 M) and HMRF (05161826)grants.
RNAseq in fat pads from Alstrom syndrome 1 (ALMS1) Knockout rats support a role for ALMS1 in leptin release and fat metabolism in adipocytes.
Authors: Ortiz Victoria C. , Liao Tandong , Mendez Mariela , Ortiz Pablo A.
Published: April 2020

Inactivating mutations in the ALMS1 gene in humans cause early onset obesity and metabolic syndrome. Deletion of ALMS1 in rodents induces early onset obesity and insulin resistance on normal chow. The mechanisms by which ALMS1 cause obesity are not clear but likely involve the development of leptin resistance since adult ALMS1 KO rats are hyperphagic and hyperleptinemic. It is not known whether ALMS1 controls leptin release and expression in adipocytes prior to development of obesity. ALMS1 has been associated to ciliary function which may play a role in adipogenesis. We hypothesized that deletion of ALMS1 would alter gene pathways involved in fat metabolism and cilia formation in young ALMS1 KO rats, which should contribute to hyperleptinemia. To study this, we obtained abdominal fat pads from young ALMS1 KO rats shortly after weaning (7 weeks) and prior to changes in body weight. We found that plasma leptin levels were higher in young ALMS1 KO rats (ALMS1: 3530±300 vs. WT: 172±16 pg/ml, p<0.05) despite no differences in body weight or baseline glucose. Leptin release (normalized to fat weight) was 120% higher (wild type: 30±8, ALMS1‐KO: 56± 15 pg/mg tissue/2h) in abdominal fat pads from ALMS1 KO rats. To study the genes involved in higher leptin release we performed RNAseq in freshly isolated
abdominal fat pads. 12,860 genes were identified and quantified. In fat pads from ALMS1 KO there were 163 upregulated and 237 significantly downregulated genes. Leptin mRNA was significantly increased (1.68 Log2 fold, p<0.001, n=4) as were several genes involved in lipogenesis (Elovl6, Scd1, Scd2, Mogat2, Acly, Lpl, Dgat2, Fasn). Ingenuity pathway analysis indicated that the top canonical pathways impacted were: oleate biosynthesis, glycolysis I, hepatic fibrosis, and clathrin‐mediated endocytosis signaling. Importantly, at least 10 ciliary genes were significantly decreased (Traf3ip1, Pard6g, Cngb1, Ak7, Ttll1, Mlf1, Ccdc162, Dnah6, Ccdc187, Foxj1) suggesting a role for ALMS1 in ciliary function. To examine this, we immunolabeled cilia in abdominal adipocytes with gamma acetylated tubulin antibodies, and examined sections by confocal microscopy. Short cilia (1–2 μm) were observed in both WT and ALMS1 KO adipocytes. We concluded that ALMS1 is likely involved in leptin release from adipocytes. It is not clear whether increased leptin expression and release is secondary to neuroendocrine alterations or alterations in cilia‐induced signaling. Our data also point to an important role for ALMS1 in regulating adipocyte function and leptin physiology.

Support or Funding Information

Henry Ford Hospital Research Fund
Plasma Orexin A Level is Increased in Salt‐Sensitive Hypertension
Authors: Gao Huanjia , Bigalke Jeremy , Bloch Cassie , Chen Qinghui , Chen Bojun , Shan Zhiying
Published: April 2020

Accumulating evidence demonstrates that the brain orexin system plays a crucial role in the control of blood pressure and cardiovascular function. In addition, hyperactivity of central orexin system function has been implicated in the pathology of salt sensitive hypertension (SSH). However, whether or not peripheral orexin system activity is altered in SSH remains unknown. In this study, we compared plasma orexin A levels and adrenal orexin receptors including orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R) expression in Dahl salt‐sensitive (Dahl S) rat, an animal model of SSH, under high salt diet with their normotensive Dahl S cohorts. 7‐weekold male and age‐matched female Dahl S rats were divided into two groups of each and were fed either normal diet (NS, 0.4% NaCl) or high salt diet (HS, 4%NaCl). Six weeks following different diet treatments, all rats were euthanized and their plasma orexin A levels were measured using ELISA. Their adrenal glands were collected and subjected to real time PCR for OX1R and OX2R mRNA measurements. The results showed that HS diet increased plasma orexin A levels in both male (HS: 6.97±0.98 vs. NS 4.34±0.95 pg/ml; n=3; p=0.053); and female (HS: 6.17±0.65 vs. NS: 3.65±0.15 pg/ml; n=4; *P<0.05) rats. No significant difference was observed in orexin A level between male and female rats in both NS and HS groups. We therefore performed the rest of the experiments using female Dahl S rats. Real Time PCR results showed that OX1R mRNA level is increased by 3‐fold (n=5; *P<0.05) and OX2R mRNA level is increased by 1.9‐fold (n=5; *P<0.05) in HS diet treated rats compared to control rats. Immunohistochemistry assay showed that OX1R and OX2R are primarily expressed in the medulla of the adrenal gland and their expressions are co‐localized with tyrosine hydroxylase, an enzyme that catalyzes the first and rate limiting step in the synthesis of catecholamines including norepinephrine. This result coupled with the evidence that adrenal and plasma norepinephrine levels are increased in HS loaded Dahl S rats suggests that increased orexin system activity in the adrenal gland may increase norepinephrine production and subsequent release, therefore contributing to the development of SSH. Further studies will be performed to investigate whether blockage of the adrenal orexin receptors will decrease norepinephrine level and prevent or attenuate SSH in Dahl S rats.

Support or Funding Information

(Grant support: NIHR15HL129213 (Shan); Michigan Technological University and Portage Health Foundation Research Excellence Fund (Shan); Guangzhou University of Chinese Medicine, Graduate School international Schloarship (Gao)
DOES CARDIOPLEGIA PROVIDE CARDIAC PROTECTION BY INDUCING Nr‐E2 RELATED FACTOR‐2(Nrf2)
Authors: Diao Hongting
Published: April 2020

Background

Open heart surgery is often an unavoidable procedure for treatment of cardiovascular disease. Myocardial ischemia‐reperfusion injury (MIRI) can occur as a result of cardiopulmonary bypass. The multifaceted mechanisms of MIRI involve the generation of proinflammatory mediators and increases in reactive oxygen species (ROS), resulting in damage to proteins, lipids, and DNA, ultimately impacting postoperative cardiac function and outcomes. Nf‐E2 related factor‐2 (Nrf2), a basic leucine zipper transcription factor, is regarded as one of the most important regulators in antioxidant pathways. Nrf2 binds to and activates the Antioxidant Response Element (ARE) in the promoters of many antioxidant and detoxification genes.

Purpose

We addressed whether or not cardioplegic solutions induce the activation of Nrf2, therefore serving to protect the myocardium from MIRI. Methods. We used adult cardiomyocytes in culture to test whether or not four different types of cardioplegic solutions induce Nrf2. The conditions tested include routine laboratory cell culture at 37°C and hypothermic temperatures of 10–20°C.

Results

Our data show that certain cardioplegic solutions caused increases in Nrf2 protein levels under both cell culture and hypothermic conditions. ARE luciferase reporter assay confirmed Nrf2 activation by the cardioplegic solutions. Both High K and Del Nido showed reliable activation of Nrf2.

Conclusions

Our data supports our hypothesis that cardioplegic solutions can be cardiac protective via the activation of Nrf2.

Support or Funding Information

This work is supported by research grants of NIH R01 GM125212 (QMC), NIH R01 GM126165 (QMC), Holsclaw Endowment (QMC)
Development and Application of a New Platform for Measuring Phospholipase A2 Activity in Cells Using Mass Spectrometry
Authors: Argus Joseph P. , Dennis Edward A.
Published: April 2020

Members of the phospholipase A2 (PLA2) superfamily hydrolyze fatty acids from the sn‐2 position of membrane phospholipids. PLA2‐mediated release of n‐3 and n‐6 polyunsaturated fatty acids (PUFAs) is a highly regulated and critical step in producing eicosanoids and docosanoids. These signaling molecules play key roles in a variety of important processes including inflammation, onset of premature labor, and neural degenerative diseases. Thus, a better understanding of how PLA2 functions in cells may uncover new therapeutic avenues and lead to potent pharmacological agents. Though a rapid in vitro assay of PLA2 function has been recently published, one barrier to progress in this field is the lack of methods for determining PLA2 activity in cells. To address this issue, we have developed a new platform for measuring PLA2 activity ex vivo. Enriching cells with natural or deuterated PUFAs followed by mass spectrometry allows for tracking of hydrolysis at the sn‐2 position by both the disappearance of substrate phospholipids and the appearance of product PUFAs. An optimized cell culture procedure and lipid analysis workflow allow for significantly improved throughput. We anticipate that this platform will not only lead to a better understanding of PLA2 regulation and function in cells, but also allow for rapid screening of inhibitor candidates.

Support or Funding Information

Supported by NIH grant GM20501
In vitro physiological membrane‐on‐a‐chip and its application in cell and neuronal biology
Authors: Heo Paul , Rothman James , Pincet Frederic
Published: April 2020

Experimental setups to produce and to monitor model membranes have been successfully used for decades and brought invaluable insights into many areas of biology. However, they all have limitations that prevent the full in vitro mimicking and monitoring of most biological processes. Here, a horizontal free‐standing membrane having a physiological lipid composition is reconstituted in a 3D printing‐based microfluidic chip. Simultaneous monitoring of the membrane processes with a microscope and patch‐clamp amplifier reveals that the membrane is free‐standing, horizontal, fully fluid, stable, flat, and large enough. Dual open‐channels adjacent to the bilayer are freely used to alter each lipid monolayer composition (e.g., to form an asymmetric membrane) and oriented protein insertion. Currently, this in vitro membrane setup is integrated to investigate various molecular mechanisms in cell and neuronal biology. I will introduce the application of this setup to decipher the post‐translational insertion of tail‐anchored protein into the ER membrane, including the neuronal vesicle trafficking and α‐synucleinopathies.
The Personalized Precise Exercise Training with Holistic Integrative Protocol (PPET‐HIP) for Effective Management in Patient with Chronic Diseases (CDs)
Authors: Sun Xingguo
Published: April 2020

BACKGROUND

The patients with chronic diseases (CDs, i.e. hypertension, hyperglycemia, hyperlipidemia, hyperuricemia and obesity etc. with or without MI or Stoke) were uncured, according to the guidelines of all each one group of exports. However, based on our new Holistic Integrative Physiology and Medicine (HIPM), we might be able to use the core personalized precise exercise training and continuous functional monitoring with holistic integrative protocol (PPET‐HIP) for effective management of CDs patients.

METHODS

Ten CDs patients, who has more than 10–30year history and four of them had MI or Stoke, were recruited from 2014–2018.Before and after 12‐week intervention, general evaluations(blood pressure, glucose, and lipid profiles), cardiopulmonary exercise testing (CPET), echocardiogram, and the adjustment of drugs treated CDs patients were recorded respectively. Based on HIPM for CPET interpretation, we gave each patient an PPET‐HIP for adjusting of all life styles.

RESULTS

All patients exercised safely with any problem. After 12w HIPM‐PPT management, the all abnormal parameters of hypertensive, hyperglycemia, hyperlipidemia, hyperuricemia and obesity were become normal; CPET parameters, left ventricular function, and QoL were improved significantly than those before (P<0.05). Then we decreased drugs dosages and totally stopped drugs, the levels of blood pressure, glucose and lipid were still maintained stable. Surprisingly, the curable condition was achieved in CDs patients.

CONCLUSIONS

HIPM guiding CPET for the PPET‐HIP could safely and effectively control the conditions of patients with CDs.

Support or Funding Information

National Natural Science Foundation of China (81470204);

Fuwai Hospital, National Cardiovascular Institute of Chinese Academy of Medical Sciences (2012‐YJR02);

Research on Clinical Characteristics of the Capital (Z141107002514084);

Research and Outcome Promotion of Clinical Characteristics in the Capital (Project No. Z161100000516127)
Identifying Neural Correlates of Nicotine Withdrawal in Mice: The Neuregulin Signaling Pathway
Authors: Fisher Miranda , O'Donovan Bernadette , Adeluyi Adewale , Ortinski Pavel , Turner Jill
Published: April 2020

Addiction to nicotine and the ability to quit smoking are influenced by genetic factors. Therefore, it is important to understand how genes and drugs of abuse mechanistically impact each other. One well‐characterized protein responsible for regulating both response to drugs and gene expression is the transcription factor cAMP response element‐binding protein (CREB). Work from our lab indicates that hippocampal specific alterations in CREB signaling and synaptic plasticity underlie certain nicotine withdrawal (WD) phenotypes in a region‐specific manner. We found that CREB deletion in the ventral hippocampus (VH), a region known for regulation of mood and emotion, results in amelioration of nicotine WD‐induced anxiety‐like behaviors. High throughput chromatin immunoprecipitation sequencing (ChIP‐seq) studies determined that WD from nicotine differentially modulates CREB binding to the gene Neuregulin‐3 (Nrg3), a neural‐enriched epidermal growth‐like factor that plays a role in the formation and maintenance of mature synapses. Interestingly, genome wide association studies (GWAS) in humans have found that single nucleotide polymorphisms within the NRG3 gene and that of its cognate receptor, ERBB4, are associated with smoking cessation outcomes. In mice, qPCR and Western blotting experiments established that NRG3 and ErbB4 are upregulated at the 24h WD time point in the VH, with expression returning to baseline by 1‐week post WD. Conditional VH deletion of Erbb4 blocked WD‐induced anxiety‐like behaviors. This phenotype was accompanied by decreased levels of inhibitory GABAergic release and altered network clustering of excitatory pyramidal cells within the ventral CA1, an area enriched in Nrg3 and Erbb4 mRNAs and sensitive to nicotine WD. This data suggests that disruption of VH NRG3‐ErbB4 signaling attenuates WD‐induced anxiety‐like phenotypes through altering GABAergic modulation of CA1 pyramidal cell activity. Further examination of downstream signals of ErbB4 activation may lead to the identification of potential targets for treating nicotine withdrawal symptomology.

Support or Funding Information

This study was supported by

•NIH/NIDA grant DA032681, the PhRMA Foundation, and the USC ASPIRE Grant program (JRT).

•SPARC Grant, University of South Carolina (MLF)
The Deaf Culture Panel: Introducing Culture into Anatomy Education
Authors: Greene Sarah , Scott Jessica Armytage
Published: April 2020

The anatomy laboratory is a place where one can integrate education in basic science and professionalism in medicine. Cultural competence is one component of professionalism that has received less attention in the anatomical sciences as opposed to other competencies. Yet cultural competence is imperative in providing appropriate and effective communication and care across multiple patient populations. A lack of such competence has contributed to heath care disparities between hearing and d/Deaf and hard of hearing (DHH) patients. Therefore, this study sought out to introduce and evaluate a Deaf Culture panel presentation, which was provided as one of five Applied Anatomy sessions during the first‐year integrated medical student curriculum at Morehouse School of Medicine. This 90‐minute session was scheduled during the Head and Neck section of the course, soon after the ear and auditory system had been studied. Three panelists were invited to share their experiences in medicine during this panel; two were culturally Deaf and communicated in American Sign Language, and one utilized a cochlear implant and communicated in spoken English. Panelists shared their experiences and challenges in communication with the medical community about health care. This included incorrect assumptions made by health care professionals, challenges with access to appropriate communication, and the effect of this poor communication on their ultimate care. Although this panel was considered a lecture and therefore optional, a large majority of the class of 100 students were in attendance. This panel was evaluated in several ways. First, students were invited to complete optional pre‐ and post‐assessments to measure how this session impacted their knowledge and awareness of Deaf culture and working with DHH patients. Assessments demonstrated a significant increase in knowledge and awareness from pre‐ (n=33) to post‐assessment (n=13; P < 0.001). Next, students were asked to evaluate the session using a Likert scale of 1–5. The average rating of the session was 4.8, indicating that the session was quite well received. Next, students were given the option to write open‐ended comments on their experiences with the session. One such response included, “I learned several things that I will carry into my career and life in general.” Finally, informal observations by the authors during and after the panel suggested that students were highly engaged during the session, with several staying afterwards to ask questions of the participants one on one. These findings are promising in terms of incorporating this type of experience into medical education more broadly. This was a successful integration of a session to promote cultural competence and communication within the anatomy curriculum, which has the potential to directly impact future patient care.

Support or Funding Information

NIH, Grant number P03‐G12MD007602

U.S. Department of Education, Grant Number P03‐1B141018
Dynamic expression profiles during development of placodes in the embryonic chicken eye
Authors: Franz-Odendaal Tamara Anne , Giffin Jennifer
Published: April 2020

The conjunctival epithelium of the chicken eye develops a series of placodes beginning at embryonic day 6.5. These placodes develop into conjunctival papillae over the course of 1.5 days before disappearing. These transient structures are responsible for inducing the underlying mesenchyme to develop into a ring of scleral ossicles. Here we assess the gene expression patterns of five major signaling pathways known to be involved in the development of other epithelial appendages, namely the WNT/β‐catenin, bone morphogenetic protein (BMP), ectodysplasin (EDA), fibroblast growth factor (FGF) and hedgehog (HH) pathways. Our comprehensive qPCR analyses of distinct regions of the eye, at different time points, enabled us to assess multiple gene expression patterns. Our data show that FGF1, FGF20, sonic hedgehog (SHH), ectodysplasin receptor (EDAR) and BMP4 are amongst the most highly upregulated genes identified in the system. EDAR, FGF20 and SHH were also significantly different across different regions of the eye and over developmental time within specific regions. Correlation analyses among relative normalized gene expression values revealed the strongest correlation between EDA and lymphoid enhancer factor‐1 (LEF1) and between FGF2 and FGF10. The enhanced sensitivity of qPCR reveals a more detailed signaling pattern than previously known. The dynamic expression patterns uncovered here are consistent with a conserved patterning signaling network observed in a diverse range of other placodes, including hair follicles and feather buds.

Support or Funding Information

This study was funded by the Natural Sciences and Engineering Research Council of Canada
Cocaine‐induced locomotor stimulation is mediated by autophagic degradation of the dopamine transporter
Authors: Harraz Maged M. , Guha Prasun , Kang In Guk , Malla Adarsha P. , Semenza Evan R. , Song Young Jun , Treisman Isaac , Cortés Pedro , Coggiano Mark A. , Tanda Gianluigi , Snyder Solomon H.
Published: April 2020

Cocaine exerts its stimulant effect by inhibiting dopamine reuptake leading to increased dopamine signaling. This action is thought to reflect binding of cocaine to the dopamine transporter (DAT) to inhibit its function. However, cocaine is a relatively weak inhibitor of DAT, and many DAT inhibitors do not share the behavioral actions of cocaine. Here we show that sub‐nanomolar concentrations of cocaine elicit neural autophagy in vitro and in vivo. Pharmacologic inhibition of autophagy significantly reduces the locomotor stimulant effect of cocaine in mice. Autophagy depletes transporters for dopamine but not serotonin in the nucleus accumbens following cocaine injection. The dopamine transporter is enriched in the ventral midbrain lysosomal fraction following intraperitoneal injection of cocaine, which is reversed by inhibition of autophagy. Furthermore, autophagy inhibitors significantly reduce dopamine levels in the nucleus accumbens following cocaine injection. Taken together, these findings suggest that the behavioral stimulant effect of cocaine is mediated at least in part by autophagic degradation of DAT.

Support or Funding Information

Funding: this work was supported by U.S. Public Health Service Grants DA00266 and DA044123 to SHS and a NARSAD Young Investigator Grant (grant # 25360) from the Brain & Behavior Foundation to MMH. Support for this research was provided in part by the National Institute on Drug Abuse ‐ Intramural Research Program, NIH/DHHS (Z1A DA000611).
The Regulation of Fibroblast to Myofibroblast Activation by Zinc Finger E‐Box Binding Homeobox 1 and 2
Authors: Meikle Zach , Yu Liping , Netticandan Thomas , Wigle Jeffrey
Published: April 2020

After a myocardial infarction (MI), a dense collagen scar is formed that initially prevents the heart from rupturing. However, after the scar is formed, collagen deposits accumulate and the extracellular matrix (ECM) is remodeled in the viable areas of the heart, distal from the site of injury, which causes the entire organ to vstiffen in a process called cardiac fibrosis (CF). CF often contributes to the development of heart failure (HF) which is a major clinical problem in Canada. Cardiac fibroblasts (FBs) provide structural support and remodel the ECM in the healthy heart, but after an MI, FBs become activated, proliferate, and adopt a myofibroblast (MF) phenotype. MFs can continue to produce ECM long after they become activated which leads to CF and ultimately HF. Two transcription factors that have been identified as potential contributors to CF are zinc finger E‐box binding homeobox 1 and 2 (ZEB1/2). ZEB1 and ZEB2 are essential for epithelial‐mesenchymal transition (EMT) and, previous studies in our lab have shown that ZEB2 is also directly involved with FB activation. Based on other research we hypothesize that ZEB1 may also regulate FB activation and be functionally redundant with ZEB2. Our initial results show that ZEB1 protein and RNA levels both increase as FBs convert to MFs. By comparing samples at varying time points we have increased our understanding of the FB to MF activation process. This study show that ZEB1 may be involved in the fibrotic process and further analysis will determine the extent of its involvement.

Support or Funding Information

Supported by a grant from NSERC and from the St. Boniface Hospital Foundation Research without Borders initiative.
New‐old‐ways: where does innovation stand with technology in anatomy labs
Authors: Curcio Daniella , Laitman Jeffrey
Published: April 2020

At the Icahn School of Medicine at Mount Sinai (ISMMS), Structures is a very intensive course that encompasses and integrates the subjects of Anatomy, Histology and Embryology. In 2019, we have incorporated iPads into our course, Structures. We here report the insights and challenges we faced while planning and implementing the project. We also include the input from students and educators on the outcomes of this beta version, as well as their vision for the future. At the ISMMS, the first‐year class has 124 students, divided in 24 groups of 5 or 6 components. Each group performs a full cadaver dissection, supervised and supported by faculty, residents, surgeons, emergency attendants, clinical specialists, and teaching assistants (TAs). Our TAs take part in teaching, organize review sessions, and play a major role in Table Conferences—our in‐lab oral assessment on practical knowledge of Anatomy. The team involved in the iPad project was comprised by directors, faculty, TAs, physicians, members of the Medical Education Department, and members of the Information Technology Group: experts in software/hardware and specialists in design and content development. Our goal is to provide our students with the foundation to proceed into the second‐year, without losing sight of the step‐exams, and use the iPads to facilitate content integration. The use of technology in the anatomy lab is not a new paradigm, but our experience shows that that in the area of custom‐tailored content and purposeful selection of apps, innovation and creativity has plenty of room to grow and flourish.