bluuebunny/crossref_metadata_2025 huggingface dataset:
Examining Curricular Trends in Introductory Biochemistry Courses at Large Universities
Authors: Sharp Katherine Alberta , Everson Heather Rose , Krall Rebecca McNall
Published: April 2020

Current conversations in chemical education highlight the importance of student‐centered learning approaches and the need to implement such learning approaches in college classrooms. However, commentary and other conversations continue to suggest traditional lectures are predominantly used to teach biochemistry. The goal of this study was to determine curricular trends within introductory biochemistry courses at R1 institutions of higher education. By determining the current state of biochemistry education, strategies can be developed to promote student‐centered learning strategies, particularly in large lecture courses common in biochemistry at the introductory level. The primary focus of this study was to identify common teaching and learning strategies presented in course syllabi. Introductory‐level biochemistry syllabi were collected from 42 R1 institutions. All syllabi were approved by the American Chemical Society (ACS) or counted towards an ACS‐approved degree/program. Syllabi were assessed on course delivery methods, pedagogical strategies used within the course, forms of formative and summative assessment, activities to establish real‐world connections, and major topics presented to students. The results indicate that most courses were presented in a traditional lecture format with few integrating student‐centered strategies such as peer‐led team instruction. Similarly, few syllabi described instructional scaffolds used to construct real‐life connections with biochemistry content. Topics most frequently addressed in the courses included fundamental building blocks, biopolymers, and supramolecular structures. Not surprising was the ubiquitous use of large lecture‐style courses. Lecture has long been shown to be an efficient method of instruction that promote memorization of facts rather than cultivating complex understandings essential in problem‐solving. Results presented demonstrate the need for student‐centered strategies in biochemistry at R1 universities to promote deeper learning, develop critical thinking and problem‐solving skills, and foster connections between content and real‐world applications students could face in their future careers.
The GID E3 Ubiquitin Ligase Converts Between Anticipatory and Active States Through the Incorporation of Swappable Substrate Receptors
Authors: Langlois Christine R. , Qiao Shuai , Sherpa Dawafuti , Chrustowicz Jakub , Beier Viola , Karayel Ozge , Schulman Brenda A.
Published: April 2020

Cells must have the ability to rapidly respond to environmental cues in order to adapt to changes in nutrient availability or stress. Cellular changes at the protein level can take many forms, including modification of the activity of previously existing proteins, synthesis of new proteins, and degradation of proteins that are no longer needed. Indeed, protein turnover by E3 ubiquitin ligases provides a rapid and irreversible mechanism for cellular response. For example, yeast cells starved for sugar require gluconeogenic production of glucose. However, upon glucose availability, this energetically expensive process must be terminated. The GID (
Glucose‐
Induced
Degradation) E3 ligase plays a pivotal role in this process by targeting gluconeogenic enzymes, including Fbp1 and Mdh2, for degradation. However, the precise molecular mechanisms that govern the regulation of the GID ligase remain unclear.

To understand the mechanism of the active GID E3 complex, we combined in vivo studies in yeast cell biology and biochemistry with in vitro biochemical studies reconstituting the endogenous properties with recombinant proteins. We found that in response to sugar starvation, an inactive GID complex assembles in cells in anticipation of upcoming environmental changes. Following sugar availability, the GID complex is activated through the binding of its substrate receptor, Gid4, which allows the complex to recognize and ubiquitinate key gluconeogenic enzymes. Furthermore, the GID complex is not a singular complex, but is instead a family of multisubunit E3 ligases able to respond to a variety of cellular stresses through the incorporation of swappable substrate receptors.
Neuroinflammation and Blood Brain Barrier Injury in a Murine Model of Cardiac Arrest
Authors: Ousta Alaa , Piao Lin , Fang Yong Hu , Vera Adrianna , Nallamothu Thara , Garcia Alfredo J. , Sharp Willard W.
Published: April 2020

Background

Neurological injury is often evident following resuscitation from cardiac arrest and yet, the pathophysiology of this injury is not well understood. Moreover, treatment options are limited to targeted temperature management (TTM) and conservative care. We hypothesize that neuroinflammation and blood brain barrier (BBB) injury are associated with neurological injury following cardiac arrest and represent potential targets for therapeutic intervention.

Methods

We randomized adult C57BL/6 male/female mice to either a 12‐minute KCl‐induced asystolic CA ‐ under anesthesia and ventilation ‐ followed by CPR (N=12) or sham intervention (N=9). Neurological assessments of mice were performed using standardized neurological scoring, video motion tracking, and sensory/motor testing. BBB permeability was assessed by injection of fluorescein isothiocyanate (FITC) dextran beads. Changes to immune cell number in the brain was assessed by immunohistochemistry using the microglia marker (Iba1+).

Results

Neurological injury following cardiac arrest was evident by neurological scores (difference of 3.419 ± 0.5576, 95% CI 2.25–4.586), sensory scores (difference of −61.39 ± 16.65 seconds, 95% CI −97.10 to −25.67), motor scores (difference of −107.3 ± 6.503 seconds, 95% CI −121.2 to −93.35) and video analysis of distance (difference of 8.914 meters ± 1.411, 95% CI 4.425 −13.40) and speed (difference of 0.07428 m/s ±0.01175, 95% CI 0.03687 to 0.1117). Post‐CA brains demonstrated increased bead fluorescence compared to controls indicative of increased blood brain barrier injury. Moreover, Iba1+ cell number was approximately 2.47 times greater and integrity density was approximately 4.66 times greater post‐CA suggesting neuroinflammation following CA.

Conclusions

Global neurological injury is evident following asystolic cardiac arrest in mice but is particularly prominent in regard to motor movements. This injury is associated with BBB injury and nueroinflammation. Ongoing studies are determining the mechanisms and possible therapeutic targets of this injury.

Support or Funding Information

The project described was supported by Grant Number T32HL007381 from the National Heart, Lung, and Blood Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute or the National Institutes of Health.
Structural Analysis of Arabidopsis thaliana Cellulose Synthase A1 Catalytic Domain
Authors: Massenburg Lynnicia , Vandavasi Venu , O’Neill Hugh , Kumar Manish , Nixon Tracy
Published: April 2020

Previous evidence has shown that the Arabidopsis thaliana CESA1 catalytic domain (AtCESA1catD) can form stable monomers and oligomers. Negative stain TEM and initial cryo‐EM screens of monomer revealed a fit to the cotton CESA1 computational model (Gh506). Optimized cryo‐EM screens pushed the limits of cryo‐EM sub‐100 kDal imaging to observe domain detail in the 59.7 kDal AtCESA1catD. The first cryo‐EM optimization strategy utilized the Volta Phase Plate at low defocus to capture high contrast images with more detail. This strategy yielded problematic maps and was not ideal to yield a high resolution structure. The second cryo‐EM optimization strategy used higher electron dose over a defocus range. This strategy revealed the P‐CR and CSR domains and is very promising for high resolution structures. We purified AtCESA1catD monomer and showed that negative stain structure fits to Gh506. The 2nd cryo‐EM optimization is promising for high resolution, and future work will involve picking more protein particles followed by low‐ to high‐resolution refinement.

Support or Funding Information

The work was fully supported as part of The Center for Ligno Cellulose Structure and Formation, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science. Additional support was given by the Alfred P. Sloan Foundation’s Minority Ph.D. (MPHD) Program, awarded in 2018–19 (Grant No.: G□2016‐20166039).
Role of CD28 Signaling on T cell Phenotype following Lymphodepletion and Homeostatic Reconstitution
Authors: Sood Nitish , Crepeau Rebecca L. , Liu Danya , Ford Mandy L.
Published: April 2020

Calcineurin inhibitors, the current standard of care in immunosuppression for transplant patients, leads to nephrotoxicity and loss of graft function over time. Preclinical studies have shown selective CD28 blockade (CD28 dAb, Lulizumab) to be superior to calcineurin inhibitors in reducing toxicity and prolonging allograft survival. Emory University will soon commence a Phase 2 clinical trial testing the efficacy of a Lulizumab‐based immunosuppression regimen in kidney transplantation. Patients in the trial will be T cell depleted (TCD), rendering them lymphopenic at the time of transplant and will therefore undergo homeostatic reconstitution in the weeks following transplantation. Lulizumab prevents T cell activation by selectively targeting CD28 signaling, but it is presently unclear what effect blocking this pathway will have on T cell phenotypes following homeostatic reconstitution (HR) in these patients. Previous studies have shown that HR leads to increased differentiation of naïve T cells into memory cells and to premature senescence. We hypothesized that blocking CD28 signaling in T cells during this process could prevent memory differentiation and senescence. We investigated this question using a murine model of TCD and skin transplantation. Four groups of mice were established: no treatment control, TCD alone, CD28 dAb alone, and both TCD and CD28 dAb (n = 4). Mice were sacrificed at 6 weeks post‐depletion and transplantation, and blood and tissues were collected for flow cytometric analysis. Using both traditional flow cytometry analysis and an unsupervised machine‐learning approach, we found that TCD CD4+ T cells have significantly elevated expression of the memory markers CD27, CD103, and CD69 when compared to no treatment control CD4+ T cells, indicating HR‐induced differentiation into memory T cells. In addition, TCD CD4+ T cells also expressed significantly higher levels of PD‐1, TIGIT, and CD226 (DNAM‐1) when compared to no treatment control CD4+ T cells, indicating a higher degree of senescence. Finally, we found that treating TCD mice with CD28 dAb restored expression of both memory markers and costimulatory markers to baseline levels. As such, we believe CD28 dAb presents a viable method to control HR‐induced differentiation and senescence of CD4+ T cells. These results were consistent across the blood, lung, and spleen. Our future directions include using murine models to determine whether the selective CD28 blockade‐mediated rescue of the HR‐induced phenotypic changes significantly affects protective immunity to infection.

Support or Funding Information

Research reported here was supported by the National Institutes of Health under award number R25 DK 101390 ‐ Title: Summer Undergraduate Program in Renal Research (SUPERR).
Effect of Organosulfide Diallyl Trisulfide (DATS) from Garlic on Racially Different Triple Negative Breast Cancer Cells
Authors: Kanga Konan W. , Mendonca Patricia , Soliman Karam F.A. , Darling-Reed Selina
Published: April 2020

In triple negative breast cancer (TNBC), inflammation and immune response play a major role in the progression and metastasis of the disease. TNF‐α is a first response cytokine in the developing of the tumor and is released by macrophages to destroy the tumor due to its toxic effect on cancer cells. If TNF‐α is unable to destroy the cancer cells, macrophages begin to mimic the characteristics of cancer, eventually turning into tumor‐associated macrophages (TAM). Meanwhile, diallyl trisulfide (DATS), an organosulfide found in garlic, has previously been reported to be effective in chemoprevention, apoptosis, and cell cycle arrest in DNA damaged normal breast epithelial cells, breast cancer cells, as well as other cancers. The objective of this study is to investigate the effects of the natural compound DATS on the release of TNF‐α‐induced tumor promoting cytokines in TNBC cells, specifically MDA‐MB‐231 and MDA‐MB‐468 (representing Caucasians and African Americans, respectively), and to evaluate those specifically associated with TAM recruitment. Cell viability, cytokine arrays, and specific ELISAs were performed to investigate DATS effects on TNBC cells. In cell viability studies, DATS showed a dose‐response effect in concentrations ranging from 0–200 μM in MDA‐MB‐231 and MDA‐MB‐468 cells. Based on these results, we established a concentration of 75 μM to be used for further experimentation. The cytokine arrays showed that TNF‐α (40 ng/mL) induced the up‐regulation of monocyte chemoattractant protein‐1 (MCP‐1/CCL2) in Caucasians and African Americans cell lines; however, interleukin‐6 (IL‐6) was only up regulated in MDA‐MB‐231 cells. DATS treatment showed to be effective in inhibiting CCL2 expression in MDA‐MB‐231, but not in MDA‐MB‐468 cells. In addition, IL‐6 also showed a down regulation after DATS treatment in Caucasian cells. In order to validate the results from the arrays, ELISA quantitative assays specific for CCL2 and IL‐6 were performed. The results confirmed that TNF‐α induces expression of CCL2 expression in both breast cancer cell lines, and DATS treatment leads to the down regulation of CCL2 in MDA‐MB‐231 only, with no significant effect in MDA‐MB‐468 cells. The data from this study showed that DATS has the ability to modulate the immune response involved with TAM and cancer progression through CCL2 down regulation in MDA‐MB‐231, but not in MDA‐MB‐468 cells. These findings may also provide a better understanding of the poor therapeutic response in African American patients with TNBC.

Support or Funding Information

Supported by a grant from NIMHD U54 007582
Concentration‐ and time‐dependent effects of benzalkonium chloride in human lung epithelial cells; fate of necrosis, apoptosis, or epithelial mesenchymal transition
Authors: Kim Sou Hyun , Son Seung Won , Choi Hun Ji , Lee Wongyu , Jung Young-Suk
Published: April 2020

Benzalkonium chloride (BAC), an antimicrobial agent in inhalable medications and household sprays, has been reported to be toxic to pulmonary organs. Although cell membrane damage has been considered as the main cytotoxic action of BAC, its concentration‐ and time‐dependent cellular effects on lung epithelium has been not fully understood. In the present study, human lung epithelial (H358) cells were exposed to 0.2–40 μg/mL of BAC for 30 min to 21 days. Cell membranes were rapidly disrupted by 30 min exposure, but 24 h incubation of BAC (4–40 μg/mL) dominantly caused apoptosis rather than necrosis. BAC (2–4 μg/mL) induced mitochondrial depolarization which may be related to the increased pro‐apoptotic proteins (caspase‐3, PARP, Bax, p53, and p21), and the decreased anti‐apoptotic Bcl‐2. IRE1α, BiP, CHOP, and p‐JNK proteins were also elevated by BAC (2–4 μg/mL) suggesting the possible involvement of ER stress in the apoptosis. Long‐term (7–21 days) incubations of BAC (0.2–0.6 μg/mL) did not affect the cell viabilities but led to epithelial mesenchymal transition (EMT) as shown as the decrease of E‐cadherin, and the increases of N‐cadherin, fibronectin, and vimentin by up‐regulating the EMT transcription factors, Snail, Slug, Twist1, Zeb1, and Zeb2. Therefore, we conclude that apoptosis can be an important mechanism of acute BAC cytotoxicity in lung epithelial cells, and chronic exposure to BAC even at sub‐toxic doses can promote pulmonary EMT.

Support or Funding Information

This study was funded by Nano‐Material Technology Development Program through the National Research Foundation of Korea (NRF) funded by Ministry of Science and ICT (NRF‐ 2018M3A7B4071233).
Academic Effectiveness of a Medical Teacher as Perceived by the Educators Themselves and Their students
Authors: Jurjus Rosalyn A. , Hijal Nora , Farhat Rima , Hussein Inaya Hajj , Jurjus Abdo
Published: April 2020

Introduction

The essence of medical school education derives from not only the knowledge of the teacher, but also the effective dissemination of such knowledge to the medical student. This process constitutes the foundation of a fruitful medical education – transforming basic science information to tangible, clinical applications. There are various perspectives as to what facilitates this process, and these come from both the perspective of the medical student as well as from those of the educators. The aim of this study is to look into the qualities of an effective teacher in the medical school environment as perceived by both the educator and the learners.

Methods

An anonymous online self‐administered Likert‐scale survey in English using the Lime online survey tool was administered to the teaching faculty and first through fourth year medical students. Data was analyzed using the Chi Square Test. Perceptions on the elements of effective teaching was broken down to five main elements in the survey. Analytical/synthetic approach, Teacher Performance/Organization, Teacher‐group interaction, Teacher‐Individual student interaction, Dynamism/enthusiasm.

Results

Many attributes and characteristics are rated of same importance by both the educators and the students. However, the educators rated much higher than the learners items like “Stating Learning objectives for each class session” (p< 0.001), “Identifying what he/she considers important” (p< 0.001), “Interacting with students” (p< 0.001), and “Motivating students” (p< 0.001). Other attributes like, “making students feel comfortable” (p=0.001), “encouraging class discussion” (p=0.001), “inviting students to share their knowledge and experiences” (p< 0.001) were also ranked significantly higher by the educators. As for the desirable characteristics that make a session effective, the educator rated higher being Enthusiastic, Composed, Well and properly dressed, and Disciplined (p< 0.001).

Conclusions

Learners do not share the same outlook on what makes a great medical educator with the teachers themselves. In the future, we plan to run a multicenter, international study to compare and contrast the perceived attributes by students and teachers from multiple medical schools in different countries and from various cultural backgrounds.
In Vitro Modulatory Evaluation of Mas Receptor/Angiotensin‐(1‐7) in Doxorubicin‐induced Cardiotoxicity
Authors: Marques Juliana Romeu , Frozza Fernanda Tereza Bovi , Guecheva Temenouga , Irigoyen Maria Cláudia , Leguisamo Natalia Motta
Published: April 2020

Background

Doxorubicin (DOX) antineoplastic is considered the prototype of cardiotoxicity for inducing direct damage to the myocardium. Pharmacological blockage of Angiotensin (Ang) II action is one of the main strategies to manage DOX‐induced cardiomyopathy. The effects are also mediated by the activation of the Mas receptor (MasR)/Ang‐(1‐7) axis, which is the counter‐regulatory axis of Ang II. The modulation of MasR/Ang‐(1‐7) axis is Ang‐(1‐7) (agonist) and A779 (antagonist) and may be a potential strategy in the context of cardiotoxicity.

Objective

Evaluate the influence of pharmacological modulation of MasR/Ang‐(1‐7) axis on DOX‐induced cardiotoxicity in cardiomyoblast cells.

Methods

Murine cardiomyoblasts (H9c2) were treated with A779 (10μM) for 30′ followed by Ang‐(1‐7) (100nM) for 30′ previous to expose with 0.1μM and 0.35μM (IC50) of DOX for 24h. Cell viability (Neutral Red and Trypan Blue), cell death profile (Annexin/7AAD), DNA damage induction (Comet Assay) and mitochondrial membrane integrity (Rhodamine 123) were evaluated after treatment protocol. The experiments were performed in triplicate, and data were analyzed by ANOVA followed by Dunnet, considering p <0.05.

Results

MasR modulation did not prevent the reduction of viability of cardiomyoblasts by DOX, neither altered the pattern of cell death. Both concentrations of DOX induced DNA strand breaks (C: 27.5 ± 13.4; DOX 0.1μM: 53.5 ± 9.1; DOX IC50: 109.5 ± 17.6 U.A.). Pretreatment with peptides suggested a reduction of the frequency of nucleus damage (DOX 0.1μM + Ang‐(1‐7): 36.5 ± 6.3; DOX 0.1μM +A779: 36.5 ± 0.7 U.A.) only at 0.1 μM DOX concentration, while pretreatment with A779 or Ang‐(1‐7) preserved the mitochondrial membrane integrity.

Conclusion

Modulation by agonism or antagonism of MasR in H9c2 cells reduces the DNA damage and the mitochondrial integrity. These results seem to be independent of MasR modulation, pointing to the involvement of other receptors and their crosstalk.

Support or Funding Information

CAPES, CNPq and FAPICC/ICFUC
Microenvironment‐responsive nanoparticle platform for anticancer drug delivery.
Authors: Wlodarczyk Marek Tadeusz , Dragulska Sylwia , Poursharifi Mina , Cheng Ying , Martignetti John , Mieszawska Aneta
Published: April 2020

Chemotherapy is a leading method of anticancer treatment. However, most of the chemotherapy agents have high systemic toxicity, poor bioavailability and solubility, which limit their clinical dose and decrease therapeutic outcomes. Nanoparticle (NP)‐based drug delivery systems have a potential to address these problems. We describe the formulation of a pH sensitive NP platform suitable for anticancer drug delivery. The NP’s core is composed of polylactic‐co glycolic acid (PLGA) conjugated to a platinum‐based drug. The NP’s corona is composed of a targeting ligand against Mucin 1 – a protein overexpressed on some epithelial cancer cells, and a pH‐sensitive pegylated phospholipid shell that provides high water solubility of the NPs. PLGA is biodegradable, biocompatible, and is FDA approved. The pH sensitive coating is achieved with a hydrazone linker that connects phospholipids with hydrophilic polyethylene glycol (PEG). Hydrolysis of the PEG‐hydrazone‐phospholipid occurs faster at slightly acidic environment (pH 6.8), which is characteristic of a tumor. The purpose of PEG cleavage is to unshield the targeting ligands on the NPs and facilitate fast NPs uptake by the cancer cells through receptor‐mediated endocytosis. The DNA aptamer is used as targeting ligand against Mucin 1, and the NP was tested in ovarian cancer model where Mucin 1 was found to be highly overexpressed. Our platform has been tested in both chemo naïve and chemo resistant ovarian cancer cell lines, including A2780, CP70, ES2, TOV21G, OV90. The NPs uptake and biological activity was high in all cells tested, and the results were compared to carboplatin, a free drug analogue. Also, preliminary in vivo studies demonstrated efficient NPs accumulation within the tumor tissue. The NP design is flexible and can easily be extended to delivery of other anticancer agents, or to the treatment of different tumor types.

Support or Funding Information

Supported by NIH Grant
Case Report: A Unique Unilateral Variation of a Divided Extensor Digitorum Muscle in a Subject with Bilateral Absence of Extensor Indicis
Authors: Schettler Stephen P. , Lister James P. , Miller Joseph M.A.
Published: April 2020

Reports of variations in the extensor digitorum muscle have been documented since at least 1813. These variations have fallen into 6 groups: (1) deficiencies in tendon number – usually the tendon to the 5thdigit being absent, (2) doubling of tendons to digits, (3) difference in number of muscle bellies, (4) presence of tendinous slips that join other muscles, (5) presence and arrangement of intertendinous connections, (6) presence of separate muscle bellies for each tendon, and (7) accessory muscle bellies.

The present case report describes a unilateral variation of the extensor digitorum muscle, observed in a 73 year‐old Caucasian male with bilateral absence of the extensor indicis muscles. During anatomical dissection of this individual, we observed that the extensor digitorum muscle on the subject’s right side appeared to be divided into two distinct muscle masses. The lateral muscle mass (1 cm wide at its thickest point and 11 cm in length) originated from the medial aspect of the extensor carpi radialis brevis and provided a tendon (20 cm in length) to the index finger. The medial muscle mass had the usual origin to the lateral epicondyle via the common extensor tendon and provided tendons to the third and fourth digits. Interestingly, the extensor indicis muscles were also absent bilaterally.

While cases of absent extensor indicis muscles have been reported previously, we are not aware of any report in the literature that describes a divided extensor digitorum muscle or such a divided extensor digitorum muscle occurring in the presence of bilaterally missing extensor indicis muscles. Indeed, to our knowledge, this is the first such report of this occurrence. Thus, to the 6 groups of variant types commonly recognized for extensor digitorum as listed above, we now add a 7th new category of “divided muscle mass” (which in our case is “bipartite”, but future studies may discover other arrangements). Developmental and functional considerations of this variation will be further discussed.

Support or Funding Information

Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA
Beta3‐Adrenergic Receptor (B3AR) Activation Induces Estrogen Receptor Beta (ERb) and Improves Mitochondrial Metabolism in Adipocytes
Authors: Queathem Eric D. , Welly Rebecca , Lubahn Dennis , Rector R. Scott , Clart Laura , Fritsche Kevin , Vieira-Potter Victoria
Published: April 2020

In vivo, ERb activation protects against obesity and white adipose tissue (WAT) dysfunction. Mechanisms are unknown, but we hypothesize that it involves adipocyte‐specific mitochondrial activity via induction of OXPHOS and UCP1– proteins that characterize WAT “browning,” a process sufficient to rescue metabolic dysfunction. In cancer cells, increasing mitochondrial ERb increases OXPHOS transcription and mitochondrial activity via a mechanism requiring mitochondrial transporter, GRP75. We sought to determine whether this mechanism applies to adipocytes in vitro, and the potential in vivo implications. In wild type (WT) mice, the adipocyte‐specific B3AR ligand, CL316,243 (CL) induced WAT UCP1 and ERb protein (Western blot), and GRP75 mRNA (qPCR) (all n=10/grp, P<0.05). In primary adipocytes from WT mice (n=3/treatment), CL increased UCP1 (~2‐fold; P<0.05) and ERb protein (~4‐fold; trend, P=0.097). In vivo, we show that adipocytes harvested from mutant mice missing ERb DNA binding domain (ERbDBDKO) display suppressed mitochondrial O2 consumption (via Oroboros; n=6–8/grp, P<0.05) and lower OXPHOS protein expression (P<0.05). We next tested whether ERbDBDKO mice would have impaired exercise‐induced WAT browning. Remarkably, unlike WT (~3‐fold UCP1 increase; P<0.05), ERbDBDKO mice were nonresponsive to exercise induction of UCP1 protein in WAT. Taken together, our data support a critical role for adipocyte ERb in mediating lipolysis driven mitochondrial responses. We hypothesize that the mechanism involves GRP75. Identifying the mechanism by which ERb enhances adipocyte mitochondrial activity has enormous potential to treat a spectrum of metabolic diseases.
The Perception and Emotional Experiences of Ethiopian Medical Students during Human Cadaver Dissection
Authors: Gemechu Jickssa , Zenebe Abay , Wasserman Jason , Barremkala Malli , Hussein Inaya Hajj
Published: April 2020

Human cadaver dissection has been used as an instructional methodology in Anatomy for centuries and remains time‐honored and accepted method. However, initial exposure to cadaver dissection has been found to evoke emotional reactions in medical students, and has raised concern. Previous studies report that some students experience stress and even psychological trauma, with a concern about exposure to disease during dissection. The emotional experiences and coping strategies may also be affected by cultural differences between medical students. The main aim of this study is to assess and compare medical students’ perception, emotional experiences and the impact of cultural attachment on the dissection of the human cadaver. A longitudinal survey was conducted focusing on cultural identity, prior experience with cadaver dissection, emotional experiences, and coping strategies in a medical school of Addis Ababa University, Ethiopia in 2018. Data were collected at three time points a week before initial exposure to dissection (time 1), a week after the first encounter (time 2), and at the end of cadaver dissection (time 3). Findings indicate that self‐rated perception of evoked emotional experiences enjoyable, stimulating, exhilarating, and interesting increased across the three time points, while scary, depressing, unbearable, and anxiety‐provoking emotions significantly decreased. These results suggest that while cultural attachment appears to affect the rate of change over time in several different emotional responses, religious affiliation (Christian, Muslim) or an ethnic background (Amhara, Oromo or others) per se, do not manifest any significant differences across these emotional experiences, nor the rate at which they change for individuals in the sample.
seededclustR: Using Cluster Analysis of Mitochondria to Improve Cell Segmentation and Automated Quantification of mtDNA in Smooth Muscle Cells.
Authors: dos Santos Anita , Ng Irvin , Poburko Damon
Published: April 2020

Intro

Segmentation of fluorescently labelled cells is essential to pipelines of automated image analysis. Identifying regions of interest (ROIs) around labelled nuclei is relatively easy using tools available in ImageJ/Fiji, but determining the bounds of cells can be much harder. We asked if fluorescently labelled mitochondria could be used to improve the automated generation of cellular ROIs.

Method

The perimeter coordinates of nuclear ROIs and the centroid coordinates, area, mean intensity, angle, and solidity of ROIs around MitoTracker Orange (MTO)‐labelled mitochondria were generated in ImageJ and exported to R. Using the nuclei as seeds for a recursive k‐Nearest Neighbour search extending from each nucleus, MTO particles were assigned to their nearest‐neighbouring cells. A concave hull was created around MTO particles assigned to each cell using the concaveman package. We determined the sensitivity of this algorithm by comparing the accuracy of the generated concave hulls to the corresponding hand‐drawn ROIs, and ROIs from Voronoi expanded distance maps

Results

When we compared seededClustR ROIs to those drawn by hand, the Intersection‐Over‐Union (IOU) was 68 ± 8% (mean ± SD), Precision was 88 ± 6% and Recall 95 ± 3%. In contrast, Voronoi expansion of nuclei resulted in the IOU of 46 ± 18%, Precision of 83 ± 17%, and Recall of 91 ± 11%. Optimal IOU and Recall were observed when each MTO particle was compared to its 7–10 nearest neighbours. IOU and Recall were maximized using recursive steps of 40–200 μm, while Precision decreased from 94% to 88% as step size increased from 7–40 μm. Unexpectedly, extending seededClustR to an n‐dimensional kd‐tree calculation that included varying weights of MTO particle mean intensity, area, standard deviation, angle and solidity reduced cell tracing accuracy. Similarly, using Wilks’‐test to identify outliers decreased IOU by 17 percentage points (pp) and Recall by 20 pp, though it increased Precision by 6 pp. Finally, we tested whether if the more accurate cell ROI from seededClustR versus Voronoi ROIs could improve our ability to detect changes in optical measures of mtDNA in A7r5 smooth muscle cells treated with angiotensin II, 5,6‐dideoxycytidine, and H2O2, where results showed promising gains.

Conclusion

seededClustR is a generalizable algorithm that can use simple XY coordinates of puncta‐like features of cells to generate cellular ROIs around labelled nuclei with measures of accuracy that exceed simple dilation of nuclear ROIs. This approach should facilitate improved precision in automated screens of cellular phenotypes.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada
Role of Wnt5a in Switching Macrophage Phenotype in the Tumor Microenvironment
Authors: Hazen Alyx T. , Coschigano Karen T. , Malgor Ramiro
Published: April 2020

In 2018, cancer was the second leading cause of death in the United States. Past research has demonstrated the importance of macrophages in both the tumor microenvironment and in cancer progression. Macrophages are specialized immune cells that are differentiated into either the M1 phenotype (pro‐inflammatory/tumor‐suppressing) or M2 phenotype (anti‐inflammatory/tumor‐promoting), depending on the signals in the tumor microenvironment. Research has demonstrated that the expression of Wnt5a, a secreted glycoprotein, is correlated with a cancer’s level of aggression. Additionally, evidence shows that the treatment of M1 macrophages with Wnt5a increased the expression of IL‐10, a molecule expressed by M2 macrophages and associated with the progression of cancer. The objective of this study is to investigate the effect of exogenous Wnt5a on the expression levels of M1/M2 phenotypic markers. The hypothesis is that Wnt5a facilitates the switching of macrophages from the M1 to M2 phenotype through the induction of IL‐10. This would support the idea that Wnt5a functions as a tumor promoter and potentially contribute in the progression of cancer.

To test this hypothesis, THP‐1 and human peripheral blood monocyte derived macrophages were evaluated for expression of the M1 markers CD80 and IL‐6 and the M2 markers CD163 and IL‐10 using flow cytometry and RT‐qPCR either with or without Wnt5a treatment.

Our preliminary results confirm that, without Wnt5a treatment, M1 macrophages strongly expressed Wnt5a and, further, demonstrate that, after treatment with exogenous recombinant Wnt5a, the expression of M2 markers, including IL‐10, increased, while the expression of M1 markers decreased. Results from this study lead us to speculate that Wnt5a could play a role in switching macrophage phenotype from M1 to M2 and thus determining the overall tumor behavior. Furthermore, the manipulation of Wnt5a signaling could potentially be an attractive strategy in the development of more effective cancer treatments.

Support or Funding Information

The John J. Kopchick MCB/TBS Undergraduate Student Support Fund (Ohio University) andThe Provost Undergraduate Research Fund (Ohio University)
Characterization of the Inhibition of Allelic Variants of Cytochrome P450 2A6 with trans‐Cinnamic Aldehyde
Authors: Riehlman Emily , Mogstad Emily , Harrelson John , Chan Jeannine
Published: April 2020

Cigarette smoking and nicotine addiction continues to be a leading cause of preventable death. The enzyme cytochrome P450 2A6 (CYP2A6), which is highly polymorphic with over forty known alleles, is the primary enzyme that catalyzes the initial oxidation of nicotine in humans. Studies have shown that trans‐cinnamic aldehyde (tCA) is a mechanism‐based inactivator of this enzyme and is therefore a potential smoking cessation aid. However, the inhibitory effectiveness of tCA with the gene products of the different alleles is unknown. The goal of the work presented was to characterize inhibition of the CYP2A6 variant resulting from the *23 allele by tCA. This allele results in a point mutation (R203C). Site‐directed mutagenesis was used to engineer CYP2A6*23 from a truncated form of the human CYP2A6*1 gene. The recombinant protein was expressed in E. coli and purified. Using ligand‐binding titrations, we found that the Kd for tCA binding to CYP2A6*23 was 7.1 μM, whereas the Kd value for CYP2A6*1 was 10.6 μM. Consistent with previous reports, CYP2A6*23 had lower activity than that of CYP2A6*1; however the relative inhibition of both CYP2A6*23 and CYP2A6*1 by 80 μM tCA was similar. Therefore, while tCA has a slightly higher affinity for CYP2A6*23, the change to a cysteine at position 203 does not significantly affect the effectiveness of tCA as an inhibitor. So tCA could still be a potential smoking cessation aid for individuals with the CYP2A6*23 allele.

Support or Funding Information

National Institute on Drug Abuse Award Number 15DA042341; Pacific University Faculty Development Grant
Investigating the Molecular and Functional Impact of a Novel DLG2/PSD‐93 Mutation in Neurodevelopmental Disorders
Authors: Yang Eddy , Scott Daniel , Nithianantharajah Jess
Published: April 2020

Neurodevelopmental disorders (NDDs) such as autism spectrum disorder and intellectual disability involve aberrations in normal brain development. Human genomic studies of NDDs continue to identify mutations that converge on genes encoding postsynaptic proteins, including the synaptic scaffold DLG2 (PSD‐93 protein). Currently, we still do not fully understand the functional significance of DLG2 mutations and how the protein is impacted in these NDDs. To shed light on this, we investigated a novel DLG2 variant that has been identified in two brothers with intellectual disability and autistic‐features. We found the mouse Dlg2 R714N mutation (corresponding to human R837N) did not greatly change expression levels of PSD‐93 in HEK293 cells. We also found that binding of R714N PSD‐93 to a known interacting partner protein was largely unaffected. However, structural analyses showed that the R714N mutation may alter the conformation of the protein which results in decreased protein stability when protein‐bound. Therefore, our results show that the human DLG2 R837N missense variant likely leads to subtle changes in the shape and stability of the PSD‐93 protein. This may impact its structural role to dynamically interact and reorganise proteins at the postsynapse and thus, may impact synaptic remodelling and plasticity. Subsequent work is underway to assess this mutation in mouse neuronal cultures by examining impacts to synapse number and signalling. This work will help elucidate mechanisms of DLG2/PSD‐93 dysfunction and will increase our understanding of the functional impact of postsynaptic gene mutations in NDDs.

Support or Funding Information

Research supported by the Australian Government Research Training Program Scholarship
A Report on a Novel Variation of the Lateral Circumflex Femoral Artery Among Humans
Authors: McGarvey Terence W. , Bhattacharya Arunabh , Newman-Caro Avalouisa B. , Fajardo Roberto J.
Published: April 2020

The medial and lateral circumflex femoral arteries are critical to the proper perfusion and function of the hip joint. The lateral circumflex femoral artery (LCFA) is used a graft material in coronary artery bypass, and tissue flaps that include this artery, are harvested for plastic surgery reconstructions. Although the two circumflex femoral arteries typically originate from the deep (profunda) femoral artery (DFA), variations in their origins have been reported. Here we report on a unique variation of the LCFA in a donor body. A cadaveric dissection of the anterior thigh was bilaterally performed at the University of Incarnate Word School of Osteopathic Medicine to demonstrate the origin and branching of the LCFA. Bilateral asymmetry in the origin of the LCFA and the medial circumflex femoral artery (MCFA) was observed in this cadaver. On the left side, the LCFA originated from the common femoral artery (CFA) deep to the inguinal ligament. On the right side of the body, the LCFA originated from the DFA. While the MCFA took origin from the DFA on the left side of the body, it shared a common origin with the DFA from the CFA on the right side. In summary, we report a unique unilateral variation of the LCFA where it originates from the CFA deep to the inguinal ligament. This high origin for the LCFA, at the boundary between the external iliac and femoral arteries, has not been previously reported and is unique even among mammals. It is important to determine the branching patterns of the LCFA and the derivative branches of the femoral artery as they are critical arterial pathways for the lower limbs and because the LCFA is a target in coronary grafts and plastic surgery skin flaps.
The localization and topography of the arteries on the middle forehead region for eluding complications following forehead augmentation: Conventional cadaveric dissection and ultrasonography investigation
Authors: Phumyoo Thirawass , Jiirasutat Nuttapatch , Jitaree Benrita , Rungsawang Chalermquan , Tansatit Tanvaa
Published: April 2020

Forehead augmentation with filler injection is one of the most dangerous procedures associated with iatrogenic intravascular injection resulting in the severe complications. Nonetheless, few studies have determined the explicit arterial localization and topography related to the facial soft tissues and landmarks. Therefore, this study aimed to determine an arterial distribution and topography on the middle forehead region correlated with facial landmarks to grant an appropriate guideline for enhancing the safety injection. The research procedures of this study were approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (IRB No. 786/61, COA No. 149/2019) and conducted in accordance with the Declaration of Helsinki of the World Medical Association (WMA). Cadaveric dissection and ultrasonographic investigation in healthy volunteers were employed to study the forehead region. The cadavers were legally donated for medical education and research, and the participants have signed the informed consent prior to the ultrasound operation. Nineteen Thai embalmed cadavers were discovered with conventional dissection and 14 Thai healthy volunteers were investigated with ultrasonographic examination on the middle forehead. At the level of mid‐frontal depression point, the transverse distance from the medial canthal vertical line to the supraorbital artery were 9.1 mm (superficial branch) and 15.1 mm (deep branch). The depths from skin of the artery were 4.1 mm (superficial branch), and 4.3 mm (deep branch). Furthermore, the frontal branch of superficial temporal artery was detectable in 42.1% as an artery entering the forehead area. At the level of lateral canthal vertical line, the vertical distance of frontal branch was 31.6 mm, and the depth from skin of the artery was 2.7 mm. In conclusion, a proper injection technique could be performed based on an intensive arterial distribution and topography, and ultrasonographic examination prior to the injection is also suggested in order to restrict the opportunity of severe complications.

Support or Funding Information

This presentation was supported by Overseas academic presentation scholarship from Faculty of Medicine Vajira Hospital, Navamindradhiraj University.
Investigating Receptor Expression in Response to Wnt5a Isoforms in Urothelial Carcinoma Cell Lines
Authors: Hemmelgarn Jessica , Coschigano Karen T. , Malgor Ramiro
Published: April 2020

Urothelial carcinoma (UC), the most common form of bladder cancer, has a high rate of recurrence and a low five‐year survival rate for high grade and metastatic tumors. Expression of Wnt5a, a protein that plays a role in many critical processes in cells and is involved in many different molecular signaling pathways, has been shown to correlate with UC development and pathological stage. Recently the Wnt5a gene has been shown to encode two different isoforms (Wnt5a‐L and Wnt5a‐S). Correlation of isoform expression with tumor cell phenotype suggests opposing roles, either as tumor‐promoting or tumor‐suppressing, depending on the cancer type. The effects of each Wnt5a isoform in UC, however, are not well understood.

In order to understand how the Wnt5a isoforms function in the development of UC, it is important to understand which signaling pathways are being activated or inhibited by each isoform. This project aims to identify Wnt5a receptors that are differentially upregulated or downregulated in response to treatment with either Wnt5a‐L or Wnt5a‐S in several different UC cell lines derived from different stages of tumors, as well as in a primary human bladder cell line. The project will include identification and characterization of the Wnt5a receptors normally expressed in the untreated, basal state, information which is currently unknown. Wnt5a isoform treatment will be conducted using conditioned media collected from Chinese Hamster Ovary (CHO) cells that have been genetically modified to express either Wnt5a‐L or Wnt5a‐S. Using western blot analysis, we have already quantified the amount of each isoform present in the conditioned media so that equal amounts of each isoform can be used for cell treatments. We have also used western blot analysis to demonstrate that we can isolate membrane proteins from cell lysates. We will now be treating the cell lines with equal concentrations of Wnt5a‐L, Wnt5a‐S or commercially available Wnt5a as a positive control; isolating the membrane proteins from each cell line after treatment; and applying the isolated membrane proteins to custom antibody arrays in order to examine the expression of 17 different Wnt5a receptors. Results from these experiments will show what receptors are upregulated or downregulated in response to each Wnt5a isoform, which will help elucidate the signaling pathways regulated by each isoform in the pathogenesis of UC.

Support or Funding Information

This project was supported in part by funding from the Honors Tutorial College, Student Enhancement Award, and Heritage College of Osteopathic Medicine Research and Scholarly Activities Committee, all at Ohio University. Special thanks is given to Dr. Karl Willert, who provided the CHO cell lines expressing each Wnt5a isoform.
Anatomical Classification for the Migration of the Distal Catheter of the Ventriculoperitoneal Shunt
Authors: Allouh Mohammed Zohair
Published: April 2020

There have been many reports on migration of the distal catheter of the Ventriculoperitoneal shunt (VPS) since this complication was recognized more than 50 years ago. However, there have been no attempts to analyze its different anatomical patterns and to assess the potential risk of these patterns to the patients. We comprehensively reviewed all reports of distal VPS catheter migration indexed in PubMed and were able to identify three different anatomical patterns of migration based on the catheter extension and the organs involved: (1) internal migration, when the catheter invades any viscus inside the thoracic, abdominal, or pelvic cavity; (2) external migration, when the catheter penetrates through the body wall either incompletely (subcutaneously) or completely (to outside of the body); and (3) compound migration, when the catheter penetrates a hollow viscus and then protrudes through a pre‐existing anatomical orifice. In addition, we analyzed the association between each migration type and several key factors, which could affect the incidence of this complication such as patient’s age, sex, shunt duration, and previous shunt revision. External migration occurred mostly in infants. In contrast, internal migration occurred mostly in adults. A body wall weakness was not a risk factor for catheter protrusion in external migration. Shunt duration was a very critical factor in the migration pattern, as most newly replaced shunts tended to migrate externally. Clinicians must pay close attention to cases of large bowel perforation by the catheter, since these cases were most often associated with intracranial infections. The organ involved in the compound type of migration could determine the route of extrusion, as the bowel was involved in all trans‐anal migrations and the stomach in most trans‐oral cases.